On the influence of PI(4,5)P₂ and PI(3,4,5)P₃-enriched plasma
membrane microdomains on exocytosis
by
James R. Jackson
BS Electrical Engineering, Youngstown State University
A thesis submitted for the degree of Doctor of Philosophy to the faculty of the University of
Cincinnati
Committee Chair: David Richards, PhD
Department of Biomedical Engineering, College of Engineering and Applied Science
University of Cincinnati
2600 Clifton Ave, Cincinnati, OH 45221
Abstract
Although once thought to be a homogenous, randomly-oriented mixture of lipids and proteins, the
cellular plasma membrane has recently been shown to contain highly-ordered and heterogenous
domains which are enriched with specific proteins and lipids. Specific regions have also been shown to
exist within the membrane which are preferred sites for exocytosis to take place. There has been strong
evidence to support the idea that the organization of the lipids and protein machinery required for
exocytosis into a subset of these domains is responsible for the occurrence of preferred exocytic sites,
although the means by which these domains are organized, and the interactions between the different
molecules involved in the exocytosis process remain to be fully understood. In this thesis, I present data
from two studies which investigate the effects of different manipulations performed on the lipids
present in these domains, and their resultant effect on the process of exocytosis.
In the first study, we treated pheochromocytoma 12 (PC12) cells with the cholesterol sequestering drug
methyl-β-cyclodextrin (M-β-CDX) (2 mM) with both short term (10 min.) and long term (2 day)
treatment durations. This experiment was carried out in order to investigate whether removal of
cholesterol from the membrane would result in disruption of organized lipid-enriched domains, as well
as a resultant inhibition of exocytosis. Our results showed that both short and long-term treatment with
M-β-CDX led to an inhibition of exocytosis, however, short-term treatment did not result in a net
decrease in membrane cholesterol levels. That we did not see any significant change in the morphology
of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) or phosphatidylinositol 3,4,5-trisphosphate
(PI(3,4,5)P₃)-enriched domains following treatment seems to imply that cholesterol might not be a
requirement for the organization of these domains, as it is in others. Most significantly, that we saw a
marked inhibition of exocytosis without commensurate cholesterol depletion following short-term M-βCDX treatment seems to imply a second, previously unknown interaction between M-β-CDX and some
ii
component of the plasma membrane, aside from its previously established role as a cholesterol
sequestering agent.
In our second study, we expressed three different forms of fluorescently-tagged pleckstrin homology
(PH) domains (PH-phospholipase C delta 1 (PLCδ1), PH-Protein Kinase B (AKT), and PH-General Receptor
for Phosphoinositides 1 (GRP1)), which are commonly used to label specific phospholipids which they
bind with varying degrees of specificity in biological membranes. The purpose of this study was to
examine the influence of plasma membrane pools of PI(4,5)P₂ and PI(3,4,5)P₃ on the localization of
exocytic sites in the membrane. Our results showed that, of the PH domains tested, PH-PLCδ1 is most
specific for PI(4,5)P₂, and PH-GRP1 is most specific for PI(3,4,5)P₃. Furthermore, expression of all three
PH domains led to significant inhibition of exocytosis and an increased occurrence of secretory vesicles
dwelling at the membrane without the opening of a fusion pore. These results imply that expression of
GFP-tagged PH domains effectively inhibits the exocytic machinery, regardless of specificity for either
PI(4,5)P₂ or PI(3,4,5)P₃.
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iv
Abstract
ii
Abbreviations
ix
1: Introduction
1
1.1: Fundamental Principles of Neuroscience
1
1.1.1: The neuron
1
1.1.2: The synapse
2
1.1.3: Neuroendocrine signaling
3
1.2: The neuronal plasma membrane
5
1.2.1: Composition
5
1.2.2: Membrane dynamics
6
1.3: Lipid rafts
7
1.3.1: Morphology and organization
7
1.3.2: Role of cholesterol in lipid-enriched domains
9
1.3.3: Functional importance of lipid-enriched domains
10
1.4: Overview of exocytosis
11
1.4.1: Secretory vesicles
11
1.4.2: The SNARE proteins
12
1.4.3: Docking, priming, and fusion
12
1.5: Phospholipids in exocytosis
17
1.5.1: PI(4,5)P₂ in plasma membrane microdomains
17
1.5.2: Importance of PI(4,5)P₂ in exocytosis
18
1.5.3: An emerging role for PI(3,4,5)P₃
20
1.5.4: Pleckstrin homology domains
21
1.6: Thesis overview
22
v
2: Materials and Methods
24
2.1: Molecular biology techniques
24
2.1.1: Cell cultures
24
2.1.2: Constructs
24
2.1.3: DNA purification
25
2.1.4: Protein purification
26
2.1.5: Immunohistochemistry
27
2.2: Biochemical assays
28
2.2.1: PH domain affinity assay
28
2.2.2: Cholesterol assay
29
2.3: Microscopy techniques
30
2.3.1: Immersion oil N.A. optimization
30
2.3.2: Widefield imaging
30
2.3.3: TIRF microscopy
31
2.3.4: Digital deconvolution
32
2.4: Calcium dependence
34
2.4.1: Manipulation of extracellular calcium levels
34
2.4.2: Calcium channel blocking with CdCl₂
35
3: Methyl-β-cyclodextrin blocks secretion from PC12 cells
39
without detectable cholesterol depletion
3.1: Abstract
39
3.2: Introduction
39
3.3: Results
42
3.3.1: PIP₂ and PIP₃ antibodies efficiently identify
vi
42
membrane subregions
3.3.2: Cholesterol depletion by methyl-β-cyclodextrin
43
3.3.3: Methyl-β-cyclodextrin blocks secretion without removal
46
of cholesterol
3.3.4: Lipid clusters persist after cholesterol depletion
47
3.4: Discussion
52
4: Pleckstrin homology domains specific for PI(4,5)P₂ and
55
PI(3,4,5)P₃ are equally effective at inhibiting
exocytosis in PC12 cells
4.1: Abstract
55
4.2: Introduction
55
4.3: Results
58
4.3.1: Pleckstrin homology domains demonstrate varying
58
results for PIP₂ and PIP₃
4.3.2: PH-GFP expression inhibits exocytosis
60
4.3.3: PH-GFP expression fosters long duration exocytic events
62
4.4: Discussion
65
5: General Discussion
71
5.1: Overall summary
71
5.1.1: Effect of cholesterol on domains and exocytosis
71
5.1.2: Effects of PH-GFP expression on exocytosis
71
5.2: Influence of methyl-β-cyclodextrin on exocytosis
5.2.1: Interactions between methyl-β-cyclodextrin and
other membrane constituents
vii
72
72
5.2.2: Where do elevated cholesterol levels come from?
76
5.3: Variations in PH domain affinities
78
5.4: Effects of GFP-tagged pleckstrin homology expression
80
5.4.1: Influence of PH-GFP expression on secretion rates
80
5.4.2: PH-PLCδ1-GFP expression has previously been shown to
81
Inhibit secretion
5.4.3: Potential mechanisms of inhibition by PH-GFP expression
81
5.5: Functional implications of PIP-enriched domains in secretion
86
5.6: Are these effects unique to exocytic sites?
87
5.7: General conclusions
88
Bibliography
91
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Abbreviations
ACSF
Artificial cerebro-spinal fluid
AKT/PKB
Protein kinase B
ANF
Ankyrin family protein
BLAST
Basic local alignment search tool
BSA
Bovine Serum Albumin
CCD
Charge coupled device
dSTORM
Direct stochastic optical reconstruction microscopy
GFP
Green fluorescent protein
GLUT4
Glucose transporter 4
GPI
Glycophosphatidylinositol
GRP1
General receptor for phosphoinositides 1
GTP
Guanosine triphosphate
HA
Hemagglutinin
H-β-CDX
Hydroxypropyl-β-cyclodextrin
HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HPA
Hypothalamic-pituitary-adrenal
IPTG
Isopropyl-β-D-thiogalactopyranoside
LDCV
Large dense-core vesicles
M-β-CDX
Methyl-β-cyclodextrin
NA
Numerical aperture
NPC
Niemann Pick Type C
OD
Optical density
PBS
Phosphate buffered saline
ix
PC12
Pheochromocytoma 12
PCR
Polymerase chain reaction
PH domain
Pleckstrin homology domain
PI(3,4,5)P₃
Phosphatidylinositol(3,4,5)trisphosphate
PI(4,5)P₂
Phosphatidylinositol(4,5)bisphosphate
PLCδ1
Phospholipase C delta 1
PTEN
Phosphatase and tensin homologue on chromosome 10
ROI
Region of interest
SKIP
Skeletal muscle and kidney enriched inositol polyphosphate 5-phosphatase
SNAP
Synaptosomal-associated protein
SNARE
soluble N-ethylmaleimide-sensitive factor activating protein receptor
STED
Stimulated emission depletion
TIRF
Total internal reflection fluorescence
x
Introduction
1.1: Fundamental Principals of Neuroscience
1.1.1: The Neuron
One of the final frontiers of scientific understanding is the elucidation of the biophysical mechanisms by
which the phenomenon of cognition takes place in the human brain. The brain itself is a highly
organized and complex collection of over 100 billion individual cells, called neurons, which are
specialized to communicate with each other through chemical signals called neurotransmitters. Over
the course of the past century, our knowledge of the molecular machinery governing the function of the
nervous system has grown dramatically, much of which revolves around the function of those cells
which comprise this system.
Two different classes of cells exist within the central nervous system, one of which is known as glial cells;
the other are called nerve cells, or neurons. Glial cells predominantly act as support cells to neurons,
and also provide firmness and structure to the brain as a whole (Kandel, Schwartz et al. 1995). Another
class of glial cells is responsible for producing an insulating sheath known as myelin, which serves to
cover and insulate large axons, allowing for faster signal transmission(Sherman, Krols et al. 2012). Other
glial cells act as scavengers, removing neurotransmitters from the synapse following signaling (Barres
1991), and in other cases, aide to guide neuronal migration during development (Kandel, Schwartz et al.
1995).
Neurons themselves are a unique class of cells, possessing a cell body similar to other eukaryotic cells,
but morphologically unlike other cells of living systems for two defining characteristics, known as the
axon and the dendrite, which allow them to communicate with one another at greater distances and
with more specificity than could be afforded to cells which required spatial localization next to one
1
another in order to communicate. Axons and dendrites are both long (some axons reach lengths as
great as 1 meter in the human body) and thin, and serve as the information carriers of the neuron. A
typical neuron possesses many dendrites, each of which serves to receive information from several
other pre-synaptic cells, and one axon, which serves to transmit signals to other cells. Signals are sent in
the form of electrical impulses known as action potentials, which are transmitted along the axon to a
series of post-synaptic cells across synapses, which serve as the connection sites between neuronal cells,
and the strength of transmission across which is the basis of learning and memory (Kandel, Schwartz et
al. 1995).
1.1.2: The synapse
Although the brain contains over 100 billion cells, its real computing power stems from the connections
between these cells, known as synapses. Each neuron forms an average of 1,000 synapses with
postsynaptic cells which receive the cell’s outgoing signals, and each makes as many as 10,000
connections with other cells in which it receives incoming signals, resulting in over 100 trillion total
synaptic connections within a single brain (Kandel, Schwartz et al. 1995).
There are two major types of synapses within the central nervous system, known as electrical and
chemical synapses. Electrical synapses serve to quickly transmit signals in the form of action potentials
from one cell to another, at a rate of transmission from pre- to post-synaptic cell of 0.1-0.2 msec.
(Furshpan, Potter 1959). Electrical signals are transmitted from cell to cell via a class of ion channels
called gap junctions, which provide a direct, physical connection between cells for the purpose of quickly
transmitting information (Uehara, Burnstock 1970). While electrical synapses rely on direct connections
between cells, in the form of gap junctions, chemical synapses are divided by a region known as the
synaptic cleft. As stated previously, signal transduction at chemical synapses occurs on a slower
timescale than electrical transduction, occurring in the range of 1-2 msec (Sabatini, Regehr 1999).
2
Although they lack the speed of transmission possessed by their electrical counterparts, chemical
synapses have an advantage which lies in their ability to modulate the strength of connections between
chemically-connected cells.
1.1.3: Neuroendocrine signaling
Along with the importance of chemical signaling at the synapse, exocytosis is also a critical process in
the neuroendocrine system which acts to regulate a multitude of processes in living systems.
Endocrinology is the study of the systems within the body which produce and secrete hormones, as well
as the signaling properties of those hormones as they travel from their cells of origin through the
bloodstream to their final destination. The neuroendocrine system lies at the intersection of the
nervous and endocrine systems, and involves the interaction of both systems in achieving proper
hormonal regulation. In the human body, several processes which are critical for survival are controlled
by the neuroendocrine system, ranging from metabolism (JÉQUIER 2002) to respiration (Abelson, Khan
et al. 2010).
The first clear evidence for interaction between the central nervous and endocrine systems was
presented by Ernst and Berta Scharrer in 1945, which demonstrated through a series of histological
images of neurons from the hypothalamus that these cells exhibited properties like those of the
endocrine system (Scharrer, Scharrer 1945). Prior to this time, several experiments in laboratory
animals had also demonstrated that removal of the pituitary gland resulted in both an inhibition of
growth, as well as dysfunction of the endocrine glands (discussed in (Guillemin 2011)). Geoffrey Harris
next published findings from his research demonstrating that manipulations to the hypothalamus could
directly lead to changes in hormonal secretion from the pituitary gland (Harris 1955). In the late 1950’s,
Roger Guillemin and Andrew Schally independently extracted substances from the hypothalamus which,
when applied to pituitary tissue, led to release of pituitary hormones (discussed in (Valentinuzzi 2010)).
3
Further study led to the elucidation of specific hormonal release factors (Burgus, Butcher et al. 1971,
Matsuo, Baba et al. 1971), and led in 1977 to their being awarded the Nobel Prize for their discoveries
regarding the production of peptide hormones within the brain.
The neuroendocrine system plays a diverse set of roles within a living system, many of which are critical
for survival. For instance, hormones called leptin and ghrelin which are secreted in the digestive tract,
provide feedback to the hypothalamus which can lead to either feelings of hunger or satiation,
controlling an organism’s desire to consume energy (JÉQUIER 2002, Landgren, Simms et al. 2011). The
neuroendocrine system also interacts with the immune system to influence inflammation, acting to
maintain homeostasis between the stress and immune systems when an organism becomes infected by
a pathogen (Verburg-van Kemenade, Van der Aa et al. 2012). As we have just mentioned, there also
exists a clear connection between the neuroendocrine system and stress response, leading to activation
of the sympathetic nervous system and its associated physiological symptoms (Rodrigues, LeDoux et al.
2009).
When the delicate homeostasis maintained by the neuroendocrine system is disrupted, or malfunctions
in some fashion, several different, clinically-relevant disorders can result. For instance, both type 1 and
type 2 diabetes when pancreatic beta cells are inhibited from secreting insulin (Zhao, Ohara-Imaizumi et
al. 2010), resulting in an inability to process sugar in the affected individual. Also, many traumatic brain
injury patients experience disruptions in the hypothalamic-pituitary-adrenal (HPA) axis following injury,
which can in some cases lead to death (Behan, Phillips et al. 2008). Patients are particularly at risk for
hypopituitarism after injury, which can result in a number of resulting conditions including
glucocorticoid deficiency (Kokshoorn, Wassenaar et al. 2010). These findings and others indicate the
importance of the neuroendocrine system in a number of clinically-relevant disorders, and illustrate the
importance of neuroendocrine study to the field of biomedical research.
4
The neuroendocrine system acts to maintain the delicate balance of homeostasis between a series of
systems within living organisms. The proper functioning of this system is critical to the growth,
development, maintenance, and ultimately, survival, and dysfunction or perturbation of this system can
result in any number of medical conditions. The study of vesicular release and its mechanisms,
therefore, is of direct relevance to the field of neuroendocrine secretion, with implications covering a
range of diseases and disorders which can occur within living organisms.
1.2: The neuronal plasma membrane
1.2.1: Composition
The plasma membrane serves to provide a selectively-permeable barrier which separates and protects
the intracellular organelles from the extracellular environment. The membrane is comprised of a
mixture of phospholipids and proteins which act in unison to allow the necessary components to cross
into and out of the cell, while preventing any unnecessary and/or harmful substance from access to the
cell’s intricate interior.
All cell membranes are based on the general structure of the lipid bilayer, which consists of
approximately 10⁹ lipid molecules in a typical cell (Alberts, Johnson et al. 2008). The types of lipid
molecules which are present within cell membranes are known as amphiphillic lipids, because they have
both a hydrophilic (polar) and a hydrophobic (nonpolar) end. The most readily-available form of lipids in
the plasma membrane are the phospholipids, which have a polar head group and two non-polar tail
groups. Another key membrane lipid, known as sphingomyelin, is similar in structure to the
phospholipids, but is built from sphingosine rather than glycerol. In fact, the combined lipids of
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin comprise more
than half of the total mass of most cell membranes (Bretscher 1973). When dispersed within an
aqueous environment, the hydrophobic tails cluster together in order to minimize the free energy of the
5
net system by contacting the fewest number of water molecules possible, and this clustering together of
the hydrophobic tails results in the spontaneous formation of lipid bilayers (Alberts, Johnson et al.
2008).
Along with the lipids which contribute the fluidity and basic structure of the cell membrane, a series of
proteins, known as membrane proteins, exist within the plasma membrane and contribute between 25
and 75% of the net mass of the plasma membrane, depending on cell type (Bretscher 1973). There are
several different means by which membrane proteins can be associated with the plasma membrane,
one of which is to project a protein region, known as a trans-membrane domain, through the lipid
bilayer. Trans-membrane domains tend to be hydrophobic, like the hydrocarbon tails of the lipids in the
inner leaflet of the bilayer, and are usually surrounded by hydrophilic domains. Other membrane
proteins associate with only the cytosolic monolayer of the membrane through insertion of an
amphiphilic α helix, and in still other cases, proteins associate with the membrane by way of a
glycosylphosphatidylinositol (GPI) membrane anchor (Alberts, Johnson et al. 2008).
1.2.2: Membrane dynamics
As previously mentioned, the plasma membrane contains a series of ion channels which selectively open
and close in order to allow ions to flow across the membrane, thus varying the polarized electrical
charge across the membrane. At rest, a neuron contains an excess of negative charge on the
intracellular side of the membrane, known as resting membrane potential, and is usually within the
range of -60 to -70 millivolts, relative to the extracellular space (Kandel, Schwartz et al. 1995). The four
most common ions which traverse the membrane and influence membrane potential are potassium,
sodium, chloride, and calcium. At rest, the neuronal membrane maintains an excess of sodium ions
outside of the membrane, and an excess of potassium ions within the intracellular area. Through
passive diffusion, the number of sodium ions passing through the membrane into the cell roughly equals
6
the number of potassium ions which are released through the membrane, however, if this passive
diffusion alone were allowed to occur over a long period of time, the gradients would eventually
dissipate, thus lowering the membrane potential. The gradient is maintained by the active transport of
sodium and potassium ions across the channel by sodium-potassium pumps, which are proteins in the
membrane which serve to move three sodium ions to the extracellular space and two potassium ions to
the intracellular space at a time, thus keeping an excess of intracellular potassium and extracellular
sodium and holding the membrane potential to a relatively constant voltage level (reviewed in (Wright
2004)). During an action potential, this balance is disrupted by the opening of sodium channels, which
allows sodium ions to rush into the cell, causing the net membrane potential to rise sharply. The
membrane potential does not remain at this elevated level for long however, due to the subsequent
opening of potassium channels, which causes potassium to flow out of the cell, along its own
concentration gradient, and lowering the neuronal membrane potential again to a value nearer its own
resting potential. Following a recovery phase in which ion channels and the sodium-potassium pumps
work in concert to return the cell to its normal resting potential, the polarity becomes stabilized, and the
cell is ready to fire again at the arrival of the next action potential (Kandel, Schwartz et al. 1995).
1.3: Lipid Rafts
1.3.1: Morphology and Organization
Singer and Nicholsen suggested the fluid mosaic model of the plasma membrane in 1972 (Singer,
Nicholson 1972). According to this model, the membrane was suggested to consist of a randomlyoriented mixture of lipids which were arranged into two layers of phospholipids, with their hydrophobic
cores facing one another, and a mixture of transporters, channels, and other proteins randomly
intercalated among the lipids. This model was considered to be an accurate representation of the
plasma membrane until it was shown that clusters of lipids were present in the plasma membrane (Lee,
7
Birdsall et al. 1974), which suggested a previously-unknown heterogeneity to the composition of the
plasma membrane. Schnitzer et al., (Schnitzer, McIntosh et al. 1995), were the first to show that
different classes of ordered domains were contained within the detergent-resistant membrane
fractions. Shortly thereafter, it was shown that other parts of the plasma membrane were organized
into domains as well, only some of which were contained within detergent-resistant membrane
fractions (Pike, Han et al. 2002). Currently, the consensus is that lipid rafts are defined to be small (10200 nm.) domains that are enriched in various phospho- and sphingolipids in the plasma membrane
(Pike 2004).
