-- - - - - - - - - - - -
Chapter 1: Introduction
1.1
PREFACE
One of the most important aspects of a living cell is to sense and respond to the
external environment. The perception of an external stimulus of the cell is the
property of a set of proteins called receptors, which mediate the process of sensing
and uptake of molecules from the external environment into the cell. A living cell
besides receptor-mediated process, also indulges in processes of drinking and eating
from the extracellular environment. The overall process of uptake of an extracellular
material into a membrane-limited organelle in a living cell is called 'endocytosis',
further categorized depending on the mode of uptake of molecules (Pas/an and
Willingham, 1985). The engulfment of large particulate matter is termed as
'phagocytosis' and the organelle formed in this process is termed as a 'phagosome'
(Pastan and Willingham, 1985). Most of the bacteria and viruses are cleared from the
blood by this process. These vesicles are generally greater than 250 nm in diameter.
The uptake of fluid phase/large bubbles from extracellular environment is termed as
'pinocytosis/macropinocytosis' processed by 'Pinosome/macropinosomes' (Pas/an
and Willingham, 1985), which are around or less than 150 nm in diameter. Similarly,
the uptake via receptor-mediated pathway involving a clathrin-coated pit is termed as
a 'receptor-mediated endocytosis' and the organelle involved is called a receptosome
(Pastan and Willingham, 1985). In general, the sub-cellular organelles involved in
this overall process of endocytosis by any of the three above-mentioned routes are
termed as 'endosomes'.
In a prokaryotic cell, communication with external environment takes place
across the plasma membrane. Many enzymes are secreted to the cell exterior and
small metabolites are taken inside the cell by certain transport proteins present in the
plasma membrane. In contrast, a eukaryotic cell has evolved an elaborate mechanism
of endocytosis, by which it takes up the extracellular material, delivers it to the
digestive enzymes stored in Iysosomes, and the metabolites generated after digestion
are then delivered to the cytosol directly. For this purpose, the ligand has to travel
along the internal membrane system, which also helps in regulating the delivery of
newly synthesized proteins and carbohydrates to the cell exterior or to a specific cellsurface domain by a process called exocytosis (Alberts et aI., 1994). Hence, endocytic
and biosynthetic-secretory pathways go hand-in-hand. All compartments along both
2
the pathways are in constant communication with one another by certain transport
vesicles, which bud off from one membrane and fuse to another. The traffic in the cell
•
is highly organized. The biosynthetic-secretory pathway leads to outside of the cell
making a macromolecule move from endoplasmic reticulum (ER) to Golgi apparatus
and finally leading it to the cell surface, where a side route leads to lysosomes too.
The endocytic pathway leads the molecule inward, moving it from the cell surface to
endosomes and then to the lysosomes. Inspite of a continuous intake of
macromolecules from the external fluid to the cell interior the surface area and
volume of the cell remain unchanged, suggesting that as much membrane is being
removed from the cell surface in the endocytic process, the same is being replenished
back by the process of exocytosis. Hence, endocytic and exocytie pathways together
constitute a very complex, but an efficient transport network, which is very essential
for the biogenesis of plasma membrane, lysosomes and endosomes. It is also
necessary for the secretion of proteins and other molecules from the cell and the
uptake of molecules from the external environment (Alberts et al.. 1994).
1.2
DIFFERENT TYPES OF UPTAKE MECHANISMS
As mentioned earlier, depending on the type of molecule and the mode of its uptake,
endocytic process has been broadly classified into pinocytic, phagocytic and receptor
mediated pathways. However, further categorization of the processes has been done
depending on whether or not the process utilizes clatbrin, a molecule known to be
very important for transport of trans membrane receptors involving coated pits.
1.2.1 Clathrin-dependent pathways
The endocytic process usually begins at specialized regions of the plasma membrane
called clathrin-coated pits, which typically occupy nearly 2% of the total plasma
membrane area. Substances dissolved in the extracellular fluid are internalized by
getting trapped in clathrin-coated pits by a process called "fluid-phase endocytosis" or
"pinocytosis". In most animal cells, clathrin-coated pits and vesicles provide an
efficient pathway for taking up specific macromolecules from the extracellular fluid, a
process called "receptor-mediated endocytosis". More than 25 different receptors are
known to participate in receptor-mediated endocytosis of different types of molecules,
and they all apparently utilize the same clathrin-coated pit pathway (Smythe and
Warren. 1991).
3
While c1athrin-coated pinocytic vesicles are usually small and uniform in size,
c1athrin is also involved in the fonnation of much larger vesicles including secretory
vesicles that contain large protein aggregates and phagosomes that contain large
particles. In the case of phagocytic vesicles clathrin forms patches rather than
complete coats on the forming vesicles (Anderson, 1992).
1.2.2 Clathrin-independent pathways
Until recently, the notion has prevailed that all cell-surface receptors follow the same
pathway and are internalized by c1athrin-coated pits, with sorting at the cell surface
achieved solely through the direct or indirect binding of receptor cytoplasmic domains
to c1athrin-associated proteins. However, c1athrin-independent ways of entry into the
cell also exists (Felberbaum-corti el al., 2003). Two views have been proposed
regarding c1athrin-mediated and non-c1athrin-mediated pathways: (i) that both
pathways operate in parallel and non-c1athrin-mediated pathway is upregulated, when
the other is switched off; (ii) that both use same mechanism for budding from plasma
membrane and that c1athrin along with its adaptor proteins serve the purpose to trap
some specific membrane proteins and their ligands in the vesicle (Robinson el aI.,
1996).
1.2.2.1 Caveolae-mediated pathway
Most of the extracellular ligands are known to be internalized by using c1athrin-coated
vesicles. However, IL-2 (interleukin-2) appears to use a non-c1athrin-mediated
pathway to be internalized; IL-2 receptor in leukocytes is efficiently internalized after
K+ depletion (Robinson et al., 1996). It was shown to enter cells through lipid raft and
caveolar pathways that do not require Eps 15, a component of the clathrin machinery.
The endocytosis of the receptor still proceeds by the classical pathway; that is from
endosomes to Iysosomes for degradation. Lipid rafts are cholesterol- and
sphingolipid- rich domains in the membrane (Anderson and Jacobson, 2002; Simons
and Toomre, 2000), a subpopulation of which fonn membrane invaginations called
cavcolae that are rich in Caveolin protein. Lipid rafts and caveolae function in
vesicular and cholesterol trafficking (