One of the earliest functional forms of lipid rafts to be discovered were the caveolae, which are defined
as invaginations at the cell surface which are internalized to form pinocytic vesicles (Anderson 1998).
Caveolae generally have a diameter of 25-150 nm, and are found both individually and clustered
together into small groups within the plasma membrane, and are stabilized by the protein caveolin (Le,
Guay et al. 2002). Schnitzer et al. (Schnitzer, McIntosh et al. 1995) showed that caveolae could be
separated from a low-density Triton-X-100-resistant membrane fraction, and suggested that more than
one type of ‘raft’ might be present within the aforementioned detergent-resistant membrane fractions.
The exact composition of lipid rafts remains to be clearly defined, most likely as a result of the profuse
heterogeneity with which they occur. To date, over 200 different molecular components have been
suggested to occur within rafts (Foster, de Hoog et al. 2003). Detergent-based raft isolations have
shown that sphingomyelin, for instance, represents roughly 10-15% of the total amount of lipid
contained within rafts (Brown, Rose 1992). Glyco-sphingolipids have similarly been shown to comprise
10-20% of raft lipids. Rafts are similarly depleted in other lipids, including the glycerophospholipids
phosphatidylcholine and phosphatidylethanolamine, which compose only 30% of raft lipids, compared
with 60% of un-ordered plasma membrane (Brown, Rose 1992, Pike, Han et al. 2002, Prinetti, Chigorno
8
et al. 2000). Aside from lipid enrichment, several membrane proteins have also been shown to
associate with lipid rafts, both in fractionation and microscopy-based experiments. As previously
mentioned, the organizing protein caveolin colocalizes with, and appears to arrange caveolae . Other
proteins which have been shown to colocalize with raft fractions include the soluble N-ethylmaleimidesensitive factor activating protein receptor (SNARE) protein syntaxin (van den Bogaart, Meyenberg et al.
2011), synaptosomal attachment protein 23 (SNAP-23) (Chamberlain, Burgoyne et al. 2001), and a
number of GPI-anchored proteins (reviewed in (Pike 2004)).
1.3.2: Role of Cholesterol in Lipid-Enriched Domains
In many cases, cholesterol has been shown to be key to lipid raft formation and organization, and occurs
at levels 3-5 times greater in detergent-resistant membrane fractions compared to non-domain
membrane fractions (Pike 2003). This results from the electrostatic properties of phospho- and
sphingolipids, as well as those of cholesterol interaction with those lipids. Generally, phospholipids
contain both saturated and unsaturated acyl chains, which tend to exist in an unordered phase, and
sphingolipids, which have longer, saturated acyl chains, cluster together into tight domains, but which
lack lateral mobility within a membrane (Brown, London 2000). When cholesterol is added to model
membranes containing phospho- and sphingolipids, these sphingolipid-enriched domains become
laterally mobile, similar to the mobility found in the disordered phase (Brown, London 1998, Brown,
London 2000)).
Much of what is known about the influence of cholesterol on lipid raft formation stems from the
perturbation of cholesterol levels in the plasma membrane, and one of the most commonly-used
methods of cholesterol removal involves cells being treated with a drug called methyl-β-cyclodextrin
(Giocondi, Milhiet et al. 2004, Kabouridis, Janzen et al. 2000, Kato, Nakanishi et al. 2003). The drug
works by approaching the plasma membrane at such a close proximity that cholesterol molecules are
9
able to be exchanged from the membrane into the hydrophobic core of cyclodextrin (Yancey,
Rodrigueza et al. 1996). Cyclodextrin treatment has been shown to lead to the disappearance of
membrane caveolae (Dreja, Voldstedlund et al. 2002), as well as inducing changes to the physical
properties of detergent-resistant fractions (reviewed in (Zidovetzki, Levitan 2007)). Several studies have
examined the effect of cholesterol depletion on exocytic activity, resulting from the apparent function of
lipid-enriched domains in SNARE protein organization. Indeed, following cholesterol depletion several
studies have demonstrated changes in the distribution of the SNARE proteins syntaxin 1 (Lang, Bruns et
al. 2001, Low, Vasanji et al. 2006), syntaxin 3 (Low, Vasanji et al. 2006), and SNAP-23 (Chamberlain,
Gould 2002). Cholesterol depletion has also been shown in many cases to lead to the inhibition of
exocytosis (Kabouridis, Janzen et al. 2000, Kato, Nakanishi et al. 2003, Tarasenko, Sivko et al. 2010),
which has been interpreted as the result of raft disruption, and resultant interference with the proper
workings of the exocytic machinery.
1.3.3: Functional Importance of Lipid-Enriched Domains
Along with enrichment of membrane lipids, studies have also shown that several membrane proteins
colocalize with enriched lipids (Aoyagi, Sugaya et al. 2005, Chamberlain, Burgoyne et al. 2001), and it is
this capability to arrange and organize key proteins within close proximity to one another which seems
to be a key functional advantage afforded to cellular function by the existence of ordered domains in
the membrane. During T cell receptor activation, GPI-anchored proteins which are associated with lipid
enriched domains become immobilized into nanoscale clusters in the membrane, bringing proteins
which are required to interact as part of the activation process within close proximity to each other
(Kaizuka, Douglass et al. 2009). Other proteins, such as the influenza virus haemagglutinin (HA), have
been shown to localize to lipid domains due to interactions with other raft-associated lipids or proteins
(Scheiffele, Roth et al. 1997). Also, certain proteins have been suggested to localize to membrane rafts
10
due to the length of their transmembrane domains and the increased thickness of lipid domains (Nezil,
Bloom 1992, munro 1995). As discussed below, another key set of proteins which are arranged by lipid
rafts are the SNARE proteins, which are required for exocytosis (Salaün, Gould et al. 2005), thus the
collection of multiple SNARE proteins within the close proximity of a single raft likely plays a key role in
the efficient transmission of synaptic signals.
1.4: Overview of Exocytosis
1.4.1: Secretory Vesicles
Aside from the constitutive exocytosis which all cells undergo, neurons (and other secretory cell types)
possess a second, regulated secretory pathway which they utilize to send chemical signals to one
another in the form of neurotransmitters. In order to efficiently transfer these chemical constituents
through the intracellular environment, neurotransmitters are concentrated and packaged into small,
bilayered packages known as vesicles. In fact, two types of secretory vesicles occur in neurons: large,
dense core vesicles which are used to transmit protein and peptide signals and the smaller synaptic
vesicles which transmit smaller neurotransmitter molecules.
Large, dense-core vesicles (LDCVs) originate from and are packaged within the trans Golgi network, and
then fuse with one another outside the Golgi network, with their cargo becoming more concentrated as
a result (Traub, Kornfeld 1997). Once the dense-core vesicles have matured, meaning that they have
become tightly packed with molecular cargo, the vesicles are then delivered to the plasma membrane
by motor proteins which transport the vesicles along cytoskeletal filaments within the cell (Oheim,
Stuhmer 2000). Because of the rapid pace at which small neurotransmitters are released during a rapid
series of action potentials, the smaller synaptic vesicles are required to release their chemical signals
and then to re-load and prepare for another round of exocytosis at much faster rates than can be
achieved in transport between the trans Golgi network and the plasma membrane. This is especially the
11
case in neuronal cells, in which the dense core vesicles must traverse a long, directed path through the
axon to reach the synapse. Synaptic vesicles, however, are able to re-load with neurotransmitter and
re-enter the releasable pool on a much faster timescale as a result of synaptic vesicle recycling. This is
achieved by neuronal reuptake of the vesicular membrane following exocytosis, and subsequent reloading of the vesicle at the synapse so that the vesicle is quickly prepared to undergo another round of
exocytosis (Li, Foss et al. 2011)
1.4.2: The SNARE Proteins
In order for exocytosis to occur, the vesicular membrane has to fuse with the neuronal plasma
membrane so that the vesicular cargo can be released through the plasma membrane to the
extracellular space. The SNARE proteins syntaxin and SNAP-25, have been shown to localize
predominantly to the plasma membrane and are required for vesicular exocytosis (Salaün, Gould et al.
2005, van den Bogaart, Meyenberg et al. 2011). These proteins, along with the vesicle-associated
SNARE protein synaptobrevin, come together to form a stable complex which consists of four α-helices,
two of which are contributed by SNAP-25, with one each present from syntaxin and synaptobrevin (An,
Almers 2004). As these four proteins complex together, the vesicular membrane and the cellular plasma
membrane are brought into close proximity and the curvature of the membranes is also affected in such
a manner that the opposing bilayers fuse together, allowing a fusion pore to open between the
intravesicular and the extracellular space (Shi, Shen et al. 2012).
1.4.3: Docking, Priming, and Fusion
Once a vesicle is loaded with cargo, it must approach the membrane at a site which contains the
necessary protein machinery for vesicular fusion. This process begins with vesicular transport along
microtubules to exocytic sites on the membrane (Kamal, Goldstein 2000), which are organized by lipid
rafts as described previously (Couteaux, Pecot-Dechavassine 1970, Peters, Palay et al. 1991). The first
12
step in vesicular attachment to the active zone is known as tethering, and involves the attachment of a
loaded vesicle to the docking site (Matteoli, Takei et al. 1991, Geppert, Goda et al. 1997). Biochemical
studies have shown that tethering is a guanosine triphosphate (GTP)-ase-dependent process which
utilizes the Rab3A-Rabphilin protein (reviewed in (Szule, Harlow et al. 2012)). After the vesicle becomes
tethered to the docking site, the vesicle actually becomes docked to the membrane, which serves the
purpose of maintaining and stabilizing fusion-ready vesicles at the active zone for release.
Experimentally, a vesicle is generally considered to be docked when the vesicular membrane is held
within a certain distance of the plasma membrane, or when the presence of a ‘contact patch’ between
the vesicle and the plasma membrane occurs (Verhage, Sørensen 2008).
Two questions which remain to be fully elucidated with regard to docking are whether docking is a
required step in the vesicular fusion process, and whether all docked vesicles become primed and
undergo membrane fusion. For instance, a study in bovine chromaffin cells showed that 20% of
vesicular fusion events occurred within a span of less than 300 milliseconds, seemingly on a faster
timescale than would be required if the vesicle were to have paused to become docked to the exocytic
site prior to fusion (Allersma, Wang et al. 2004). Another study suggested that vesicles showed equal
probabilities to undergo membrane fusion regardless of their distance to the membrane prior to release
(Kishimoto, Liu et al. 2005). Verhage and Sorenson (Verhage, Sorensen 2008) suggested that these
conclusions were made based on the assumption that electron micrographs of docked vesicles taken at
resting state could be compared to TIRF microscopy-visualized fusion events after stimulation, and that
these two conditions cannot be reliably compared due to the speeding of the priming and fusion
reactions in the high-calcium environment that follows stimulation. The authors also cited several
studies in which the number of docked vesicles was much greater than the number of vesicles which
underwent exocytosis (Parsons, Coorssen et al. 1995, Steyer, Horstmann et al. 1997, Plattner, Artalejo et
al. 1997), and proposed a model of vesicle docking in which some vesicles can become ‘dead-end’
13
docked, meaning that the vesicles become attached and stabilized at the membrane, but are for an
unknown reason unable to undergo exocytosis.
Several proteins have been suggested to play a role in the machinery responsible for docking, probably
the most commonly-cited of which are the sec1/munc18 genes. Interactions between these genes and
syntaxin have been shown to occur in several experiments with cleavage or mutation of either gene
resulting in a significant drop in the number of docked vesicles at the membrane (Voets, Toonen et al.
2001, Toonen, Kochubey et al. 2006, Wit, Cornelisse et al. 2006) Studies have also shown that the other
SNARE proteins, unlike syntaxin, do not appear to form any critical docking complexes with syntaxin
(Sørensen, Nagy et al. 2003, Borisovska, Zhao et al. 2005). Granuphilin also appears to be involved in
docking, as studies using pancreatic beta cells have demonstrated a significant decrease in the number
of docked vesicles following granuphilin knockout (Gomi, Mizutani et al. 2005). Expression of rabphillin
in chromaffin cells also led to an increased number of docked vesicles, although expression in neurons
led to an inhibition of secretion, implying that rabphilin may lead to a form of ‘dead-end’ docking as
described above (Tsuboi, Fukuda 2005, Deak, Shin et al. 2006). The rab proteins rab3A, rab27A, and
rab27B have also been shown to influence docking in several different cell lines (van Weering, Toonen et
al. 2007, Martelli, Baldini et al. 2000, Gomi, Mori et al. 2007), in the case of rab3A, potentially in the
form of direct interaction with the syntaxin/Munc18 complex (Graham, Handley et al. 2008), or else
indirectly through an interaction with granuphilin (Coppola, Frantz et al. 2002).
Finally, several studies
have shown that synaptotagmin has a strong and direct influence on the number of apparently docked
vesicles at the membrane (Geppert, Goda et al. 1994, Littleton, Stern et al. 1993, Jorgensen, Hartwieg et
al. 1995, Reist, Buchanan et al. 1998).
Once a vesicle has become docked at an exocytic site, the next step in the fusion process, called priming,
involves at least a partial assemblage of the fusion machinery and preparation of the vesicle for fusion
14
immediately upon receipt of an increase in intracellular calcium levels (Verhage, Sorensen 2008).
Although the process of vesicular priming appears to be a requisite for fast neurotransmitter release
after stimulation, the exact mechanism and machinery by which priming takes place remains to be fully
characterized.
Several studies have shown that the complexins, small, soluble adaptor proteins, are required for
vesicular priming to take place (Cai, Reim et al. 2008). There are four different forms of complexins
which are expressed in mammals; complexin-1 and complexin-2, which are present in all cells
(McMahon, Missler et al. 1995), and complexin-3 and complexin-4, which are predominantly expressed
in retinal cells (Reim, Wegmeyer et al. 2005). Complexins bind via a central helix to a groove on the
surface of the SNARE complex (Chen, Tomchick et al. 2002, Bracher, Kadlec et al. 2002), apparently at a
step after the zippering of the SNARE complex has begun, and apparently act to prevent complete
zippering of the SNARE complex until the arrival of a calcium signal, and subsequent removal of
complexin from the SNARE complex, most likely by synaptotagmin (as described below). Other studies
have alternatively suggested that complexin might act to stabilize partially zippered SNARE complexes,
and to prime these complexes for activation by synaptotagmin following an increase in local intracellular
calcium concentration (Xue, Craig et al. 2010). According to Maximov et al., (Maximov, Tang et al.
2009), it is the N-terminal of complexin which is responsible for activation of the fusion machinery. Yang
et al., (Yang, Kaeser-Woo et al. 2010) showed that the N-terminal is also apparently responsible for
complexin’s priming function. Kaeser-Woo et al, (Kaeser-Woo, Yang et al. 2012), showed that deletion
of the C-terminus of complexin resulted in a significant depletion of the readily releasable pool of
vesicles in cortical neurons, apparently indicating that the C-terminus is required for complexin’s priming
functionality.
15
As we have stated above, complexin apparently works in concert with another key priming protein
known as synaptotagmin, commonly known to act as a calcium sensor which drives fast exocytosis
following membrane depolarization (Chapman 2002). Synaptotagmin possesses a membrane-spanning
domain, an intralumenal domain, and a large cytoplasmic region which contains two calcium-binding C2
domains, known as C2A and C2B (Fukuda, Mikoshiba 2001, Sugita, Hata et al. 1996). The C2 domains
themselves consist of 8-stranded β-barrels, and have two calcium binding pockets (Augustine, Charlton
et al. 1987, Elferink, Peterson et al. 1993). Synaptotagmin is integrated into the vesicular membrane by
its membrane-spanning domain, and draws the vesicle close to the plasma membrane by association of
the C2B domain with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) in the plasma membrane, and
interactions between the C2 domains and the plasma membrane protein syntaxin (Chapman 2008).
More specifically, the C2A domain appears to directly penetrate the lipid bilayer at the location of two of
its calcium binding sites (Bai, Earles et al. 2000, Chapman, Davis 1998, Davis, Bai et al. 1999). Because
the nature of this interaction is dependent on electrostatic interactions between positively charged
residues in the calcium binding domains and negatively charged polar headgroups in lipid bilayers, the
strength of the C2 domain’s binding affinity is, in many cases, stronger in the presence of a plasma
membrane (Brose, Petrenko et al. 1992, Davis, Bai et al. 1999, Nalefski, Wisner et al. 2001). The isolated
C2B domain, unlike the C2A domain, does not appear to exhibit a strong binding affinity for
phospholipid bilayers; however when linked to the C2A domain, the C2B domain does display a strong
binding affinity for phosphatidylserine on a similar scale to that demonstrated by the C2A domain
(Fergestad, Broadie 2001). The isolated C2 domains of synaptotagmin have also been shown to
promote fusion even without its trans-membrane domain, likely as a result of membrane de-formation
which is caused when the C2 domains insert into the plasma membrane following calcium entry
(Chapman 2008). Chapman et al., (Chapman 2002) have proposed a model in which synaptotagmin
aides the vesicle priming process by binding the vesicular membrane via its transmembrane domain, and
16
binding PI(4,5)P₂ in the plasma membrane with its C2B domain. Following membrane depolarization,
local intracellular calcium levels rise and the C2 domains penetrate the plasma membrane, acting to aide
in pulling the vesicular membrane closer to the plasma membrane, and also inducing a local “bulging” in
the membrane at the vesicle fusion site, which probably aides in fusion of the vesicular and plasma
membranes (reviewed in (Chapman 2008)). At the same time, another protein, which has been
suggested to be synaptotagmin, acts to remove complexin from the partially-zippered SNARE complex,
allowing the four alpha barrels to fully zipper for fusion to take place (reviewed in (Jahn, Fasshauer
2012)).
Munc13, a critical SNARE-interacting protein, was initially discovered as a gene which, when mutated,
resulted in an ‘uncoordinated’ phenotype (Maruyama, Brenner 1991), however little else was known
about its function, or the molecular basis for its phenotypic effects. The N-terminal of Munc13 contains
a C2A domain, but unlike synaptotagmin, this C2 domain does not appear to possess any calcium
binding sites. Following the C2 domain is a long sequence whose purpose remains to be understood,
followed by a calmodulin binding sequence and a C1 domain (Sudhof 2012). Munc13 appears to serve a
two-fold purpose at the exocytic fusion site, first serving to prime the SNARE protein machinery for fast
assembly and subsequent vesicle fusion pending calcium influx, and secondly, influencing synaptic
plasticity through modulation of its priming influence on presynaptic signals (Sudhof 2012). Several
studies have pointed to Munc13 as playing a critical role in changing syntaxin from a closed to an open
conformation, thus making it available to complex with the other SNARE proteins for fusion to occur
(Gerber, Rah et al. 2008, Ma, Li et al. 2011). Munc13 modulation is achieved primarily through
interaction with the RIM proteins, which serve to break up dimerized C2A domains of Munc13 proteins,
thus activating the un-dimerized proteins to exert their priming influence on local syntaxin molecules
(Dulubova, Lou et al. 2005).
17
1.5: Phospholipids in exocytosis
1.5.1: PI(4,5)P₂ in plasma membrane microdomains
The phospholipid phosphatidylinositol (4,5) bis-phosphate (PI(4,5)P₂) has been shown to play a critical
role in the organization of several components of the protein machinery which are required for
exocytosis. That it is able to play a key role in exocytosis despite its relatively low concentration in the
membrane most likely results from its tendency to occur in enriched domains within the membrane. As
has been discussed previously, the plasma membrane consists in part of lipid-enriched microdomains, at
least a sub-set of which have been shown to be enriched in PI(4,5)P₂. PI(4,5)P₂-enriched microdomains
most likely originate from recruitment of kinases such as phosphatidylinositol-4-phosphate 5-kinase to
the membrane, where they act on local pools of PI(4)P to increase the concentration of PI(4,5)P₂, and/or
through lateral sequestering of PI(4,5)P₂ by proteins which bind and interact with PI(4,5)P₂ (reviewed in
(Wen, Osborne et al. 2011). Wang and Richards (Wang, Richards 2012) showed that PI(4,5)P₂ was
present in enriched microdomains in the plasma membranes of PC12 cells at an average size of 64 nm,
using an antibody which was specifically targeted to PI(4,5)P₂ and a super-resolution microscopy
technique known as Direct Stochastic Optical Reconstruction Microscopy (dSTORM). These results were
in agreement with another study which was conducted by van den Bogaart et al., (van den Bogaart,
Meyenberg et al. 2011), which showed PI(4,5)P₂-enriched domains to be present, also in PC12 cell
membranes, at an average size of 73 nm using Stimulated Emission Depletion (STED) microscopy.
1.5.2: Importance of PI(4,5)P₂ in exocytosis
Phosphoinositides consist of a long, fatty-acyl chain which is typically oriented within a lipid bilayer, and
is linked by a glycerol backbone to a myo-inositol headgroup. This headgroup can be phosphorylated at
the 3’, 4’ and 5’ hydroxyl positions, which then generates seven different possible phosphoinositides
(Sasaki, Takasuga et al. 2009). Following the discovery that type 1 phosphatidylinositol-4-phosphate 5kinase was an essential protein for exocytosis priming, the importance of PI(4,5)P₂ in exocytosis came
18
into knowledge (Hay, Martin 1992, Hay, Martin 1993, Hay, Fisette et al. 1995). Although multiple
PI(4,5)P₂ populations have been suggested to exist throughout the cell (Watt, Kular et al. 2002), the
plasma membrane pool appears to be the only one which has importance with regard to exocytosis
(Holz, Hlubek et al. 2000, Micheva, Holz et al. 2001, Lawrence, Birnbaum 2003, Hammond, Dove et al.
2006).
PI(4,5)P₂ microdomains have been shown to colocalize with and exert effects on several proteins which
play key roles in exocytosis. Possibly the most significant of these is its co-localization with the SNARE
protein syntaxin in microdomains, which was first demonstrated by Aoyagi et al (Aoyagi, Sugaya et al.
2005), and more recently, by van den Bogaart et al., (van den Bogaart, Meyenberg et al. 2011). The
former study also showed that PI(4,5)P₂ / syntaxin-enriched domains also co-localized with docked large,
dense-core vesicles. In the latter study, it was demonstrated that microdomains enriched in PI(4,5)P₂
co-localized with syntaxin-enriched domains, with PI(4,5)P₂ enriched approximately 1.9-fold and
syntaxin enriched 5.5-fold over non-domain membrane levels. Importantly, when the authors
expressed a phosphatase which de-phosphorylated the 5’-hydroxyl group of PI(4,5)P₂, they found a 3.7fold reduction in the number of syntaxin-enriched clusters; thus implying that PI(4,5)P₂ not only colocalizes with, but is a requirement for the clustering of syntaxin molecules at exocytic sites. As was
previously mentioned, PI(4,5)P₂ has also been shown to be a molecular binding target of the C2 domains
of synaptotagmin, and that the prongs of these domains apparently insert into PI(4,5)P₂ in the plasma
membrane following calcium entry (Kuo, Herrick et al. 2011).
Along with its interactions with secretion-associated proteins, PI(4,5)P₂ has also been shown to aide in
the coordination and organization of the actin cytoskeleton. This phenomenon was first shown through
PI(4,5)P₂’s ability to bind the actin-capping proteins profilin and gelsolin (Janmey, Stossel 1987), and
several studies have since shown greater evidence of PI(4,5)P₂ influence on actin organization. For
19
instance, the protein moesin, which acts in connecting the actin cytoskeleton to the plasma membrane,
has been shown to require PI(4,5)P₂ binding at two unique binding sites in order for moesin to be
activated (Ben-Aissa, Patino-Lopez et al. 2012). Neural Wiskott Aldrich Syndrome Protein (N-WASP), a
protein which acts to promote actin polymerization, also becomes activated in an actin-dependent
manner (Benesch, Lommel et al. 2002). The Rho GTP-ase cell division control protein 42 (Cdc42) has
also been shown to induce actin polymerization in concert with PI(4,5)P₂ in a number of studies (Chen,
Ma et al. 2000, Higgs, Pollard 2000, Tomasevic, Jia et al. 2007), and a study in adrenal chromaffin cells
demonstrated that actin dynamics were mediated by PI(4,5)P₂ levels in the plasma membrane and that
disruption of actin polymerization exerted an immediate effect on ATP-dependent exocytosis (Bittner,
Holz 2005).
1.5.3: An emerging role for PI(3,4,5)P₃
Few studies have examined the influence of PI(3,4,5)P₃ directly on exocytosis, however several studies
have shown that kinases which lead to increases in plasma membrane PI(3,4,5)P₃ have emerged which
suggest a potential role for PI(3,4,5)P₃ in secretion. For instance, phosphatase and tensin homologue on
chromosome 10 (PTEN), acts on PI(3,4,5)P₃ to hydrolyze the 3’-phosphate, and overexpression of PTEN
has been shown to inhibit the uptake (and presumably release) of glucose (Nakashima, Sharma et al.
2000). Another study in adipose tissue cells demonstrated that knocking out PTEN resulted in both
hypersensitivity to insulin, and also resistance to streptozotocin-induced diabetes, presumably as a
result of increased PI(3,4,5)P₃ levels in the plasma membrane. A study in mouse and rat myoblasts
demonstrated that silencing of skeletal muscle and kidney enriched inositol polyphosphate 5phosphatase (SKIP), another PI(3,4,5)P₃ phosphatase, although one that acts on the 5’-phosphate, led to
increased levels of insulin signaling, apparently through the PI(3,4,5)P₃-mediated translocation of the
glucose transporter 4 (GLUT4) to the membrane, and its subsequent effect on glucose release.
Interestingly, the same study examined the timescale of influence of PTEN and SKIP, and determined
20
that they act on different pools of PI(3,4,5)P₃, specifically, PTEN acts to control PI(3,4,5)P₃ levels during
resting conditions while SKIP acts on PI(3,4,5)P₃ in an insulin stimulation-dependent manner, although
both phosphatases had an inhibitory effect on insulin secretion (Ijuin, Takenawa 2012).
1.5.4: Pleckstrin homology domains
Although rafts have been successfully visualized using a number of immunohistochemical methods, cells
in these studies typically are required to undergo fixation during the immunostaining process. A useful
means for visualizing plasma membrane lipids in living cells has been developed through the use of
fluorescently tagged forms of a protein motif known as a pleckstrin homology domain. Pleckstrin
homology domains are highly conserved domains which are contained in many proteins, and which aide
in the targeting of those proteins to the membrane, at least in part through the affinity of the PH
domain for a certain phospholipid within the membrane (Falasca, Logan et al. 1998). PH domains are
useful tools for lipid visualization, in part, because domains from different proteins have varying
affinities and levels of specificity for certain phospholipids. For instance, the PH-domains from the
general receptor for phosphoinositides 1 (GRP1) has been used as a specific marker for plasma
membrane PI(3,4,5)P₃ (Pilling, Landgraf et al. 2011). Alternatively, van den Bogaart et al. (van den
Bogaart, Meyenberg et al. 2011), used the PH domain from phospholipase C delta 1 to visualize PI(4,5)P₂
in the plasma membranes of pheochromocytoma 12 (PC12) cells. Expression of these domains and their
subsequent localization to the membrane is generally utilized as a marker for membrane lipids,
however, little is known about what influence, if any, their presence at the membrane and specifically at
exocytic release sites might have on the actual exocytic machinery. In fact, Holz et al., (Holz, Hlubek et
al. 2000) showed that overexpression of PH-PLCδ1-GFP in chromaffin cells led to a significant drop in
secretion rates, suggesting that PH-GFP overexpression might have a negative impact on the intricate
workings of the exocytic machinery. Due to the prevalent use of GFP-tagged PH domains in membrane
21
lipid visualization, the further elucidation of such an effect could have significant impact on future
experimental design in the field of synaptic transmission and membrane lipid research.
1.6: Thesis overview
In this thesis, I present the results of two studies which examined different aspects of lipid-enriched
membrane domains in exocytosis. In the first study, we examined the influence of the cholesterol
sequestering drug methyl-β-cyclodextrin (M-β-CDX) on cholesterol removal and exocytosis. Our results
showed that treatment of PC12 cells with 2 mM M-β-CDX for ten minutes led to significant inhibition of
exocytosis without any substantial removal of cholesterol from the membrane. Treatment for two days,
however, did lead to membrane cholesterol removal along with secretion inhibition.
Immunocytochemistry labeling of PI(4,5)P₂ and PI(3,4,5)P₃-enriched lipid microdomains showed that
they were not, at least morphologically, affected by M-β-CDX treatment. These findings suggest a
previously unknown secondary interaction between M-β-CDX and some component of the plasma
membrane, which leads to inhibition of secretion independently of cholesterol.
Our second study examined the influence of overexpressing GFP-tagged pleckstrin homology domains
on exocytosis. For this study, we expressed PH domains from three different proteins (PH-AKT, PHGRP1, and PH-PLCδ1), tagged with the fluorophore GFP, then examined the influence of PH-GFP
expression on secretion by visualizing individual exocytic events using Total Internal Reflection
Fluorescence (TIRF) Microscopy. We also conducted an assay which tested the binding affinities of each
of the three PH domains in our study for several different membrane phospholipids, in order to
determine the relative affinities for domains enriched in PI(4,5)P₂ and PI(3,4,5)P₃ in the membrane. Our
results demonstrated that expression of all three PH domains led to inhibition of secretion, and that PH
domains specific for PI(3,4,5)P₃ were as efficient as those specific for PI(4,5)P₂ in doing so. We also
found a significant increase in the number of vesicles which exhibited long-duration dwell times at the
22
membrane, implying that exocytosis is inhibited at a step prior to the opening of a fusion pore between
the intravesicular lumen and the extracellular space.
23
Materials and Methods
2.1: Molecular Biology Techniques
2.1.1: Cell Cultures
For our experiments, we selected PC12 cells (purchased from ATCC, Manassas, VA) as our model cell
line. PC12 cells provide several advantages over other commonly-used neuroendocrine cell models in
that they can be easily cultured, respond well to pharmacological manipulation, and are less susceptible
to the variability inherent in other cell lines which are generally derived from laboratory animals
(Westerink, Ewing 2008). Another advantage to the use of PC12 cells is that they do not develop into
synapse-forming post-mitotic neurons unless treated with nerve growth factor to induce neural
differentiation (Oberdoerster, Rabin 1999). By using undifferentiated PC12 cells, we are able to assess
exocytosis directly as a result of stimulation, without the added complexity of feedback information
from other cells synaptically connected to the cell under study. We maintained our PC12 cells in 25 cm.
flasks in F12 medium from Invitrogen, supplemented with 15% horse serum (ATCC, Manassas, VA), and
2.5% Fetal Bovine Serum (Invitrogen, Grand Island, NY) at 37 degrees Celsius in a 5% CO₂ environment.
For our microscopy experiments, cells were split and transfected with 20 μg of DNA in 400 μL of
electroporation buffer (BioRad, Hercules, CA). Transfection was carried out by electroporation in a Gene
Pulser Xcell electroporation system (BioRad), with a single 15 millisecond, 250 volt pulse.
2.1.2: Constructs
To label vesicles, we constructed ANFPmCherry, a fluorescent protein which is packaged into vesicles,
then released following exocytosis. The construct was based on ANF-EGFP, which was a gift to our
laboratory from Dr. Ed Levitan, of the University of Pittsburgh. We purchased a pmCherry plasmid from
Clontech (Mountain View, CA) and amplified it, along with pre-pro-ANF by hi-fi polymerase chain
24
reaction. In a second reaction, we fused both DNA strands together to create a single ANFPmCherry
construct. Next, we removed the GFP tag from the PACGFP-N1 vector by digesting the vector at the
xho1 and not1 dynamic cut sites, and then inserted the ANFPmCherry construct into the PACGFP-N1
backbone (minus GFP). We sequenced the insert and vector as two overlapping sequences, which were
examined for the presence of stop codons and mutations by basic local alignment search tool (BLAST)
alignment with previously published sequences. pHluorin was a generous gift from the laboratory of Dr.
Gero Miesenbock, and ANFpHluorin was constructed in the same manner as ANFPmCherry. Full cDNA
of GRP1, and PLCδ1 were purchased from Thermo-Open Biosystems (Waltham, MA), and cDNA of AKT
was purchased from Origene (Rockville, MD). PH-domains were isolated and amplified by PCR, then
cloned into the GFP or pmCherry vectors.
2.1.3: DNA Purification
To generate DNA plasmids for purification, we transformed plasmid DNA for each construct into BL21
competent cells, and stored the cells in LB medium + 50% glycerol at -80˚C until they were needed for
DNA growth. When DNA purification was required, stock transformed BL21 cells were added to a 100
mL volume of LB medium, supplemented with ampicillin or kanamycin, depending on the antibiotic
resistance conferred by the expression vector. Cells were shaken overnight at 37˚C, then purified using
the Plasmid Midi-Prep kit from QIAGEN (Venlo, The Netherlands), and a modified version of the
QIAGEN-supplied protocol. The Midi-Prep kit works by specifically binding plasmid DNA with a QIAGEN
Anion-Exchange resin, which maintains bound plasmid DNA while allowing proteins, dyes, RNA, and low
molecular weight impurities to be washed from the resin by a low-salt wash solution. Once the
impurities are removed from the resin, a high-salt elution solution is used to remove the bound, isolated
plasmid DNA from the resin. The plasmid DNA can then be harvested and dissolved in a storage solution
of choice (sterilized H₂O in our experiments).
25
We pelleted the BL21 cells after overnight growth, after which the cells were lysed for five minutes, then
precipitated for fifteen minutes, and again pelleted by centrifugation at 20,000g for 20 minutes. The
pellet at this point contained genomic DNA, nuclei, and other cellular material, while the supernatant
contained the targeted plasmid DNA. We passed the supernatant through a QIAGEN filter tip filter by
gravity flow, then washed the filter twice with kit-supplied wash buffer. We eluted plasmid DNA from
the filter with kit-supplied elution buffer, which we then precipitated using Isopropanol. Plasmid DNA
was pelleted by centrifugation at 15,000g for 15 minutes, after which the pellets were dried and resuspended in purified H₂O. Plasmid DNA concentration was measured by spectrophotometery, after
which the DNA was stored at 4˚C until it was needed for transfection.
2.1.4: Protein Purification
To perform our PH-domain affinity assay, we prepared samples of each of the three PH domains which
were purified using the Profinity eXact Protein Purification kit from BioRad. Essentially, this kit works by
inserting DNA coding for each PH domain into the BioRad-supplied pPAL8 expression vector, which
expresses an affinity tag attached to the desired protein during expression. This affinity tag is selectively
bound (Kd < 100 pM) by an immobilized protease which performs a specific and controlled cleavage of
the affinity tag from the recombinant protein. Un-tagged protein, DNA, and other cellular debris is first
washed out of the binding resin with application of a wash buffer. Next, an elution buffer is added to
the resin, activating the protease and cleaving the affinity tag from the target protein, allowing it to flow
through the purification column while the affinity tag remains tightly bound to the resin.
After DNA coding for each PH domain was subcloned into the pPAL8 expression vector, the subcloned
vector was transformed into Top10 competent cells. A seed culture of transformed cells was started by
adding the transformed cells to 2mL LB medium + 100 μg/mL ampicillin in LB medium, after which the
culture was shaken overnight at 37˚C. Next, we inoculated a 50 mL expression culture with 1 mL from
26
the starter culture, again using LB medium supplemented with 100 μg/mL ampicillin. The expression
culture was shaken at 37˚C and monitored for growth until the cell density reached an OD₆₀₀ value of
0.5-0.7. We then initiated protein expression by adding 2mM isopropyl-β-D-thiogalactopyranoside
(IPTG), and shaking the expression culture overnight at 27˚C.
Protein-expressing cells were then pelleted by centrifugation, and lysed by addition of kit-supplied lysis
buffer. The lysate was centrifuged at 10,000g for 30 minutes, and the supernatant was subsequently
passed through a Profinity eXact Mini Spin Column by centrifugation at 1,000g for 30 seconds. Following
passage of all lysate through the column resin, we washed the column twice with wash buffer, and the
protein samples were eluted by cleavage of the PPAL8 affinity tag as described previously, allowing the
complete proteins to be removed from the binding resin.
Protein concentration was measured using the DC Protein Assay from BioRad, which works in a two-step
reaction resulting in color development. In the first step, cupric (Cu²⁺) cations react with the peptide
bonds which are present in proteins, reducing the cupric ions in solution to cuprous (Cu⁺) ions and also
forming a tetradentate-copper complex. In the second reaction, electrons are transferred from the
tetradentate-copper complex to a folin reagent (phosphomolybdic-phosphotungstic acid), leading to
reduction of the Folin reagent. The reduced species of Folin reagent takes on a bluish hue which
intensifies as the reaction completes over a 15 minute incubation period at room temperature.
Absorption was compared against a standard curve in order to ascertain quantitative values for the
concentration of protein yielded from each purification procedure.
In order to determine the optimal temperature for maximum protein yield, we also tested several
different temperatures at which the expression cultures were maintained overnight. Expression was
tested at 37, 30, and 27, and 25˚C, and the net protein yield was measured in each case following
27
isolation. The optimal protein yield was found to occur at 30˚C, which was then used as the expression
temperature for all subsequent protein isolations.
2.1.5: Immunohistochemistry
For immunohistochemistry experiments, cells were plated on coverslips (25 mm) which had been
washed in HCl, and coated with Geltrex basement membrane (purchased from Invitrogen) and
incubated overnight at 37 degrees Celsius in a 5% CO₂ environment. The following day, cell-coated
coverslips were washed twice with serum-free medium (to remove material contained in the incubation
medium), and were then fixed in a 4% paraformaldehyde + serum-free medium mixture for ten minutes
at room temperature. Cells were then permeabilized with 0.5% Igepal in sterilized phosphate buffered
saline (PBS), again while rocking for ten minutes at room temperature. Anti-PI(4,5)P₂ and AntiPI(3,4,5)P₃ mouse primary antibodies (Echelon Biosciences, Salt Lake City, UT) were applied at 10 μL/mL
PBS, and a secondary Alexafluor (Invitrogen) rabbit-anti-mouse secondary antibody was applied at 1
μg/mL in separate steps. Between each step, cells were washed three times with PBS, rocking for 5
minutes at room temperature. Three-dimensional images of the immunostained cells were constructed
following digital deconvolution using Slidebook 5.0 (Intelligent Imaging Innovations, Denver, CO) as
described below.
2.2: Biochemical Assays
2.2.1: PH Domain Affinity Assay
Because the literature involving the binding affinities of the three pleckstrin homology domains for
PI(4,5)P₂ and PI(3,4,5)P₃ has not been consistent, we elected to conduct our own affinity assay to discern
relative affinities of each domain for multiple membrane lipids. To this end, PIP Strips were purchased
from Echelon Biosciences. PIP Strips are hydrophobic membranes which have been spotted with fifteen
28
biologically-active lipids at 100 pmol per spot. Pleckstrin homology domains were labeled with the HA
epitope tag by PCR, and the recombinant proteins were purified using the Profinity eXact Protein
Purification System (BioRad, Hercules, CA) as described above. Strips were blocked with 10 mL of
blocking buffer (PBS-T(0.1% v/v Tween-20)+3% bovine serum albumin (BSA)) and rocked for one hour at
room temperature, after which they were washed in TBS-T+ 3% fatty acid-free BSA three times, for ten
minutes each time (this wash step was also completed after each step that follows). Then, either PIP2
or PIP3 antibodies or else a PH domain were added at 0.5 μg/mL in 5 mL PBS-T 3% BSA, and rocked at
room temperature for two hours. Finally, anti-HA antibody and anti-mouse HRP antibody were added at
concentrations of 1 and 3 μg/mL, respectively, and incubated for one hour at room temperature so that
the relative amount of bound protein could be detected for each spot. A digital image of each strip was
taken using Canon Powershot G9 digital camera with 12.1 megapixel resolution, and the resultant image
was analyzed for intensity ImageJ (NIH).
2.2.2: Cholesterol Assay
Plasma membrane extracts were prepared by repeated passage through a 1 mL Dounce-type
homogenizer (10 strokes at loose tolerance) in an extraction buffer containing 0.25M sucrose, 5mM TrisHCl (pH 7.4), and 1mM MgCl₂, and were then centrifuged at 220g for 5 minutes. After removing the
supernatants, the pellets were again passed through the homogenizer and centrifuged at 220g to
extract any remaining membrane. The supernatants from both extraction steps were then combined
and centrifuged at 100,000g, and the pellets were re-suspended in a 10 mM NaPO₄ storage solution.
Cholesterol assays were performed using the Amplex Red Cholesterol Assay Kit (Invitrogen), following
the manufacturer’s instructions. This kit utilized cholesterol oxidase to break cholesterol down into
hydrogen peroxide and ketones. The hydrogen peroxide then reacted with 10-acetyl-3, 7dihydroxyphenoxazine (Amplex Red reagent) to produce resorufin, which could be fluorescently excited
29
at 550 nm and emits at 590 nm. Fluorescence was measured using an F-7000 fluorescence
spectrophotometer (Hitachi, Tokyo, Japan), and results were quantified using the FL Solution 2.1
computer program, also from Hitachi. Fluorescence traces of known cholesterol concentrations were
also prepared, and the subsequently-generated standard concentration vs. fluorescence curve was used
to determine cholesterol concentrations in our samples. Cholesterol concentrations were normalized
relative to protein concentration for each sample, measured as previously described, in order to account
for variability in the total amount of cellular material among different samples.
2.3: Microscopy Techniques
2.3.1: Immersion Oil N.A. Optimization
In order to maximize the resolution of our widefield microscope, we also systematically tested a series
of immersion oils with refractive indices which varied from N.A. 1.500 to 1.534 (Applied Precision,
Issaquah, WA) in increments of 0.002, in order to determine which refractive index would most closely
match the refractive index of our fixation medium, thus providing the smallest point spread function,
and subsequently, the greatest resolution. 580/605 nm coated fluorescent beads (Invitrogen) were
mounted on slides in the same mounting medium used for our immunohistochemistry experiments and
illuminated with a 10 mW 561 nm laser, directed into the periphery of the back aperture of a 1.49 NA,
oil immersion objective (Olympus, Tokyo, Japan). Following capture of a three-dimensional image of a
single bead, the point spread function was directly visualized in the X-Z plane, and was compared against
the point spread function of beads imaged in the same fashion, but with varying immersion oil refractive
indices. Determined in this way, the least powerful point spread function (and therefore, the highest
resolution) was provided by immersion oil with a refractive index of 1.516 to 1.518. Oils above and
below these indices showed significantly greater diffraction, thus an immersion oil with an N.A. of 1.518
was used for all deconvolution and immunohistochemistry experiments.
30
2.3.2: Widefield Imaging
In order to produce maximal quality images, cells were grown on 25 mm coverslips which were then
mounted in an imaging chamber (Warner Instruments) such that the coverslip formed the base of the
chamber. Cells were then bathed in artificial cerebro-spinal fluid (ACSF) solution consisting of 105 mM
NaCl, 5 mM KCl, 0.7 mM MgCl₂, 2 mM CaCl₂, and 1 mM NaH₂PO₄, and 10 mM HEPES buffered to 7.2 pH
using NaOH. An Olympus ix80 inverted microscope with integrated high-precision focus drive was used
for imaging, and image acquisition was controlled by a computer running Slidebook version 5.0
(Intelligent Imaging Innovations, Denver, CO). Fluorescence excitation was produced by a rapidswitching DG4 light source (Sutter Instruments, Novato, CA) attached by liquid light guide. Emitted light
was filtered by a Sutter filter wheel which was controlled by a lambda 1 controller. For threedimensional imaging, a series of two-dimensional images were taken beginning with the focal depth
above the cell, and then decreased in increments of 0.1 μm through the depth of the cell. A
Hammamatsu ORCA R2 cooled charge-coupled device (CCD) camera communicated with the host
computer through a firewire interface.
2.3.3: TIRF Microscopy
Total Internal Reflection Fluorescence(TIRF) Microscopy provides an extremely useful tool for visualizing
events at or near the plasma membrane, including the study of the membrane itself, as well as the
approach and fusion of secretory vesicles with the membrane. The advantage to TIRF over other forms
of microscopy lies in its selective fluorescent excitation of only a small portion of the cell, generally the
membrane immediately attached to the coverslip and only a very small part of the adjacent intracellular
space. In widefield imaging, a laser excitation source is shone directly through the coverslip and into
the cell in its entirety, thus illuminating all of the fluorophores within the cell which can be excited by
the particular wavelength being used. TIRF imaging utilizes the principle of total internal reflection,
31
which states that incident light striking a dielectric interface from a higher refractive index to a lowerindex medium, at an acute enough angle(called the critical angle), relative to the interface, will be totally
reflected without passing beyond the interface. The critical angle is dependent primarily on the
refractive indices of the two media comprising the dielectric interface, and is also marginally affected by
the wavelength of light which is being reflected.
When light is reflected in this manner, only a small portion of the electromagnetic field generated by the
excitation source passes through the coverslip, in the form of an evanescent field whose strength decays
exponentially with distance above the coverslip. Because of its rapid decay, the evanescent wave
effectively illuminates a region approximately 100 nm above the coverslip, which in the case of a cell
affixed to the coverslip, means that the membrane and its immediately adjacent cytosolic region is
illuminated, while the rest of the cell remains unperturbed.
For our experiments, a 10 mW 561 nm laser was directed into the periphery of the back aperture of a
1.49 NA, oil immersion objective (Olympus). Transfections were carried out via electroporation in a
Gene Pulser (Bio Rad) using 20 μg DNA. Cells were imaged three days after transfection, in saline
consisting of 140 nM NaCl, 5mM KCl, 2mM CaCl₂, 2mM MgCl₂, 5.5mM glucose, 20mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered to pH 7.3 using NaOH. Imaging was
carried out on an Olympus ix80 inverted microscope incorporated into a Slidebok system (Intelligent
Imaging Innovations, Boulder, CO). For TIRF experiments, we generated a minimum intensity projection
image for each cell, and subtracted this image from each frame. This subtracted any permanent
features of the image from the stack, allowing arriving vesicles to be clearly seen. A maximum intensity
projection of this new stack was then used to identify regions of exocytosis, and each spot was
surrounded by a nine pixel region of interest (ROI). This ROI was then extracted as an average intensity
32
over time plot, and analyzed for peaks using ImageJ (National Institutes of Health) and Excel (Microsoft).
Number and amplitude of peaks were then compared between conditions.
2.3.4: Digital Deconvolution
Digital deconvolution is a means by which the contrast and resolution of digital images can be improved
upon, and is achieved through the use of computationally-intensive algorithms which serve to remove
or reverse the blurring effects which result from out of focus light entering the microscope aperture.
Essentially, digital deconvolution achieves similar optical resolutions to those obtained using confocal
microscopy. In both cases, out-of-focus light is removed in order to provide greater clarity at the focal
point; in the case of confocal microscopy, this is achieved through filtering light through a pinhole which
removes out-of-focus diffraction patterns prior to image capture by a CCD camera. Digital deconvolution works to also remove out-of-focus diffracted light, but does so at a point after the digital
images have been captured. The first step in the deconvolution process involves constructing a threedimensional image of the specimen, which is done by taking a stacked series of images in which the
focal plane is varied along the Z-plane (vertical, with respect to the objective).
The pattern of diffracted light originating from a single point source can be modeled mathematically by
a function which describes the series of diffraction rings which widen with distance above and below the
point source, known as the point spread function. In the X-Y plane, the point spread function resembles
a series of concentric rings, and an hourglass in the X-Z and Y-Z planes. The pattern of diffraction
described by the point spread function, convolved across all of the point sources contained within the
image in all Z-planes, leads to the net blurring effect which is seen in the final image at each diffractionlimited point source. Deconvolution acts to essentially reverse the effects of the point spread function
on individual points mathematically at each focal plane, thus removing diffracted light from the focal
33
planes surrounding the specific point source, in order to cut down on total blur and to increase the
resolution and clarity of the image as a whole.
Several algorithms are available for use in deconvolving digital images which take advantage of the
information provided by a three dimensional image stack to produce a maximal quality image. In our
Slidebook software, both a Nearest Neighbor and a Constrained Iterative algorithm are available for
deconvolution. Nearest neighbor is a class of deblurring algorithm which operates on individual twodimensional planes within a three-dimensional image, while constrained iterative algorithms operate
simultaneously on each pixel of a three-dimensional image as a whole. Both algorithms were tested,
and as expected, the constrained iterative algorithm resulted in the least blurring in our final images,
and was selected for use in our imaging experiments.
The first step in a typical constrained iterative algorithm involves the making of an estimation of the
object being imaged, which typically occurs in the form of the raw image. The three-dimensional
estimate of the imaged object is then convolved with the point spread function, and the result of this
convolution is compared against the raw image itself. Based on the differences between the convolved
estimate and the actual raw image, a new estimate is formed which, when convolved with the point
spread function, should provide a more accurate depiction of the raw image. This process continues
with new iterations until the difference between the convolved estimate and the raw image either
reaches a minimum, or else a threshold value. At this point, the estimate is output as the deconvolved
image, based on the evidence that it provides the most similar likeness to the raw image that can be
attained by the algorithm following convolution with the point spread function.
2.4: Calcium Dependence
2.4.1: Manipulation of Extracellular Calcium Levels
34
To ensure that the stimulation-driven exocytosis we saw in our experiments did demonstrate a
dependence on calcium, we tested the effect of manipulations to extracellular calcium concentrations,
as well as of blocking calcium channels with CdCl₂, a commonly-used broad-spectrum calcium channel
blocker. For both experiments, PC12 cells were grown in F12K medium in a 5% CO₂ environment at
37˚C. Cells were transfected with ANFPmCherry via electroporation as described previously, and were
then plated on Geltrex-coated coverslips in 6-well, 3 mL plates. After three days, cells were imaged on
an Olympus ix80 inverted microscope as follows:
We first tested to see whether secretion rates showed a direct relationship to extracellular calcium
concentrations by testing secretion rates in the conditions of 0, 2, and 3 mM calcium. Cells were
washed in ACSF prior to imaging in order to remove extracellular calcium and other debris from
incubation, and were then placed into an imaging chamber in ACSF without calcium (140 nM NaCl, 5mM
KCl, 2mM MgCl₂, 5.5mM glucose, 20mM HEPES buggered to pH 7.3 using NaOH). Calcium concentration
was increased by the addition of 0, 2, or 3 mM CaCl₂ as previously stated. Individual secretory events
were designated by at least a two-fold increase in fluorescence over the noise range, as in our previous
secretion experiments. As shown in Figure 2-1, we did see a calcium-dependent exocytic response, with
greater extracellular calcium levels leading to a greater amount of total secretion per unit area (0.063
Events / μm² (0 mM Ca²⁺), vs. 0.178 Events / μm² (2 mM Ca²⁺), p < 0.005, n = 5) and (0.063 Events / μm²
(0 mM Ca²⁺), vs. 0.202 Events / μm² (3 mM Ca²⁺), p < 0.05, n = 5).
2.4.2: Calcium Channel Blocking with CdCl₂
We also treated cells with cadmium chloride (CdCl₂), a broad-spectrum calcium channel blocker, in order
to ensure that blocking calcium entry would inhibit secretion. PC12 cells were grown, transfected, and
incubated as before, and were washed and bathed in ACSF, then treated with 500 μM CdCl₂ for five
minutes prior to imaging. Cells were stimulated by application of high-potassium ACSF, and exocytosis
35
was measured via TIRF excitation of ANFPmCherry-labeled vesicles as described previously. As shown in
Figure 2-2, cells treated for 5 minutes demonstrated a decrease in basal secretion levels compared to
control levels (0.038 (Control) vs. 0.024 (5 min. CdCl₂) Events / μm², p < 0.05, n = 5). Interestingly, a
further decrease in secretion occurred following stimulation with high potassium ACSF. (0.038 (Control)
vs. 0.020 (5 min CdCl₂ + Hi K⁺), p < 0.05, n = 5).
Taken together, these results demonstrate that the exocytic events visualized in our experiments are at
least to a large degree dependent on calcium concentrations. We were surprised to not see a more
complete decrease in secretion in the absence of calcium, and following blockage of calcium by CdCl₂.
Secretion following CdCl₂ treatment might be explained by incomplete blockage of the different calcium
channels present in the cell membrane, although this does not explain the secretion which we saw in
the absence of extracellular calcium. This secretion is most likely a result of intracellular calcium stores,
which are present predominantly in the smooth endoplasmic reticulum.
36
Figure 2-1: Secretion Rate vs. Extracellular Calcium Concentration. Secretion rates in PC12 cells
decrease with decreasing extracellular calcium levels. (0.063 Events / μm² (0 mM Ca²⁺), vs. 0.178
Events / μm² (2 mM Ca²⁺), p < 0.005, n = 5) and (0.063 Events / μm² (0 mM Ca²⁺), vs. 0.202 Events
/ μm² (3 mM Ca²⁺), p < 0.05, n = 5)
37
Figure 2-2: Secretion rates decrease following 5 min. CdCl₂ treatment. PC12 cells were
treated for 5 minutes with 500 μM CdCl₂, after which basal and stimulated secretion rates were
measured. In both cases, secretion was inhibited by application of CdCl₂ (0.038 (Control) vs.
0.024 (5 min. CdCl₂) Events / μm², p < 0.05, n = 5) and (0.038 (Control) vs. 0.020 (5 min CdCl₂ +
Hi K⁺), p < 0.05, n = 5).
38
Methyl-β-cyclodextrin blocks secretion from PC12 cells without detectable cholesterol depletion
3.1: Abstract
Cholesterol has been shown to aid in the organization of lipid-enriched domains within the plasma
membrane, a sub-set of which have been suggested to play a critical role in the aggregation of the
protein machinery necessary for vesicular fusion. Cholesterol depletion from the membrane, commonly
performed by application of methyl-β-cyclodextrin, has been suggested to lead to various changes in
both the morphology and function of lipid domains, as well as changes in cellular secretion levels. In this
study, we utilized methyl-β-cyclodextrin (M-β-CDX) to deplete cholesterol levels within the plasma
membrane, and examined its effect on PI(4,5)P₂ and PI(3,4,5)P₃-enriched domains and rates of vesicular
secretion. Treatment of PC12 cells with 2mM M-β-CDX for both short and long-term durations resulted
in a significant decrease in vesicle fusion without affecting the morphological properties of PI(4,5)P₂ or
PI(3,4,5)P₃ domains. Short-term treatment, however, did not lead to a measurable change in
cholesterol levels despite the lack of measurable cholesterol depletion or changes in domain
morphology. These results indicate that M-β-CDX may exert an influence on cellular signaling in
addition to its commonly accepted role as an agent of plasma membrane cholesterol depletion.
3.2: Introduction
Although the central nervous system makes up only 2% of human body mass, it contains approximately
25% of the unesterified cholesterol in the human body (Tarasenko, Sivko et al. 2010, Dietschy, Turley
2001), underlining the significance of cholesterol in the function and organization of the cell types
unique to that system. The plasma membrane contains small (10-200 nm.) domains enriched in
39
phospho- and sphingolipids and cholesterol, which serve to organize and compartmentalize cellular
processes (Pike 2004, Schmitz, Grandl 2008), and require cholesterol for their own organization and
stabilization within the plasma membrane (Mahammad, Dinic et al. 2010). The removal or perturbation
of membrane cholesterol has been shown to have numerous effects on cellular functions and processes,
including effects on the distribution of SNARE proteins (Salaün, Gould et al. 2005), ion channel
permeability (Kato, Nakanishi et al. 2003), and synaptic vesicle recycling (Wasser, Ertunc et al. 2007) .
Total internal reflection fluorescence (TIRF) microscopy studies in PC12 cells have demonstrated that
lipid rafts containing the SNARE protein syntaxin became dissociated following removal of cholesterol
from the membrane (Lang, Bruns et al. 2001). Also, reductions in both the number of docked vesicles
and total exocytic response were found in pancreatic β cells following cholesterol depletion from the
membrane, as well as migration of the SNARE protein SNAP-25 from clustered microdomains in the cell
membrane into the cytosol (Vikman, Jimenez-Feltström et al. 2009). Another study on PC12 cells also
showed a reduced exocytic response following cholesterol depletion, as well as a reduction in quantal
size and slower fusion kinetics (Zhang, Xue et al. 2009). A study in Jurkat T-cells, however, showed that
limited cholesterol depletion leads to aggregation of lipid rafts and T-cell activation as indicated by
tyrosine and MAP kinase ERK phosphorylation (Mahammad, Dinic et al. 2010).
Methyl-β-cyclodextrin (M-β-CDX) is the most commonly-used agent for removal of cholesterol from the
plasma membrane (Zhang, Xue et al. 2009, Vikman, Jimenez-Feltström et al. 2009, Tarasenko, Sivko et
al. 2010). Cyclodextrins are cyclic oligosaccharides which possess a hydrophobic cavity capable of
encapsulating various hydrophobic molecules (Zidovetzki, Levitan 2007). Although there are many
forms of cyclodextrins available, the size of the hydrophobic cavity in β-cyclodextrins and the increased
water solubility of the methylated cyclodextrin makes M-β-CDX the most ideal of the cyclodextrins for
cholesterol removal under typical laboratory conditions (Zidovetzki, Levitan 2007). M-β-CDX does not
penetrate the plasma membrane and is thought to exclusively remove cholesterol from the membrane
40
itself without affecting intracellular cholesterol levels (Vikman, Jimenez-Feltström et al. 2009). A study
in mouse pancreatic β-cells showed significant reductions in the amount of insulin secreted after cells
were treated with 0.1 mM M-β-CDX (Vikman, Jimenez-Feltström et al. 2009). M-β-CDX has also been
used in PC12 cells, which also demonstrated a significant inhibition of membrane depolarization-induced
exocytosis (Zhang, Xue et al. 2009) TIRF microscopy experiments in the same study also showed a drop
in the number of docked vesicles following M-β-CDX treatment.
One potential link between M-β-CDX-mediated cholesterol depletion and secretory inhibition is the
disruption of lipid-enriched domains involved in the aggregation of the protein machinery required for
synchronous exocytosis. These domains were first detected as regions of the membrane which were
resistant to detergent-driven dissociation (Lindner, Knorr 2009), however later studies showed that
other domains also existed (Eckert, Igbavboa et al. 2003), suggesting that not all domains are enriched in
the same molecular components. A sub-class of lipid domain enriched in PI(4,5)P₂ has been suggested
to play a critical role in the aggregation of the proteins required for vesicular fusion to take place
(Chamberlain, Burgoyne et al. 2001). PI(4,5)P₂-enriched domains were shown to colocalize with the
SNARE protein syntaxin as well as docked large dense-core vesicles, and a PIP5 kinase-driven increase in
PI(4,5)P₂ in the same study demonstrated an increase in total levels of secretion (Aoyagi, Sugaya et al.
2005). Another study showed that secretion levels dropped significantly after PI(4,5)P₂ microdomains
were bound by a GFP-tagged pleckstrin homology domain, further indicating the importance of PI(4,5)P₂
in the process of vesicular exocytosis (Holz, Hlubek et al. 2000). The membrane penetration activity of
synaptotagmin, another protein responsible for speeding the vesicular fusion process in response to
calcium entry, has been shown to be steered toward exocytic sites by PI(4,5)P₂ (Bai, Tucker et al. 2004) .
Increases in PI(4,5)P₂ levels in bovine chromaffin cells have also been shown to increase the number of
vesicles which are readily released following stimulation (Milosevic, Sørensen et al. 2005). Blocking
hydrolysis of PI(3,4,5)P₃ in mast cells has also been shown to lead to increased levels of de-granulation
41
(Damen, Ware et al. 2001), illustrating the potential importance PI(3,4,5)P₃ might also have in exocytic
processes.
In this study, we have used methyl-β-cyclodextrin to examine the role of cholesterol depletion on the
organization and subsequent function of lipid domains enriched in PI(4,5)P₂ and PI(3,4,5)P₃. We saw a
significant decrease in levels of secretion following treatment with 2mM M-β-CDX when treated for two
days. We also found that M-β-CDX treatment for two days led to significant cholesterol depletion in the
membrane, while ten minute treatment did not have a significant effect on cholesterol levels.
Interestingly, ten minute M-β-CDX treatment led to a comparable inhibition of secretion. These findings
imply an interaction between M-β-CDX and some component of the plasma membrane aside from its
commonly-accepted role interacting with cholesterol.
3.3: Results
3.3.1: PIP₂ and PIP₃ Antibodies Efficiently Identify Membrane Subregions
A previous study showed that antibodies against both PI(4,5)P₂ and PI(3,4,5)P₃ could be effectively used
in visualization of PI(4,5)P₂ and PI(3,4,5)P₃-enriched rafts within the cell membrane (Wang, Richards
2012). To clearly identify regions within the plasma membrane enriched in either PI(4,5)P₂ or
PI(3,4,5)P₃, PC12 cells were fixed and immunostained with antibodies against those lipids. Punctate,
clearly-defined clusters enriched in both PI(4,5)P₂ and PI(3,4,5)P₃ were shown to be present in the cell
membrane as seen in Figure 3-1 A and B, respectively. To restrict analysis to a single membrane plane,
we used only the bottom five slices of each three-dimensional image stack for raft size analysis. In the
case of both PI(4,5)P₂ (Figure3- 1C) and PI(3,4,5)P₃ clusters (Figure 3-1D), domains were indicated by
diffraction-limited spots in the plasma membrane. These immunostained regions are within the range
of sizes for lipid-enriched domains as determined by atomic force microscopy (Yuan, Furlong et al. 2002)
42
suggesting that the clusters being visualized are in fact PI(4,5)P₂ and PI(3,4,5)P₃-enriched lipid rafts in
the membrane.
3.3.2: Cholesterol Depletion by Methyl-β-Cyclodextrin
To deplete cholesterol levels within the plasma membrane, we treated cells with methyl-β-cyclodextrin
(2mM, 10 min or 2 day incubation), a commonly-used agent of membrane cholesterol removal. Cells
were also treated with 2mM M-β-CDX which had been saturated with 2 mM cholesterol to test whether
the drug’s effects specifically resulted from plasma membrane cholesterol removal. Following
cyclodextrin treatment, we conducted a biochemical assay to determine the efficiency with which M-βCDX removes cholesterol from the membrane. Cells were treated with M-β-CDX as described above,
after which plasma membranes were extracted by differential centrifugation and cholesterol levels were
assessed using the Amplex Red Cholesterol Assay Kit from Invitrogen. Sample fluorescence curves in
which an excitation wavelength of 540nm was applied and emission wavelengths were scanned
between 570 and 670 nm are shown for cells either left un-treated (Figure 3-2A) or treated for two days
(Figure 3-2B) with 2mM M-β-CDX. Standardized curves were attained for each reaction using known
cholesterol concentrations as a means of calibrating cholesterol concentration to fluorescence intensity
(Figure 3-2C). Cellular cholesterol concentrations were then assessed by examining fluorescence levels
attained in cell extractions under the above-stated conditions, and comparing these values against the
standard curves. Four separate reactions were carried out, and the cholesterol concentration values
were then averaged to yield the results shown in Figure 3-2D. Protein concentrations were also
measured for each cell extract in order to control for variability in total cellular material, and
concentrations under each condition were normalized against control values. As shown, cholesterol
43
Fig. 3-1: PI(4,5)P2 and PI(3,4,5)P3 antibodies efficiently identify membrane subregions.
Cultured PC12 cells were fixed and treated with (A,C) Anti-PI(4,5)P2 and (B,D) AntiPI(3,4,5)P3 mouse IgM primary antibodies, and then labeled with Alexafluor568 Rabbitanti-mouse IgG secondary antibodies to visualize membrane subregions enriched in either
PI(4,5)P2 or PI(3,4,5)P3. Average size for PI(4,5)P2 subregions were consistent with
previously published values for lipid raft sizes, indicating that the subregions being labeled
are in fact lipid-enriched subregions frequently referred to as lipid rafts.
44
A
B
C
D
3000
Cholesterol Concentration
/ Protein Concentration
Fluorescence
2500
2000
1500
1000
500
0
0
1
2
3
Cholesterol Concentration (μg / mL)
3.5
3
2.5
2
1.5
1
0.5
0
*
*
Control 10m
CDX
10m 2d CDX 2d CDX
CDX +
+ chol.
chol.
Fig. 3-2: Cholesterol depletion by methyl-β-cyclodextrin. (A and B) Fluorescent emission spectra for cells
(A) left un-treated or (B) treated for two days with M-β-CDX. (C) Cholesterol concentration standard
curve demonstrating linear relationship between cholesterol concentration and fluorescence. (D)
Cultured PC12 cells were treated with 2mM M-β-CDX for 10 minutes or two days, and with 2mM M-β-CDX
which had been saturated with 2mM cholesterol, also for 10 minutes or two days; control cells were left
untreated. Cellular extracts were prepared as described and cholesterol levels were assessed using the
Amplex Red Cholesterol Assay Kit from Invitrogen. Cholesterol concentrations were normalized to control
values for each experiment. Cholesterol levels were unaffected by 10 min and 2 day cholesterol-saturated
M-β-CDX treatment (1.704 and 2.274 vs. control (1) , n = 4), but increased following ten minute unsaturated M-β-CDX treatment (1.865 vs. control (1), p < 0.05, n = 4) and decreased following 2 day
treatment (0.581 vs. control (1), n=4, p<0.05).
45
levels were not affected by ten minute and two day cholesterol-saturated M-β-CDX treatment (1.704
and 2.274 vs. control (1), n = 4). Cells treated for two days with 2mM MBCDX showed a significant
decrease in overall cholesterol concentration compared with control cells (0.581 vs. control (1), n=4,
p<0.05). Surprisingly, those cells treated for ten minutes at the same M-β-CDX concentration showed an
increase in cholesterol levels (1.865 vs. control (1), p < 0.05, n = 4).
3.3.3: Methyl-β-Cyclodextrin Blocks Secretion Without Removal of Cholesterol
To determine the effect of both short and long-term methyl-β-cyclodextrin treatment on the rate of
occurrence of vesicular fusion events in PC12 cells, we conducted a TIRF microscopy analysis of vesicle
secretion following methyl-β-cyclodextrin treatment as defined above. To visualize individual vesicular
fusion events, PC12 cells were transfected via electroporation with a construct encoding pre-pro-ANF
fused to the fluorophore mCherry, which can be excited by a 560 nm. excitation laser source, and were
then either left un-treated (Figure 3-3 A-D) or treated with 2mM M-β-CDX for ten minutes (Figure 3-3 EH) or for two days (Figure 3-3 I-L). A 25 second timeseries of TIRF images was taken at an imaging
frequency of 4 Hz. to determine baseline secretion levels In cells either left un-treated (Figure 3A,B), or
treated with 2mM M-β-CDX for either ten minutes (Figure 3-3 E,F) or two days (Figure 3-3 I,J). Sample
traces for un-treated, ten minute M-β-CDX-treated, and two day M-β-CDX treated cells are given in
Figures 3-3 B, F, and J, respectively. Under each treatment condition, cells were then stimulated via
membrane depolarization with a high-potassium solution, following which release levels were again
measured (Figure 3-3 C,D, G,H,K, and L).
Treatment with 2mM M-β-CDX for both ten minutes and two days resulted in a marked decrease in both
un-stimulated and stimulated levels of secretion (Figure 3-3M). Specifically, unstimulated secretion fell
from control levels of 0.138 fusion events / μm² (n= 5 [cells], # fusion events = 895), to 0.055 events /
46
μm² in cells treated for ten minutes (n = 5, # fusion events = 128, p < 0.005), 0.044 events / μm² in cells
treated for ten minutes with cholesterol-saturated M-β-CDX (n = 5 [cells], # fusion events = 362, p <
0.005) and 0.050 events / μm² in cells treated for two days (n =5, # fusion events = 251, p <= 0.007).
Stimulated secretion levels also dropped, from control levels of 0.188 events / μm² (n = 5 [cells], # fusion
events = 1397) to 0.069 events / μm² following treatment for ten minutes (n = 5, # fusion events = 160, p
<= 0.01), 0.066 in cells treated with cholesterol-saturated M-β-CDX (n = 5, # fusion events = 537, p <
0.006) and 0.049 events / μm ² in cells treated for two days (n = 5, # events = 240, p <= 0.002).
3.3.4: Lipid Clusters Persist After Cholesterol Depletion
To determine what effect methyl-β-cyclodextrin treatment had on PI(4,5)P₂-enriched lipid rafts, we
again immunolabeled PC12 cells as described for the experiments in figure 3-1 following either ten
minute (Figure3-4 D) or two day(Figure 3-4 G) M-β-CDX treatment, or else left un-treated (Figure 3-4 A).
Because cholesterol has been shown to play a vital role in the structure and function of lipid rafts
(Kannan, Barlos et al. 2007) , its removal from the membrane was expected to lead to disruption of raft
function, and possibly also changes in raft morphology. A comparison of means for cells treated for
both ten minutes and two days showed no significant difference versus control cells with regard to raft
size, although an examination of size distributions following ten minute (Figure 3-4E) and two day
(Figure 3-4 H) treatment suggests a possible decrease in size after two-day treatment versus control
cells (Figure 3-4 B). Next, we tested whether cyclodextrin might have an effect on the amount of
PI(4,5)P₂ within individual rafts by quantifying the total amount of fluorescence from each raft after
immunostaining, and comparing the averages of these values for rafts under each test condition. As with
raft size, we saw no difference between mean intensities before and after M-β-CDX treatment, although
distributions in control cells (Figure 3-4 C) compared with cells treated for ten minutes (Figure 3-4 F),
47
and for two days (Figure 3-4 I) suggests a possible decrease in raft intensity following two day
treatment.
To examine whether cyclodextrin lead to a net decrease in the number of rafts present in the cell
membrane, we also quantified the number of PI(4,5)P₂-enriched rafts present per square micron of cell
surface area, as shown in Figure 3-4 J. In this case, cells treated with M-β-CDX for ten minutes showed
no variation from controls(0.370 rafts / μm² [10 min. M-β-CDX (n = 11, p <=0.32)] vs. 0.444 rafts/μm²
[control (n = 11)]). Cells treated for two days showed a slight, though statistically insignificant decrease
compared with controls, having an average of 0.321 rafts/μm² (n = 10, p <= 0.08).
In figure 3-5, similar results are shown for rafts enriched in PI(3,4,5)P₃. Cells were immunostained
against PI(3,4,5)P₃ after ten minute (Figure 3-5 B), two day, (Figure 3-5 C) or no treatment (Figure 3-5 A)
with 2 mM M-β-CDX. As with PI(4,5)P₂-enriched rafts, no differences were shown in mean size or
intensity compared with control cells, although distributions suggest a possible decrease in raft size
(Figure 3-5 B, E) and possibly also raft intensity (Figure 3-5 C, F) in cells treated for ten minutes as well
as those treated for two days (Figure 3-5 H,I). No significant difference in the total number or rafts
present per square micron was observed in cells treated for ten minutes (Figure 3-5 J [n = 6 (control,10
min. M-β-CDX), p <= 0.279]). Also similar to our findings in PI(4,5)P₂-enriched rafts though, we observed
no significant change in the number of PI(3,4,5)P₃-enriched rafts in cells treated for two days (0.339 rafts
/ μm², n = 9, p <= 0.667).
48
Fig. 3-3: M-β-CDX blocks secretion without removal of cholesterol. (A,C) Maximum intensity projections of cells
labeled with ANFPmCherry under TIRFM visualization. (B,D) Fluorescence traces plotting fluorescence vs. time
for individual 9 pixel regions of interest under both unstimulated (B) and stimulated (D) conditions. Vesicular
fusion events are indicated by a rise above baseline in level of fluorescence. Cells were treated with 2mM M-βCDX for 10 minutes (E,G), 2 days (I,K) or left untreated (control). The value of the total number of secretory
events per cell was divided by the cell surface area in order to account for the effect of variation of cell surface
area on the number of events visualized. (M) Following 10 minute M-β-CDX treatment, cells demonstrated a
significant decrease in secretion levels in the case of both unstimulated (0.055 vs. 0.139 (control) Events / Area
(μm²), n = 5, p < 0.005) and stimulated secretion (0.069 vs. 0.188 (control) Events / Area (μm²), n=5, p<0.01). Cells
treated with cholesterol-saturated M-β-CDX for 10 minutes also showed significant decreases in unstimulated
(0.044 vs. 0.139 (control) Events / μm² n = 5, p < 0.005) and stimulated secretion (0.066 vs. 0.188 Events / μm², n
= 5, p < 0.01). Cells treated for two days with 2 mM M-β-CDX also showed significant decreases in secretion levels
before (0.050 vs. 0.139 Events / μm², n = 5, p < 0.01) and after secretion (0.049 vs. 0.188 Events / μm², n = 5, p <
0.005).
49
Fig. 3-4: PI(4,5)P2-enriched rafts persist after M-β-CDX treatment. Cells were treated for 10 min or 2
days with M-β-CDX, following which they were immunostained for PI(4,5)P2-enriched lipid rafts as
described previously. Histogram plots comparing raft size show no difference between distributions of
control (B), 10 min M-β-CDX treated (E) cells, and in 2 day treated cells (H). Histogram distribution plots
were also prepared comparing the amount of fluorescence given off by individual rafts as a means of
examining concentration of phospholipids within those rafts, and again no difference was shown
between control (C) and 10 min. M-β-CDX treated cells (F), although raft distribution is skewed toward
lower fluorescence in 2 day-treated cells (I). (J) We also examined the total number of PI(4,5)P2
enriched rafts present in cells with and without M-β-CDX treatment. The value for the total number of
lipid rafts visualized per cell was divided by the cellular surface area to account for the effect of variation
in cell area on the number of rafts which were possible to visualize at the cell-coverglass interface.
Again, treatment with 2mM M-β-CDX for 10 minutes and for two days showed no effect on the number
of lipid rafts per square micron (0.370 vs. 0.444 [control], n = 11, p < 0.32).
50
Figure 3-5: PI(3,4,5)P3-enriched rafts also persist after M-β-CDX treatment. In experiments
analagous to those in Figure 4, PC12 cells were either (A) left untreated or (D) treated for 10
minutes or (G) 2 days with M-β-CDX and then immunostained for PI(3,4,5)P3-enriched lipid rafts.
As with PI(4,5)P2-labeled rafts, no difference was shown between (B) control and (E) 10 minute
treated cells with regard to raft size, and raft size in 2 day-treated cells also appeared not to be
affected (H). PI(3,4,5)P3 concentration within rafts, as indicated by fluorescent intensity of
immunostains in individual rafts, showed no difference between (C) control and ten minute (F)
and two day (I)-treated conditions. (J) Finally, the total number of PI(3,4,5)P3-enriched rafts per
unit area did not change following either ten minute M-β-CDX treatment (0.303 vs. 0.404, n = 6,
p<0.28) or two day treatment (0.321 vs. 0.404, n = 10, p <= 0.08).
51
3.4: Discussion
Although methyl-β-cyclodextrin has become commonly accepted as an agent of cholesterol removal, the
rate of removal is apparently subject to variation depending upon experimental conditions. Yancey et
al., (Yancey, Rodrigueza et al. 1996) proposed that individual cyclodextrin molecules remove membrane
cholesterol by approaching the vicinity of the plasma membrane in such a manner that cholesterol
molecules can transfer directly from the membrane to the cyclodextrin’s hydrophobic core without the
added step of diffusion through the aqueous phase. Several studies have demonstrated the presence of
two different pools of cholesterol which are extracted from the cell; a fast pool which is extracted at a
half-time of 15-30 seconds, and a slow pool which is extracted at a half-time of 15-30 minutes
(Zidovetzki, Levitan 2007, Haynes, Phillips et al. 2000). Another study in T-lymphocytes showed that the
fast pool cholesterol was associated with lipid raft domains, whereas the slow pool was composed of
non-raft cholesterol (Rouquette-Jazdanian, Pelassy et al. 2006). THP-1 macrophages also demonstrated
fast and slow cholesterol pools where the fast pool was associated with raft cholesterol and the slow
pool was associated with non-raft cholesterol (Gaus, Kritharides et al. 2004). Steck, Ye, and Lange
(Steck, Ye et al. 2002) saw only a fast kinetic pool of cholesterol in erythrocyte membranes however,
indicating that not all cell lines apparently possess both a fast and a slow cholesterol pool. Yancey et al.
(25), suggested that these two distinct pools are caused by either cholesterol present in the cytoplasmic
monolayer of the membrane, a separate lateral domain within the plasma membrane, or else differing
extraction times for intracellular vs. membrane cholesterol. As we did not see a drop in cholesterol
levels following 10 minute incubation with M-β-CDX, we do not see evidence to support the presence of
a fast extraction pool of cholesterol in PC-12 cells. The best explanation for this phenomenon seems to
be that intracellular cholesterol stores are trans-locating to the plasma membrane to maintain
homeostatic membrane cholesterol levels. This would explain why membrane cholesterol levels
decreased after two days of M-β-CDX treatment, which should be more than enough time to deplete
52
intracellular cholesterol stores if these are responsible for the slow cholesterol pools as hypothesized
previously.
That we saw a reduction in secretion levels following our ten minute M-β-CDX treatment was surprising
due to the lack of a significant decrease in membrane cholesterol levels following MBCDX treatment for
the same duration. This seems to suggest that M-β-CDX may be exerting an influence on the membrane
beyond its commonly accepted role as a cholesterol binding agent. Because of calcium’s critical role in
the process of synchronous exocytosis, an effect of M-β-CDX on calcium channel function or
permeability seems to be worth consideration. Indeed, a study using isolated rat nerve terminals did
show a decrease in calcium-driven, but not transporter-mediated (calcium independent) glutamate
release after ten minute treatment with 1mM M-β-CDX. This same study also showed a decrease in
evoked vesicular exocytosis after ten minute cyclodextrin treatment, however there did not appear to
be a significant change in intra-synaptosomal calcium levels (Teixeira, Vieira et al. ). A study using Jurkat
T-cells showed significant inhibition of CD-3-driven calcium influx following ten minute pre-incubation
with M-β-CDX, and noted that the same inhibition was seen immediately following M-β-CDX application
and therefore prior to cholesterol removal. The authors also noted that M-β-CDX for both two and ten
minutes led to significant plasma membrane depolarization, which they suggested could result from an
unknown side-effect of M-β-CDX treatment (Rouquette-Jazdanian, Pelassy et al. 2006). Another study
suggested that the reduction of calcium-dependent exocytosis following M-β-CDX treatment actually
resulted from the application of lethal dosage levels of M-β-CDX to cells. When cells were treated with
2.5mM M-β-CDX treatment for nine minutes, a non-lethal M-β-CDX dosage, results showed an increase
in intracellular calcium levels as well as increased aggregation of lipid rafts and activation of T-cell
signaling processes (Mahammad, Dinic et al. 2010).
53
Aside from calcium, M-β-CDX has been suggested to interact with several other components of the
plasma membrane which might also contribute to its effect in reducing exocytic processes. One study
using atomic force microscopy showed the formation of holes in dioleoylphosphaditylcholine
(DOPC)/sphingomyelin bilayers following M-β-CDX treatment, suggesting that it might also be capable of
directly removing phospholipids from the plasma membrane (Giocondi, Milhiet et al. 2004). M-β-CDX
has also been shown to interact with a variety of proteins, including ubiquitin and insulin (Aachmann,
Otzen et al. 2003). Un-methylated β-CDX has also been shown to interact with a number of cell surface
proteins (Ilangumaran, Hoessli 1998), suggesting yet another potential means by which M-β-CDX might
influence secretion levels in addition to cholesterol removal from the membrane. That we did not see a
reduction in size or intensity of PI(4,5)P₂ -enriched rafts following two day M-β-CDX treatment seems to
suggest that cholesterol may not play a crucial role in the organization and stabilization of PIP-enriched
rafts.
In conclusion, we have shown that PC12 cells treated for both ten minutes and two days with 2mM
methyl-β-cyclodextrin exhibit a marked decrease in levels of vesicular secretion compared with cells left
un-treated. We have also shown that lipid rafts enriched in either PI(4,5)P₂ or PI(3,4,5)P₃ appear to be
morphologically unaffected by ten minute cyclodextrin treatment, nor by treatment for two days, with
resulting depletion of cholesterol. Most significantly, our results demonstrate that net plasma
membrane cholesterol levels do not decrease significantly following cyclodextrin treatment for ten
minutes despite a significant drop in the rate of secretion, and that secretion rates drop roughly the
same amount in the case of both two day and ten minute treatment. Taken together, these findings
seem to imply another influence of methyl-β-cyclodextrin aside from its frequently accepted role as an
agent of membrane cholesterol depletion.
54
Pleckstrin homology domains specific for PI(4,5)P₂ and PI(3,4,5)P₃ are equally effective at inhibiting
exocytosis in PC12 cells
4.1: Abstract
Expression of fluorescently-tagged pleckstrin homology (PH) domains is a technique which is commonly
used to visualize certain phospholipids to which they are targeted, particularly in biological membranes.
In this study, we expressed three different PH domains with varying affinities for the phospholipids
PI(4,5)P₂ and PI(3,4,5)P₃ (PH-PLCδ1, PH-AKT, PH-GRP1) to determine the influence of these
phospholipids on localized sites of exocytosis. We also conducted an assay to determine the relative
affinities with which each PH domain selectively binds both PI(4,5)P₂ and PI(3,4,5)P . Our results
demonstrated that, of the three PH domains tested, PH-PLCδ1 has the highest affinity for PI(4,5)P₂, and
PH-GRP1 is highly-specific for PI(3,4,5)P₃. When each of the PH domains was expressed in PC12 cells,
exocytic rates were significantly reduced, and there was a concomitant increase in the number of
vesicles which dwelled for long periods at the membrane. These results suggest that GFP-tagged PH
domains specific for both PI(4,5)P₂ and PI(3,4,5)P₃ are equally effective at inhibiting exocytosis, and also
demonstrate that both PI(4,5)P₂ and PI(3,4,5)P₃ could play important roles in the exocytic process.
4.2: Introduction
Cellular exocytosis is the process by which the cell releases chemical signals to the extracellular space,
chiefly for the purpose of interacting with neighboring cells at the molecular level. Also, secretory cells
within the neuroendocrine system release chemical signals to regulate a diverse set of physiological
processes ranging from metabolism (Birketvedt, Geliebter et al. 2012) to immune system control
55
(Bellavance, Rivest 2012). Several studies have suggested that the regions within the plasma membrane
where such cellular signals are released are spatially regulated, and that these regions are enriched in a
specific sub-set of proteins and lipids which are required for exocytosis to take place (van den Bogaart,
Meyenberg et al. 2011, Chamberlain, Burgoyne et al. 2001, Aoyagi, Sugaya et al. 2005).
Prior to release, secretory vesicles are translocated to the plasma membrane by transporter proteins
moving along microtubules and the actin cytoskeleton (reviewed in (Kamal, Goldstein 2000)). Once they
reach the membrane, vesicular proteins interact with others, including Munc18 (Oh, Kalwat et al. 2012)
so that the vesicle becomes “docked” at a site on the membrane where the requisite proteins for fusion
are localized (Szule, Harlow et al. ). After the vesicle becomes docked, another set of less wellcharacterized biochemical reactions takes place in which the vesicle becomes “primed” for fusion with
the plasma membrane, followed by membrane fusion and release of the vesicular cargo (Jahn,
Fasshauer 2012).
The fusion process is highly-dependent on a set of proteins known as the soluble N-ethylmaleimidesensitive factor activating protein receptor (SNARE) proteins, which are required for exocytosis to take
place (Salaün, Gould et al. 2005). These proteins include the cell membrane proteins syntaxin and
SNAP-25, and the vesicle-associated protein synaptobrevin, and join together to make a four-helix
complex which drives the membrane fusion process (Melia, Weber et al. 2002). In order for fusion to
take place, multiple SNARE complexes are required to stabilize the fusion pore (Sinha, Ahmed et al.
2011, Shi, Shen et al. 2012), which can only be efficiently accomplished by specific clustering of proteins
and lipids within the plasma membrane. Indeed, studies have shown that syntaxin does occur in
enriched domains within the plasma membrane, which appear to be sites on the membrane which are
specifically geared toward exocytic release (Aoyagi, Sugaya et al. 2005, Chamberlain, Burgoyne et al.
2001, Lang 2007). It has also been shown that syntaxin-containing microdomains are also enriched in
56
the phospholipid PI(4,5)P₂, and furthermore, that PI(4,5)P₂ is a requirement for syntaxin clustering
(Aoyagi, Sugaya et al. 2005, van den Bogaart, Meyenberg et al. 2011). Aside from organizing syntaxin
microclusters in the membrane, PI(4,5)P₂ has also been shown to interact with synaptotagmin, another
protein which is critical to synchronous exocytosis, at vesicular release sites within the plasma
membrane (Schiavo, Gu et al. 1996, Bai, Wang et al. 2004). As a result of these interactions with key
elements of the exocytic machinery, PI(4,5)P₂-enriched microdomains have been suggested to play a
critical role in the process of organizing the presynaptic fusion sites (Milosevic, Sørensen et al. 2005,
Murray, Tamm 2009).
Previously, we have visualized PI(4,5)P₂-enriched lipid rafts using monoclonal antibodies targeted to
PI(4,5)P₂ (Wang, Richards 2012). A drawback of immunohistochemical labeling, however, is the
requirement for fixation of the target cells prior to labeling, thus preventing any examination of
secretion in living cells. Several studies have overcome this limitation by overexpression of a
fluorescently-tagged protein known as a pleckstrin homology (PH) domain, which occurs in several
phospholipid-targeted proteins. The affinity of a particular PH domain for its lipid target depends on the
required protein-lipid interactions, so that different PH domains can be used to target different
phospholipids. When a fluorescent tag is attached to a specific PH domain, it becomes a useful tool for
membrane lipid visualization in living cells. For instance, the PH domain from the General Receptor for
Phosphoinositides (GRP1) protein has been used to visualize PI(3,4,5)P₃ microdomains in living cells
(Pilling, Landgraf et al. 2011). The PH domain of PLCδ1 has likewise been used to label PI(4,5)P₂ lipid
rafts in the plasma membrane, although these studies did not determine whether PH-GFP
overexpression had any effect on normal secretion rates (Nawaz, Kippert et al. 2009, Kavran, Klein et al.
1998). A study by Holz et al., (Holz, Hlubek et al. 2000) has suggested that overexpression of GFP-tagged
PH-PLCδ1 exerted an inhibitory effect on exocytosis, as evidenced by decreased hormone release, as
well as membrane capacitance, in chromaffin cells.
57
Despite their frequent use as phospholipid markers in the plasma membrane, little is known about the
effects which PH-GFP expression might have on the intricate workings of the exocytic machinery, and
subsequent effects of expression on natural exocytic behavior. To answer this question, we have
expressed three different GFP-tagged PH domains and examined the effects of expression on vesicular
release. Furthermore, we have measured the relative affinity of each PH domain under study in order to
elucidate a clearer picture of which membrane phospholipids each domain is targeted toward. Our
results indicate that expression of all three PH-domains (PH-GRP1, PH-AKT, PH-PLCδ1) appear to inhibit
exocytosis, apparently at a step after docking but prior to priming and fusion of the vesicular and plasma
membranes.
4.3: Results
4.3.1: Pleckstrin Homology Domains Demonstrate Varying Affinities for PIP₂ and PIP₃
GFP-tagged pleckstrin homology (PH) domains are frequently used to visualize plasma membrane
phospholipids, however the literature does not provide consistent data for the relative phosphoinositide
affinities of the different PH domains. To answer this question, we used HA-tagged PH-domains and
membranes spotted with a variety of phosphoinositides and related lipids (Echelon) to experimentally
determine the relative binding of three PH domains which have been suggested to be specific binding
partners of PIP₂ and/or PIP₃. Figure 4-1A shows the location of each phospholipid spotted onto a strip.
The spotted strips contained all of the phosphoinositides phosphorylated at the 3, 4, and 5 positions
from the phosphatidylinositol (PI) to phosphatidylinositol(3,4,5)trisphosphate, plus lysophosphatidyic
acid (LPA), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), phosphatidylcholine (PC),
phosphatidic acid (PA), phosphatidylserine (PS), and sphingosine-1-phosphate (S-1-P). Figure 4-1 B
shows the relative binding affinities for the three PH domains tested; PH-GRP1 (left), PH-PLCδ1 (center),
and PH-GRP1(right). PH-GRP1 showed exceptional specificity for PI(3,4,5)P₃, with a roughly ten-fold
58
Figure 4-1: Relative phosphatidylinositide affinities of PH-AKT, PH-PLCδ1, and PH-GRP1.
Three different pleckstrin homology domains were applied to PIP Strips (Echelon
Biosciences), which are spotted with various phosphatidylinositols (A), to test the binding
affinity of each pleckstrin homology domain. The pleckstrin homology domain for AKT
(PH-AKT) showed a predominant affinity for PI(3,4,5)P₃, with lower affinities for PI(4,5)P₂
and PI(3,5)P₂, as well as all three monophosphosphorylated forms of phosphatidylinositols
(PI(3,4,5)P₃: p < 0.01 vs. PI(3,4)P₂, PS, PA, S1P, PC, PE, PtdIns, LPC, LPA) (B). PH-PLCδ1 also
showed a preference for PI(3,4,5)P₃, but showed nearly equivalent affinity for the bis- and
mono-phosphorylated forms (PI(3,4,5)P₃: p < 0.01 vs. PS, PA, S1P, PC, PE, PtdIns, LPC, LPA)
(C). PH-GRP1 demonstrated a significantly greater affinity for PI(3,4,5)P₃, compared with
all other forms tested (PI(3,4,5)P₃: p < 0.001 vs. all; PA, PI(3,4)P₂ p < 0.01 vs. all) (D). All
treatments showed significant difference by ANOVA, and post-hoc analysis was done using
Student’s t-test.
59
increase in binding affinity for PI(3,4,5)P₃ relative to the bis-phosphorylated forms (p < 0.001). PH-PLCδ1
has been previously shown to have preferential specificity for PI(4,5)P₂ (Nawaz, Kippert et al. 2009), due
to PLCδ1’s functional role as a PI(4,5)P₂ modulator in the plasma membrane. Surprisingly, PH-PLCδ1
showed a strong binding affinity for all of the phosphatidylinositols under study in mono, bis-, and trisphosphorylated forms, although the affinity for PI(3,4)P₂ was significantly reduced from the affinities
shown for the others. PH-AKT, which has been used to target PI(3,4,5)P₃ (Gassama-Diagne, Yu et al.
2006), did show a weak preference for PI(3,4,5)P₃ over the other poly and mono phosphorylated PIPs,
however this relationship was not as specific as we had expected it to be. Taken together, these results
demonstrate that the pleckstrin homology domain from GRP1 can be used as a highly-specific indicator
for PI(3,4,5)P₃, and that PH-PLCδ1 can be used to indicate PI(4,5)P₂ within the plasma membrane. PHAKT is a less specific indicator which prefers all tested forms of phosphatidylinositols to the other lipids
tested, possibly with the exception of PI(3,4)P₂.
4.3.2: PH-GFP expression Inhibits Exocytosis
Holz et al., (Holz, Hlubek et al. 2000) showed that expressing PH-PLCδ1-GFP led to a decrease in
secretion rates as indicated by a reduction in membrane capacitance events. To determine what effect
each of our GFP-tagged PH domains would have on secretion, we fluorescently labeled vesicles with the
peptidergic fluorophore ANFPmCherry, then visualized individual exocytic events using Total Internal
Reflection Fluorescence (TIRF) Microscopy. Figure 4-2 shows that PH-PLCδ1-GFP (Figure 4-2A) and PHAKT-GFP (Figure 4-2B) both inhibited stimulated secretion, but had no effect on basal secretion levels
(PH-PLCδ1: 0.178 (control) vs. 0.047 events / μm², n = 5, p < 0.05) (PH-AKT: 0.127 (control) vs. 0.062
events / μm², n = 5, p < 0.05). PH-GRP1-GFP expression (Figure 4-2C) also led to significant decreases in
stimulated exocytosis (0.178 vs. 0.023 events / μm², n = 5, p < .05), and unlike PH-PLCδ1 and PH-AKT,
also inhibited un-stimulated exocytosis (0.128 vs. 0.034 events / μm², n = 5, p < 0.05). In each case, PH-
60
Figure 4- 2: PH-GFP expression decreases secretion levels without fusion site enrichment.
PC12 cells were co-transfected with one of three GFP-tagged PH Domains and ANFPmCherry.
Secretion rates were determined by examining change in fluorescence of the ANFP
phluorophore over time using TIRF microscopy. Cells transfected with PH-PLCδ1-GFP
showed a significant decrease in secretion levels following stimulation (0.047 vs. 0.178
(control) Events / Area (μm²), n = 5, p < 0.001) (A). Cells expressing PH-AKT GFP also showed
significant decreases in secretion following sitmulation (0.060 vs. 0.178 (control) Events /
Area (μm²), n = 5, p < 0.001) (B). PH-GRP1-GFP expression led to significantly decreased
levels of exocytosis in both unstimulated (0.033 vs. 0.128 (control) Events / Area (μm²), n =
5, p < 0.0001) and stimulated secretion (0.021vs. 0.177 (control) Events / Area (μm²), n=5,
p<0.001) (C).
61
GFP expression did appear to inhibit secretion as indicated by our TIRF experiments, which is in
accordance with previously published findings (Holz, Hlubek et al. 2000).
4.3.3: PH-GFP Expression Fosters Long-Duration Exocytic Events
We also examined the effect PH-GFP expression had on vesicle dwell time at the plasma membrane.
Figure 4-3A shows cumulative probability plots for stimulated events with regard to time beginning at
event onset. Following PH-GFP expression with all three PH-domains, our results show an increase in
the number of fusion events which are greater than 2.5 seconds, as indicated by the decreased
curvature gradient in the PH-overexpresses compared to controls. Figure 4-3B shows an example of a
long-duration dwelling event as visualized using TIRF visualization of ANFPmCherry. In Figure 4-3C,
these results were quantified, and show significantly increased numbers of events which occur for
longer than 2.5 seconds.
The above results demonstrate that a sub-set of vesicles were approaching the plasma membrane as if
to undergo exocytic fusion and then apparently being hindered at some point prior to release, although
it was also possible that the vesicles were undergoing kiss-and-run exocytosis. To answer this question,
we used the pH-sensitive fluorophore pHluorin, tagged to ANF as we had done previously with mCherry.
When contained within the acidic intravesicular lumen, pHluorin is quenched and therefore will not
exhibit fluorescence until formation of a fusion pore, which leads to de-acidification of the vesicles and
allows the pHluorin molecule to de-protonate and fluoresce (Miesenbock, De Angelis et al. 1998).
Figure 4-4A shows our cumulative probability plots as measured using pHluorin, as opposed to mCherry.
Preliminary results In this case do not appear to demonstrate the extended-duration events that were
shown with ANFPmCherry, implying that these long-duration events resulted from vesicles docking at
the plasma membrane, but without actually fusing with the plasma membrane (PH-AKT, PH-GRP1, PH-
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Figure 4-3: GFP-tagged PH expression leads to increased frequency of long-duration fusion events.
Cells expressing a GFP-tagged PH domain as well as ANFPmCherry showed several cases where a
vesicle would approach the plasma membrane and remain at close proximity to the membrane for an
extended period prior to disappearing, due either to release or else moving back into the intracellular
space beyond the excitation range of the evanescent field. A cumulative duration plot shows that cells
expressing GFP-tagged PH domains have a greater ratio of long-duration events compared to cells only
expressing ANFPmCherry, where the plasma membrane remains un-labeled (A). An example of a longduration event is shown in (1 pixel = 215 nm) (B). Under each condition, the percentage of events
which lasted greater than five seconds was calculated (C). Results show a significant increase in the
percentage of long-duration events after overexpression of all three GFP-tagged PH domains (PHPLCδ1: 10.6% vs. 0.78% control events lasting greater than 5 s., n = 5, p < 0.01; PH-AKT: 17.2% vs.
0.78% control events lasting greater than 5 s., n = 5, p < 0.01; PH-GRP1: 11.04% vs. 0.78% control
events lasting greater than 5 s., n = 5, p < 0.05).
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Figure 4-4: Cells expressing ANFP-pHluorin show fewer long-duration events. Cells were transfected
with PH-AKT and PH-PLCδ1, and PH-GRP1 as before, but ANFP-pHluorin was used to label vesicles.
pHluorin is a pH-sensitive fluorophore which is quenched in the acidic intravesicular environment, and
fluoresces upon opening of a fusion pore and subsequent de-acidification of the intravesicular area.
Preliminary data in the form of cumulative plots of event duration do not show the significant increase
in the number of long-duration events which was seen using ANFPmCherry as a vesicle marker (PH-AKT,
PH-GRP1, PH-PLCδ1, n = 2) (A). An example of a shorter-duration ANFP-pHluorin fusion event is shown
in (1 pixel = 215 nm) (B).
64
PLCδ1, n = 2). An example of an exocytic event visualized by TIRF visualization using ANFpHluorin is
shown in Figure 4-4B.
4.4: Discussion
In this study, we aimed to determine what effect, if any, was exerted on the exocytic processes by
expression of GFP-tagged Pleckstrin homology domains, and to further discern the relative binding
affinities of each of the domains under study for the various phosphoinositides found within the plasma
membrane.
In our affinity experiments, we found that all three of the PH domains under study exhibit a
predominant affinity for PI(3,4,5)P₃. This was particularly surprising in the case of PH-PLCδ1, which has
been widely published as a marker for PI(4,5)P₂ within the plasma membrane (Aoyagi, Sugaya et al.
2005, Holz, Hlubek et al. 2000, Kavran, Klein et al. 1998). Although PH-PLCδ1 showed a preference for
PI(3,4,5)P₃, it did also have the highest affinity of the PH domains tested for PI(4,5)P₂, being nearly equal
to that shown for PI(3,4,5)P₃.
It is important to note that PI(4,5)P₂ and PI(3,4,5)P₃ do not both exist at equal levels within the plasma
membrane, with PI(4,5)P₂ having a significantly higher prevalence. A study which analyzed PI(4,5)P₂ and
PI(3,4,5)P₃ levels by gas chromatography of acidic lipid extracts from sciatic and optic nerves indicated
that PI(4,5)P₂ is present at levels two to six times greater than is PI(3,4,5)P₃ in the plasma membrane.
Other studies have likewise demonstrated that PI(4,5)P₂ is present at much higher levels than PI(3,4,5)P₃
in the membrane (Vanhaesebroeck, Leevers et al. 2001, Corbin, Dirks et al. 2004). Because PI(4,5)P₂ is
significantly more prevalent in the plasma membrane than is PI(3,4,5)P₃ , it is possible that the PLCδ1 PH
domain does not require extreme specificity to PI(4,5)P₂ in order to remain functionally efficient. That
we saw a significantly higher affinity for PI(3,4,5)P₃ relative to all other membrane phospholipids for PHGRP1 seems to agree with the requirement of a highly-specific binding partner for the less prevalent
65
PI(3,4,5)P₃ membrane target, and is also in agreement with previously published data which show PHGRP1 as a prevalent binding partner of PI(3,4,5)P₃ (Pilling, Landgraf et al. 2011). PH-AKT is also widely
known to interact with PI(3,4,5)P₃ (Kavran, Klein et al. 1998, Gassama-Diagne, Yu et al. 2006), and did
show a predominant affinity for PI(3,4,5)P₃ in our experiments, although not to the degree of specificity
seen in PH-GRP1.
Holz et al., (Holz, Hlubek et al. 2000), showed that expression of PH-PLCδ1-GFP inhibited stimulated
exocytosis in chromaffin cells, as indicated by a decrease in net hormonal release after stimulation, and
also decreased change in membrane capacitance. Our TIRF secretion experimental results are in
agreement with those findings, but indicate that even a PH-domain with high affinity for PI(3,4,5)P₃ is an
efficient inhibitor of exocytosis.
The process of exocytosis is primarily driven by the soluble N-ethylmaleimide-sensitive-factor accessoryprotein receptor (SNARE) proteins, which consist of the membrane proteins syntaxin and SNAP-25, and
the vesicle membrane protein synaptobrevin (An, Almers 2004). These proteins combine together to
form a four-helix complex which “zippers” together with the helices aligning lengthwise, bringing the
vesicular and cell membranes close enough together to enable fusion of these membranes (An, Almers
2004). When the membranes fuse, a fusion pore opens between the intravesicular lumen and the
extracellular space, allowing a vesicle to expel its contents from the cell (Shi, Shen et al. 2012).
In order to undergo exocytosis, a vesicle must first be delivered, most likely by microtubules (Kamal,
Goldstein 2000), to an exocytic site within the plasma membrane where the intricate protein machinery
required for exocytosis is localized (Lang 2007). The first step in vesicle attachment to the exocytic
machinery, called docking, is the process by which vesicles are maintained and stabilized to interact with
the SNARE proteins and undergo release at the active zone (Verhage, Sorensen 2008). Several proteins
have been suggested to function in the docking process, including sec1/munc18, the downregulation of
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which results in a significant decrease in the number of docked vesicles present at exocytic sites
(Toonen, Kochubey et al. 2006, Wit, Cornelisse et al. 2006). The vesicle membrane protein
synaptotagmin has also been suggested to play a role in docking, most likely resulting from the affinity
of its C2 domains for PI(4,5)P₂, which is enriched at exocytic sites (Geppert, Goda et al. 1994, Reist,
Buchanan et al. 1998). Other proteins which have been suggested to be involved in docking include
granuphilin (Gomi, Mizutani et al. 2005)and the rab proteins (van Weering, Toonen et al. 2007, Gomi,
Mori et al. 2007).
After docking, the vesicle becomes primed for exocytosis in a process which has yet to be clearly
understood, although it seems to involve at least a partial assemblage of the SNARE proteins, and which
serves to make vesicular cargo ready for release almost immediately upon receipt of a signal resulting in
intracellular calcium increase (Verhage, Sorensen 2008). There is strong evidence to support a role for
complexin in vesicular priming, in which it binds to a central groove in the SNARE complex at a step after
zippering of the complex has begun (Bracher, Kadlec et al. 2002, Chen, Tomchick et al. 2002). Following
an increase in intracellular calcium levels, the SNARE complex is relieved of complexin by another
protein, suggested to be synaptotagmin (Jahn, Fasshauer 2012), so that the SNARE complex can fully
zipper, driving membrane fusion. Synaptotagmin itself also aides in vesicular priming through the
interaction of its C2 domains with PI(4,5)P₂ in the membrane. Following the arrival of a calcium signal,
synaptotagmin’s C2 domains both penetrate the plasma membrane and aide in bringing the vesicular
and plasma membranes into close proximity (Chapman 2002), probably while also removing complexin
from the SNARE complex as described previously. Munc13 has also been implicated as a priming
protein, functioning predominantly in changing closed syntaxin molecules to an open conformation,
making them available to form SNARE complexes with other local SNARE proteins (Gerber, Rah et al.
2008, Ma, Li et al. 2011). Our results also showed increased occurrence of long-duration events (event
duration greater than 2.5 seconds) in which loaded vesicles approached the plasma membrane as if to
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Figure 4-5: A Model of exocytic interference by PH-GFP overexpression. We propose a model
by which overexpression of GFP-expressing pleckstrin homology domains interferes with the
machinery required for synchronous exocytosis. Vesicles appear to be localized to exocytic sites
on the membrane, so actin/microtubule localization does not appear to be the cause of
inhibition {1}. Also, vesicles maintain fairly stationary positions once they appear at the
membrane, indicating that docking is not being hindered {2}. The possibility remains that steric
hindrance by the GFP-tag prevents vesicular proteins from interacting with membrane SNAREs
{3}, or alternatively, that the PH-GFP chimera somehow prevents late-acting proteins from
reaching the exocytic site {4}.
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fuse, but were then hindered at one of the exocytic steps described above prior to release. We have
formulated a model, shown in Figure4- 5, to suggest several possibilities by which such interference
could be taking place. Several studies have shown that the actin cytoskeleton and microtubules play a
critical role in delivering vesicles to exocytic sites (as reviewed in in (Kamal, Goldstein 2000)). An
advantage of using TIRF microscopy to visualize secretion (as opposed to electrophysiological or
extracellular net release quantification) is that we are able to directly visualize vesicles which appear to
approach exocytic sites along the membrane. Because vesicles appear to be properly localizing to the
membrane, our results do not seem to suggest any interference with the actin cytoskeleton localization
(Figure 4-5 {1}). Alternatively, it is possible that the comparatively large size of the GFP tag may be
sterically hindering vesicles from interacting with the plasma membrane proteins necessary for either
docking (Figure 4-5{2}) or priming (Figure 4-5{3}) to take place. When vesicles were labeled with the
neuropeptide fluorophore ANFPmCherry, they were shown to approach the membrane and then to
remain stationary at the fusion site for a prolonged period. That these long-duration events were not
seen when vesicles were labeled with the pH-sensitive fluorophore ANFpHluorin indicates the lack of an
open fusion pore during the long-duration events. It seems most likely, therefore, that the vesicles are
able to become docked at the exocytic site, and that the exocytic process is being interrupted at either
the priming stage, or else at the actual point of fusion/release.
There are a few different cases which could likely explain exocytic hindrance at the point of priming and
fusion with the membrane. First, it is possible that vesicles are able to approach the exocytic sites and
to interact with the protein machinery necessary for vesicular docking (i.e. Munc), but that the size of
the GFP tag interferes with the ability of vesicular and plasma membrane proteins to interact for priming
actual membrane fusion to take place. Alternatively, inhibition at the priming step could be caused by
an inability for the exocytic machinery to recruit other late-acting proteins to the membrane, which also
play important roles in the efficient function of the exocytic machinery, as shown in Figure 4-5{4}. As
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suggested by (Holz, Hlubek et al. 2000), changes in calcium buffering across the membrane could also
lead to exocytic inhibition at the priming step, although this didn’t appear to be the case when they
conducted experiments specifically testing this possibility.
In conclusion, our results demonstrate that overexpression of membrane phospholipid-targeted
fluorescent proteins can lead to inhibition of natural exocytic activity. That a highly-specific PH domain
for the phospholipid PI(3,4,5)P₃ was as, if not more efficient at blocking exocytosis than PI(4,5)P₂targeted PH domains, further suggests a role for PI(3,4,5)P₃-enriched lipid rafts in the exocytic process.
Finally, we have shown that the inhibition caused by PH-GFP overexpression occurs at a step after
vesicles become docked to exocytic sites on the plasma membrane, but prior to vesicular priming and
fusion with the membrane.
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General Discussion
5.1: Overall Summary
5.1.1: Effect of cholesterol on domains and exocytosis
One of the most commonly-used means of cholesterol removal from the plasma membrane is the
cholesterol-sequestering drug methyl-β-cyclodextrin (reviewed in (Zidovetzki, Levitan 2007)). Previous
studies have shown M-β-CDX to be effective in binding individual cholesterol molecules within its
hydrophobic core (Aachmann, Otzen et al. 2003) and in removing cholesterol from plasma membranes
in living cells (Dreja, Voldstedlund et al. 2002, Haynes, Phillips et al. 2000). While significant progress
has been made in characterizing the interactions that occur between M-β-CDX and cholesterol,
interactions between M-β-CDX and other membrane components have seldom been discussed or tested
experimentally. Such interactions, however, could influence cellular function to an extent which is
beyond that of mere cholesterol removal, and could therefore significantly affect experimental
outcome. In our studies utilizing the cholesterol-sequestering drug methyl-β-cyclodextrin, we
demonstrated that treatment of PC12 cells with M-β-CDX led to a decrease in secretion levels, both
before and after stimulation. The inhibitory effects which we saw on secretion did not appear to result
from any morphological changes brought about to PIP-enriched domains, as indicated by
immunohistochemistry experiments. These results imply that the drug might be exerting some other
influence on the constituents of the plasma membrane, aside from its well-known role as an agent of
cholesterol removal in the plasma membrane.
5.1.2: Effects of PH-GFP expression on exocytosis
As with the use of M-β-CDX as a cholesterol removal agent, fluorescently-tagged pleckstrin homology
domains are commonly used biomarkers used to label lipids in living cells. Several studies have utilized
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GFP-tagged PH domains to label lipids, particularly PI(4,5)P₂ in the plasma membrane to examine various
questions regarding localization of the lipids and proteins which are active in the process of exocytosis.
Again, expression of PH-GFP constructs in living cells is generally used with the assumption that its
presence at the membrane does not influence the exocytic machinery, and the occurrence of such an
influence could exert a high level of influence on experimental outcome, particularly with regard to
exocytosis. Because the study of exocytosis frequently involves examination of membrane lipids present
at sites of exocytosis in the plasma membrane, determination of an effect of PH-GFP expression on
exocytosis is of significant importance to the field. Also of importance, literature regarding the affinities
of PH domains originating from different proteins shows that different domains possess varying
affinities for the assorted lipids present in the membrane, although published levels of specificity have
not been terribly consistent. In this study, we have expressed three different GFP-tagged PH domains
(PH-AKT, PH-PLCδ1, PH-GRP1), and shown that expression in each case leads to inhibition of exocytosis
in PC12 cells. In several cases, after PH-GFP expression vesicles appeared to sit docked at the plasma
membrane for a long period of time prior to disappearing, either as a result of exocytic release or else
from the vesicle moving back into the intracellular space. As a result of these findings, we have
proposed a model in which PH-GFP expression interferes with the exocytic machinery at a step after
vesicle docking, but prior to vesicular fusion with the plasma membrane.
5.2: Influence of Methyl-β-cyclodextrin and cholesterol on exocytosis
5.2.1: Interactions between Methyl-β-cyclodextrin and other membrane constituents
When we treated cells for ten minutes with methyl-β-cyclodextrin, we saw a decrease in cell secretion
levels without a commensurate drop in the amount of cholesterol contained in the membrane, as well
as no observable morphological changes brought about in PIP-enriched lipid domains. These findings
seem to imply that methyl-β-cyclodextrin must exert some other influence on the plasma membrane,
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aside from its well-established role chelating and removing cholesterol from the membrane. Several
possibilities exist by which M-β-CDX might exert such an influence, predominantly through interactions
with other membrane constituents, some of which have been previously suggested in literature.
One interaction which has been suggested for M-β-CDX which is critical to the questions asked in this
study is that of a possible interaction between M-β-CDX and plasma membrane phospholipids. For
instance, Leventis and Silvius (Leventis, Silvius 2001) showed that the transfer of
dipalmitylphosphatidylcholine (DPPC) between large unilamellar vesicles (LUVs) was significantly sped
by the addition of M-β-CDX, implying that its addition somehow affected phospholipid transfer
dynamics. Interactions between M-β-CDX and phospholipids were also suggested by Giocondi et al
(Giocondi, Milhiet et al. 2004), as a result of the formation of holes in dioleoylphosphatidylcholine
(DOPC)/sphingomyelin bilayers following treatment with M-β-CDX. These phenomena are not unique to
model membranes, as studies in cell membranes have also demonstrated effects of M-β-CDX treatment
on phospholipids, as was found in the above mentioned experiments using model membranes (Ottico,
Prinetti et al. 2003, Singh, Kishimoto 1983, Shiraishi, Hiraiwa et al. 1993). Considering that the
hydrophobic cavity of a single M-β-CDX molecule is only approximately 0.8 nm in diameter (Anderson,
Tan et al. 2004), phospholipids, which are typically larger in size than cholesterol molecules, would
require a slightly different mechanism of interaction with M-β-CDX than that which has been proposed
for cholesterol. Accordingly, Anderson et al. (Anderson, Tan et al. 2004) suggested that single
phospholipids might be extracted from the membrane by a single M-β-CDX molecule, and then
sequestered by three more M-β-CDX molecules to form a total fusion pore diameter of 6 nm, which is
more along the size which would be required for phospholipid sequestration. Other studies, however,
have suggested that the effects of M-β-CDX are predominantly exerted upon membrane cholesterol,
with only weak interactions occurring between cyclodextrins and phospholipids (Ohvo, Slotte 1996).
Our experiments don’t appear to demonstrate any significant interactions between M-β-CDX and
73
PI(4,5)P₂ or PI(3,4,5)P₃, as evidenced by the fact that we did not see a distinguishable change in raft
immunostain intensity following treatment with M-β-CDX. If such an interaction were to have occurred,
we would have expected to see a decrease in overall raft intensity following treatment, resulting from
removal of the phospholipids from the membrane and a subsequent drop in the amount of antibody
bound to the membrane, relative to controls. Because such a drop was not seen, we can conclude that,
at least in the case of PI(4,5)P₂ and PI(3,4,5)P₃, there do not appear to be any significant interactions
with M-β-CDX which could be the underlying cause of the secretion decrease we found in the absence of
cholesterol depletion. Furthermore, that we did not see any significant change in the size or intensity of
PI(4,5)P₂ or PI(3,4,5)P₃-enriched rafts following cholesterol depleting long-term M-β-CDX treatment
seems to imply that cholesterol might not be a requirement for the formation and/or stabilization of the
class of lipid domains enriched specifically in PI(4,5)P₂ or PI(3,4,5)P₃.
Several interactions have also been suggested between M-β-CDX and proteins localized to the plasma
membrane, most likely through interactions between the hydrophobic cavity and hydrophobic domains
within those proteins. For example, M-β-CDX was shown in one study, to interact at least partially with
ubiquitin and insulin, among others (Aachmann, Otzen et al. 2003), and with glucoamylase 1 in another
(Williamson, Le Gal-Coëffet et al. 1997). Interaction between cyclodextrins and membrane proteins
has also been shown to be a main cause of cellular hemolysis after cyclodextrin treatment (Irie, Otagiri
et al. 1982). Inhibition of tyrosine phosphorylation of the high affinity immunoglobin receptor (IgE)
FcepsilonRI by M-β-CDX treatment has been linked to loss of receptor-bound IgE molecules and of the
ganglioside GD1b (Sheets, Holowka et al. 1999), which in that case, subsequently led to vesicle loss. Mβ-CDX has also been shown to be capable of inserting into the pore of the gap junction protein connexin
(Locke, Koreen et al. 2004), which may have implications for an inhibition of vesicular release.
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A possible connection between dropping secretion levels and cyclodextrin-protein interactions in PC12
cells could be that of an interaction which occurs between M-β-CDX and ion channels in the plasma
membrane. Little is known about the rate of occurrence of such interactions, however, cholesterol itself
has been tied to properties of several ion channels. For instance, the activity of voltage-dependent
potassium channels has been shown to be affected by membrane cholesterol content (Hajdu, Varga et
al. 2003), as has the coupling of L-type calcium channels and adjacent membrane proteins (Tsujikawa,
Song et al. January 2008). Chang et al., 1995 (Chang, Reitstetter et al. 1995) also showed an effect of
cholesterol content on the gating of calcium-activated potassium channels. Although specific
interactions between M-β-CDX and membrane ion channels have not been reported, the fact that we
have seen a decrease in secretion levels without a commensurate drop in membrane cholesterol levels
seems to suggest this as a possible mechanism. This phenomenon could result from perturbation of
membrane cholesterol by the M-β-CDX to enough of an extent to disrupt the dynamic interaction
between membrane cholesterol and these ion channels, but prior to the timescale required for actual
removal of the cholesterol from the membrane. Because we actually saw an increase in membrane
cholesterol levels following M-β-CDX treatment, along with a drop in secretion levels, it seems possible
also that some membrane cholesterol is being removed by cyclodextrin treatment, but is quickly being
replaced by new cholesterol from intracellular cholesterol stores. If this is the case, some time may be
required in order for the replacement cholesterol to re-stabilize the balance of ion channel activation
which is controlled by membrane cholesterol. In the interim between the time the replacement
cholesterol arrives at the membrane and when the membrane reaches dynamic equilibrium, and
inhibition of exocytosis is a possible artifact of a disruption to this balance during the de-stabilized
phase. In order to learn more about the potential for such an effect, more will need to be known about
the timescale of the dynamic interactions occurring between cholesterol and the ion channels
responsible for calcium entry and resultant exocytosis. Alternatively, the potential for a direct
75
interaction between M-β-CDX and ion channels is worth consideration, particularly in light of the
published data suggesting that individual M-β-CDX molecules are capable of inserting directly into the
connexin protein pore, as mentioned previously.
5.2.2: Where do elevated cholesterol levels come from?
It was surprising that we saw a net increase in plasma membrane cholesterol levels following ten minute
treatment with 2mM M-β-CDX, mainly because the cyclodextrin itself had no means by which to add
cholesterol to the membrane unless it was previously saturated with cholesterol (which we did perform,
and which did lead to elevated membrane cholesterol levels). Seeing such an increase is not unheard of,
however, as a similar phenomenon was seen by Fulop et al (Fülöp Jr., Douziech et al. 2001) when they
treated T-lymphocytes with 0.5 mM M-β-CDX for 30 minutes. When the treatment time was increased
to 1 hour, they did see a decrease in plasma membrane cholesterol, which is in agreement with our
long-duration treatment results. Interestingly, this finding only held true in cells from young subjects, as
cells from older subjects showed no change in membrane cholesterol following 30 minute incubation
with M-β-CDX, and did show a decrease following 1 hour incubation. The authors suggested that this
might result from the cyclodextrin having disrupted membrane raft cholesterol without having enough
time to actually remove the cholesterol from the membrane, and suggested that new cholesterol
molecules moving to the membrane from intracellular stores to replace the disrupted raft-associated
cholesterol might lead to the elevated levels found.
The study of the effect of cyclodextrins on cholesterol levels has also been examined in clinical studies
centered around Niemann Pick Type C (NPC ) Disease, a fatal disorder that affects 1 in 150,000
individuals, with most of these cases presenting symptoms before adulthood (Chang, Reid et al. 2005).
NPC disease results from mutations in proteins NPC1 and NPC2, and results in excessive accumulation of
cholesterol in lysosomes, leading to clinical symptoms ranging from swelling of the spleen and liver in
76
early stages to dementia and loss of voluntary movement in the terminal stage (Vanier 2010). There are
currently no known cures for NPC, however recent studies have shown that hydroxypropyl-βcyclodextrin (H-β-CDX) can be effective at removing cholesterol from lysosomes, and leads to at least
mild alleviation of symptoms associated with NPC disease (Matsuo, Togawa et al. 2013, Pontikis,
Davidson et al. 2013). Another study conducted in 2010 showed M-β-CDX to be even more effective
than H-β-CDX at lowering lysosomal cholesterol levels in NPC-infected fibroblasts (Rosenbaum, Zhang et
al. 2010), suggesting that M-β-CDX might be a more powerful clinical tool in the treatment of NPC.
Interestingly, this study also demonstrated an increase in intracellular cholesterol levels following shortterm treatment with M-β-CDX, similar to our findings in the plasma membrane, and displayed evidence
that cholesterol synthesis might be initiated following depletion by M-β-CDX. If the results of this study
should lead to further clinical use of M-β-CDX, our finding of a possible, previously undetected side
effect will need to be taken into consideration during clinical trial design.
In normal cells, as much as 80-90% of the total cholesterol contained within the cell has been suggested
to exist in the plasma membrane (Lange 1991), and intracellular cholesterol levels are considered to be
much lower (Severs 1982). In order to elucidate information regarding the distribution of cholesterol in
living Chinese hamster ovary cells, Mukherjee et al., (Mukherjee, Zha et al. 1998) utilized a fluorescent
cholesterol analog to visualize localization and dynamics of cholesterol in these cells. Their results
demonstrated that although the vast majority of cholesterol does exist in the plasma membrane,
significant intracellular pools were found in both the endocytic recycling compartment and the transgolgi network. The authors had also shown in a previous study that both of these sites continuously
exchange cholesterol with the plasma membrane (Mukherjee, Ghosh et al. 1997), and suggested that
they might be responsible for maintaining cholesterol homeostasis in the membrane. It seems likely,
therefore, that the increase in plasma membrane cholesterol levels which can result from short-term
77
application of M-β-CDX would originate from these intracellular cholesterol pools, the depletion of
which after longer duration treatment would lead to decreased net membrane cholesterol.
5.3: Variations in PH-domain affinities
Pleckstrin homology domains are highly-conserved protein domains which bind various
phosphoinositols, and aid in targeting their associated proteins to these phosphoinositols with varying
specificities, depending on the nature of the protein’s interaction with these lipids. The first study to
show that PH domains can bind to lipids utilized the PH domains from pleckstrin itself, as well as several
other proteins, and showed that these domains would target and bind PI(4,5)P₂ found in lipid vesicles
(Lemmon, Ferguson et al. 1995). Subsequently, it was shown that PH domains from several other
proteins were also targeted to phosphoinositides, and that the affinity of a certain proteins PH domain
for a specific phospholipid might vary depending on the phospholipid which the protein targets (Kavran,
Klein et al. 1998).
The general structure of pleckstrin homology domains is highly-conserved across all forms, consisting of
an amphipathic α-helix at its C-terminal, followed by a core β-sandwich of two anti-parallel β sheets, and
closed off by three interstrand loops (Ferguson, Lemmon et al. 1995). It is primarily at these loops that
most of the variation in binding affinities of the different PH domains occurs, due to variability in the
length of the loops themselves, as well as in the amino acid sequences of the loops, which themselves
comprise a positively charged face for each domain, at which binding to negatively-charged
phosphoiniositol headgroups generally occurs (Ferguson, Lemmon et al. 1995).
The results of our binding affinity experiments showed that, in the case of PI(3,4,5)P₃, the relative
binding affinities for each of the PH-domains tested were, from strongest to weakest affinity, PH-GRP1,
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PH-AKT, and PH-PLCδ1. These findings are generally in agreement with previously published data, which
has shown that both PH-GRP1 and PH-AKT are targeted predominantly to PI(3,4,5)P₃ (Kavran, Klein et al.
1998, Pilling, Landgraf et al. 2011, Landgraf, Pilling et al. 2008). Both PH-GRP1 and PH-AKT, according to
studies utilizing charge-reversal mutations, possess a specific glutamate residue which acts to exclude
binding of PI(4,5)P₂, thus granting highly specific targeted binding to PI(3,4,5)P₃ for those proteins. A
primary function of AKT is to act in silencing AKT1 kinase, which it does by binding and remaining docked
to an inhibitory site on the kinase. Following the arrival of a PI(3,4,5)P₃ signal, AKTs PH domain is
recruited to the plasma membrane, thus relieving inhibition of the AKT1 kinase. GRP1 acts as an ARF
nucleotide exchange factor, which requires a similar interaction with plasma membrane PI(3,4,5)P₃ for
proper functioning (Pilling, Landgraf et al. 2011). Notably, although both PH-AKT and PH-GRP1 possess
glutamate residues which greatly increase their specificity to PI(3,4,5)P₃, those residues do not occur at
the same place within the domain. The GRP1 PI(3,4,5)P₃-specific sentry residue is localized on a
different loop and on the opposite side of the inositol ring relative to the AKT residue, which could
potentially explain the difference we found between PI(3,4,5)P₃ binding specificity between these two
domains (Pilling, Landgraf et al. 2011).
In the case of PI(4,5)P₂, we saw a pattern opposite to that which was found for PI(3,4,5)P₃ with respect
to those PH-domains under study, with PH-PLCδ1 showing the highest specificity, PH-AKT intermediate,
and PH-GRP1 the lowest specificity for PI(4,5)P₂. Again, these results agree with previously published
data which have shown PH-PLCδ1 to be a consistent biomarker for PI(4,5)P₂ (Milosevic, Sørensen et al.
2005, Aoyagi, Sugaya et al. 2005, Holz, Hlubek et al. 2000). PLCδ1 acts at the plasma membrane to
regulate the hydrolysis of PI(4,5)P₂, and has been shown to require its PH domain for proper function
within the cell (Paterson, Savopoulos et al. 1995). That we did not see the specificity for PI(4,5)P₂ might
suggest that PH domains are naturally targeted to PI(4,5)P₂ with weaker affinities for PI(3,4,5)P₃, but
79
that as is the case for AKT and GRP1, residues can be used to block the natural PI(4,5)P₂ binding affinity
in order to cause a predominant affinity for PI(3,4,5)P₃ in those domains.
5.4: Effects of GFP-tagged pleckstrin homology expression
5.4.1: Influence of PH-GFP expression on secretion rates
The use of GFP-tagged pleckstrin homology domains to label membrane phospholipids provides several
advantages over other means of visualization. Primarily, GFP-tagged PH domains can be expressed in
living cells, allowing the lipid composition of the membrane to be visualized while the dynamic
processes of a living cell take place, as opposed to the requisite fixation step which is used in other lipid
visualization techniques such as immunohistochemistry. For this reason, several studies have utilized
fluorescently-tagged PH domains to label plasma membrane lipids, and have shown them to be highly
useful to this end. Van den Bogaart et al., (van den Bogaart, Meyenberg et al. 2011), for example, used
a fluorophore-tagged form of PH-PLCδ1 to visualize PI(4,5)P₂-enriched lipid domains in PC12 cells, and
showed that these PI(4,5)P₂-enriched domains co-localized significantly with domains enriched in the
SNARE protein syntaxin. Pilling et al., (Pilling, Landgraf et al. 2011), likewise showed that the PH-domain
of general receptor for phosphoinositides 1 (GRP1) could be used for targeting PI(3,4,5)P₃ in the
membrane. Specifically with regard to exocytic release, Milosevic et al., (Milosevic, Sørensen et al.
2005) used PH-PLCδ1-GFP to show that PI(4,5)P₂ levels in the plasma membrane were directly related to
the number of releasable vesicles present in the cell, however this study did not examine whether
overexpression of PH-GFP proteins in living cells exerted any influence on secretion rates. Similarly,
Aoyagi et al., (Aoyagi, Sugaya et al. 2005) used PH-PLCδ1-GFP to examine PI(4,5)P₂-enriched
microdomain co-localization with syntaxin-enriched domains and docked vesicles in PC12 cells. The
results of this study showed again that PI(4,5)P₂-enriched microdomains did co-localize with syntaxin
and with docked vesicles, and interestingly, also showed that vesicles docked to syntaxin-enriched
80
domains which were not also enriched in PI(4,5)P₂ did not undergo exocytosis, while those docked at
PI(4,5)P₂-containing domains did. As with the previously mentioned study, however, the authors did not
examine whether expression of the PH-GFP protein exerted any effect versus un-labeled cells with
regard to secretion.
5.4.2: PH-PLCδ1-GFP expression has previously been shown to inhibit secretion
One previous study has suggested an influence of PH-GFP expression on exocytosis rates, in which PHPLCδ1-GFP was expressed in rat chromaffin cells and exocytic activity was measured both as changes in
bulk release of human growth hormone and electrophysiologically as changes in membrane capacitance
(Holz, Hlubek et al. 2000). Their results demonstrated that overexpression of the PH-PLCδ1-GFP protein
led to significant inhibition of secretion by both methods used to measure it, which is in agreement with
our own results in this study. The authors proposed and tested the hypothesis that the inhibition of the
secretory response could result from changes to membrane ion channel permeability, and examined
whether directly controlling calcium entry would have a similar effect on secretion rates. After treating
permeabilized cells with various calcium concentrations, the inhibition of secretion was still present,
implying that the inhibitory effects did not result from calcium channel permeability, at least under the
conditions used in the experiment. Further electrophysiological experiments, however, did show a
reduction in net calcium inflow during stimulatory pulses, although apparently not as a result of changes
to the kinetic behavior of the calcium channels. This finding implied that PH-GFP overexpression might
somehow lead to the opening of fewer calcium channels following stimulation, however the channels
which do open apparently do so in a normal manner.
5.4.3: Potential mechanisms of inhibition by PH-GFP expression
Because of the frequent use of fluorescently-tagged PH domains for labeling membrane phospholipids,
an effect on membrane secretion is important to future work in the field of synaptic transmission. Using
81
these domains to label cell membranes without accounting for such an effect could lead to erroneous
interpretation of results in future studies, as well as serving to create an improper understanding of the
molecular exocytic machinery. Our TIRF secretion study using GFP-tagged PH domains is the first to
provide clues as to the potential step at which the exocytosis process is being hindered. Because
vesicles appear to be approaching the membrane at exocytic sites, and then remain at those fusion sites
for an extended period, it does not appear that the actin/microtubule cytoskeletal component of vesicle
delivery to the membrane is being negatively affected. Likewise, that the number of vesicles
approaching the membrane is unchanged from control conditions, and that the vesicles exhibit little, if
any movement once they dock at the membrane, demonstrates that at least some of the docking
mechanism is also able to function properly in these cells. The step at which the exocytic process is
being hindered, therefore, is likely one either in the priming step, or early in the actual fusion process, as
evidenced by the lack of long-duration events following expression of the pH-sensitive fluorophore. It is
important to note that we presume the long-duration events to still be occurring in our pHluorin
labeling experiments, because no other experimental conditions are changed from our mCherry labeling
experiments with the exception of the fluorophore used to label the vesicles for TIRF visualization. That
we no longer visualized long-duration events following pH-sensitive labeling indicates that the vesicles
are most likely remaining in a docked stage at the membrane without any opening of the fusion pore
until the point at which exocytosis takes place. If the long-duration events persisted following pHsensitive labeling, it would have indicated that the exocytic process were being hindered at a stage after
opening of the fusion pore. There are a few possibilities which remain as potential causes for the
secretory inhibition which we have demonstrated following PH-GFP overexpression. One possibility is
that the PH-GFP construct is simply creating a steric hindrance, which is interfering with the ability of the
vesicle-associated and membrane-associated SNARE proteins to interact, thus prohibiting proper
formation of the completed SNARE complex which is required for vesicle fusion. Alternatively, it is also
82
possible that the PH-GFP construct’s presence at exocytic sites somehow affects recruitment of latent
proteins to the exocytic site which are required for the priming step.
A number of these proteins have been shown to be essential for calcium-dependent exocytosis, and
most act in concert with the SNAREs in order to speed the exocytic process following an influx of calcium
to the intracellular space. One such protein is complexin, which appears to bind the partially-assembled
SNARE complex, preventing it from completely “zippering” the four alpha helices together until its
removal by a secondary protein (Chen, Tomchick et al. 2002). Alternatively, the diaglycerol-binding
protein Munc13 has also been implicated for a role in vesicular priming (Sudhof 2012). Munc13
appears to be predominantly charged with the task of changing syntaxin from its closed to its open
conformation, thus making it available to form SNARE complexes with SNAP-25 and synaptobrevin.
Synaptotagmin has also been shown to be critical for a fast exocytic response to calcium influx through
the interaction of its C2 domains with the lipid bilayer (Chapman, Davis 1998). As was mentioned
earlier, synaptotagmin’s trans-membrane domain is inserted into the vesicular membrane, and its C2A
domain is specifically targeted to PI(4,5)P₂, the vast amount of which is present at exocytic sites in the
plasma membrane.
Because the priming process as a whole remains to be fully understood, it is difficult to speculate on
whether the recruitment of any of these priming proteins in particular could be the cause of the
decrease we found in vesicle fusion. Kaeser-woo et al., (Kaeser-Woo, Yang et al. 2012) showed that an
shRNA-induced knockdown of complexin led to a loss of complexin’s clamping function in the SNARE
assembly process, as evidenced by an increase in spontaneous exocytic events and a commensurate
decrease in calcium-driven exocytosis due to depletion of the pool of primed vesicles which would
normally undergo release only after calcium entry and subsequent complexin removal from the SNARE
assembly. In our experiments, we saw decreases in both spontaneous (control) and stimulated
83
exocytosis. If PH-GFP overexpression were preventing complexin recruitment, we would have expected
to see an increase in spontaneous release, similar to that which was found in the above-mentioned
complexin knockdown experiments. It would therefore appear that recruitment of complexin to the
exocytic sites is not responsible, at least on its own, for the decreased exocytic rates which we saw,
however because there is some other form of interference which could be preventing the SNARE
complex from even beginning to take shape, we cannot rule out the possibility that complexin
recruitment is also being interfered with, and is just not apparent due to the interference of SNARE
complex formation at a step prior to complexin binding. To test this possibility, an experiment would
need to be conducted which would allow an accurate assessment of whether complexin is efficiently
being recruited to exocytic sites following PH-GFP expression. An experiment which might yield those
results could be accomplished by expressing PH-GFP, then using an antibody against complexin to
determine whether it is being efficiently recruited to the membrane. These results could be made even
more significant with the use of a third fluorophore to visualize docked vesicles, allowing us to
determine whether complexin is being specifically recruited to the membrane at exocytic sites.
Inhibited recruitment of synaptotagmin seems extremely unlikely, because synaptotagmin is integrated
into the vesicle membrane via its transmembrane domain, and the vesicles themselves are clearly
present at the membrane, therefore implying that synaptotagmin is also present and properly localized.
Although synaptotagmin does appear to be properly localized to the exocytic site, it is possible that the
binding of the PH-GFP molecule to PI(4,5)P₂ at the plasma membrane might be interfering with the
interaction of synaptotagmin’s C2 domains with PI(4,5)P₂. In the case where complexin recruitment to
the exocytic site is not affected by PH-GFP overexpression, an inability of synaptotagmin to bind
PI(4,5)P₂ in the plasma membrane might affect its ability to rescue the SNARE complex from complexin’s
clamping effect, which could lead to decreases in both spontaneous and evoked exocytosis similar to
that which was seen in our study. Inhibited recruitment of Munc13 seems also to be a plausible cause
84
of the inhibited secretion rates, which could result from failure of Munc13 to open closed syntaxin
proteins at the release site. In this case, the closed syntaxin molecules would be unable to form new
SNARE complexes with docked vesicles, again leading to a situation in which vesicles sit docked at the
membrane, but unable to become primed for release as we have found. Of these three possibilities, the
most likely cause for our observed inhibition of exocytosis is therefore either interference with
synaptotagmin-PI(4,5)P₂ binding and a resultant inhibition of synaptotagmin-mediated complexin
rescue, or else inhibition of Munc13 recruitment, and subsequent failure to provide open syntaxin
molecules at the fusion site for the formation of new SNARE complexes with arriving vesicles.
A third potential cause of exocytic inhibition is altered dynamics of the calcium channels which allow
calcium influx, a critical step which is required for the activation of synaptotagmin. Lack of
synaptotagmin activation by calcium influx could also lead to docked vesicles not fusing with the
membrane, resulting in a decrease in both spontaneous and evoked exocytosis also. Indeed, Holz et al.,
(Holz, Hlubek et al. 2000) showed that intracellular calcium levels were decreased following PH-GFP
expression, which does argue that PH-GFP overexpression does apparently affect the behavior of at
least some calcium channels. In fact, studies have shown that plasma membrane PI(4,5)₂ binds and
directly regulates the activity of both calcium (Suh, Kim et al. 2012)and potassium channels. (Kruse,
Hammond et al. 2012)It must also be noted, however, that exocytic inhibition occurred in permeabilized
cells even when calcium levels were controlled experimentally, thus implying another mechanism of
interference possibly acting in concert with, but independently of decreased opening of calcium
channels. Taken together, therefore, the strongest argument for the mechanism of both spontaneous
and evoked exocytic inhibition seems to be either PH-GFP interfered binding of the C2 domain of
synaptotagmin to PI(4,5)P₂ at the exocytic site, or else failed recruitment of Munc13 and subsequent
perpetually-closed conformation syntaxin at exocytic sites.
85
5.5: Functional implications of PIP-enriched domains in secretion
It might also be asked what functional purpose would be served to cellular function by the occurrence of
PIP-enriched domains. First and foremost, they appear to play a critical role in organizing the
presynaptic exocytic machinery which is required for chemical signaling in both neuroendocrine and
neuronal cells. Both pre- and post-synaptically, there are several advantages to organization of the
molecular machinery required for exocytosis. As has been previously mentioned, both syntaxin and
SNAP-25 are required for exocytosis, and must be able to form complexes with the vesicle-associated
protein synaptobrevin. It would stand to reason, then, that these proteins should be localized within
close proximity to each other in the membrane so that vesicle fusion does not depend on both vesicle
localization to the membrane and also having to seek out sections of the membrane which may or may
not possess all of the protein machinery required for vesicle release. Arguing further toward the
importance of organized exocytic release sites, several studies have shown that multiple SNARE
complexes are required to stabilize and dilate the fusion pore that forms between the vesicular and
cellular membranes for exocytosis to take place (Shi, Shen et al. 2012, An, Almers 2004, Sinha, Ahmed et
al. 2011). It seems critical for the cellular ability to quickly release chemical signals, therefore, to have
the presynaptic exocytic machinery localized within close proximity for fast complex formation following
the arrival of a vesicle at the membrane. As a result of the apparent requirement of PI(4,5)P₂ for the
clustering of syntaxin and SNAP-25, as well as its known interaction with synaptotagmin, it seems
likewise imperative that PI(4,5)P₂-enriched domains exist in the plasma membrane, and that these
domains contain the aforementioned SNARE proteins syntaxin and SNAP-25. In this manner, all of the
core pre-synaptic exocytic constituents are efficiently organized to specific domains within the plasma
membrane and are capable of complexing immediately upon vesicle arrival.
86
In the case of neuronal connections, the organization of the presynaptic exocytic machinery into specific
domains would also be useful for efficient transmission of chemical signals from the presynaptic to the
postsynaptic cells. Due to the requirement that chemical signals released from the presynaptic cell must
traverse the synaptic cleft in order to reach postsynaptic receptors, the organization of postsynaptic
receptors in close proximity to presynaptic release sites would serve to further enhance the efficiency of
chemical signaling across the synapse. Taken together, it is apparent that several functional advantages
are afforded to chemical signaling, both pre and postsynaptically, as a result of organized domains which
are enriched in phospholipids such as PI(4,5)P₂ and also in the protein machinery required for
exocytosis.
5.6: Are these effects unique to exocytic sites?
We have shown that treatment of the plasma membrane with the drug Methyl-β-cyclodextrin (with and
without cholesterol removal) and expression of three different pleckstrin homology domains can lead to
the inhibition of exocytic activity in PC12 cells. In both of these cases, it remains to be seen whether the
effects of these conditions are uniquely exerted on exocytic sites, or whether they affect the cell as a
whole, with effects only being made visible at secretion sites. For instance, several studies have
suggested different rates of cholesterol removal in domain vs. non-domain sections of the plasma
membrane (reviewed in (Zidovetzki, Levitan 2007)). In the case of cholesterol removal, we cannot
speculate as to whether there was a preference for domain vs. non-domain cholesterol, as our assay
examined the plasma membrane as a whole. Perhaps an interesting future study might involve
examining high-density vs. low-density fractions from the membrane, in order to discern some idea of
whether or not such a preference is present.
In the case of PH-GFP expression, it appears that the plasma membrane as a whole is effectively labeled
by each of the three PH-GFP molecules used in this study. Other experiments have shown punctate
87
distribution of PH-GFP constructs in living cells (van den Bogaart, Meyenberg et al. 2011, Aoyagi, Sugaya
et al. 2005), however our own results seem to show a more general labeling of the membrane as a
whole. Again, it is difficult to speculate on any other cellular functions which might be affected by
labeling, and thus possibly interfering with the interactions of, membrane phospholipids. We can say
with certainty, however, that this homogenous membrane binding was to sufficient enough of a degree
to interfere with the exocytic machinery, and therefore, may well also be capable of interfering with
other delicate protein machinery on the inner leaflet of the plasma membrane.
5.7: General Conclusions
In this thesis, I have presented the results of two studies which examined different aspects of lipidenriched membrane domains in exocytosis. In the first study, we examined the influence of the
cholesterol sequestering drug methyl-β-cyclodextrin (M-β-CDX) on cholesterol removal and exocytosis.
Our results showed that treatment of PC12 cells with 2 mM M-β-CDX for ten minutes led to significant
inhibition of exocytosis without any substantial removal of cholesterol from the membrane. Treatment
for two days, however, did lead to membrane cholesterol removal along with secretion inhibition. We
also examined the size, intensity, and number of PIP-enriched rafts following M-β-CDX treatment using
fluorescently-tagged antibodies against both PI(4,5)P₂ and PI(3,4,5)P₃, and the results of these studies
suggest that there were no distinguishable morphological changes in domains enriched in these two
phospholipids following M-β-CDX treatment. These findings suggest a previously unknown secondary
interaction between M-β-CDX and some component of the plasma membrane, which leads to inhibition
of secretion independently of cholesterol.
Our second study examined the influence of expressing GFP-tagged pleckstrin homology domains on
exocytosis. For this study, we expressed PH domains from three different proteins (PH-AKT, PH-GRP1,
and PH-PLCδ1), tagged with the fluorophore GFP, then examined the influence of PH-GFP
88
overexpression on secretion by visualizing individual exocytic events using Total Internal Reflection
Fluorescence (TIRF) Microscopy. We also conducted an assay which tested the binding affinities of each
of the three PH domains in our study for several different membrane phospholipids, in order to
determine the relative affinities for domains enriched in PI(4,5)P₂ and PI(3,4,5)P₃ in the membrane. The
results of this assay demonstrated that, in the case of PI(4,5)P₂, the relative binding affinities of the
three PH domains under study is, from greatest to least affinity, PH-PLCδ1 > PH-AKT > PH-GRP1. In the
case of PI(3,4,5)P₃, however, the relative affinities are exactly the opposite, PH-GRP1 > PH-AKT > PHPLCδ1. Of the domains tested, PH-GRP1 appeared to possess the largest degree of specificity, likely
resulting from the relatively lower concentration of PI(3,4,5)P₃ present in the membrane, and the
subsequent requirement of a high degree of specificity for that lipid so that binding to the other
phospholipids present in the membrane doesn’t interfere with GRP1’s necessary interaction specifically
with PI(3,4,5)P₃.
Our results demonstrated that overexpression of all three PH domains led to inhibition of secretion, and
that both PH-GRP1 and PH-AKT, the PH domains most specific for PI(3,4,5)P₃, were as efficient as PHPLCδ1, the domain most specific for PI(4,5)P₂ in blocking secretion. Also, there was a significant increase
in the number of vesicles which exhibited long-duration dwell times at the membrane, as indicated by
vesicles visibly dwelling close to the membrane in our TIRF secretion experiments. When we labeled
vesicles using the pH-sensitive fluorophore ANFpHluorin, the long duration dwelling vesicles were no
longer visualized. These results seem to imply that expression of GFP-tagged pleckstrin homology
domains leads to inhibition of exocytosis, apparently at a step after vesicular docking at the exocytic
site, but prior to fusion with the membrane.
These studies suggest previously unknown roles for two commonly-used tools in the study of lipidenriched domains and exocytosis. The possibility of a secondary interaction between M-β-CDX and the
89
plasma membrane will have a significant impact on future studies which utilize this drug for cholesterol
removal. Likewise, the inhibitory effect of PH-GFP overexpression on exocytosis will need to be
addressed in any future studies which aim to utilize PH-GFP as a membrane lipid marker during any
studies involving the exocytic machinery. In both cases, the knowledge gained from these experiments
will contribute to further clarity and increased awareness of possible secondary effects associated with
the use of these tools for all future experiments in which they are utilized.
90
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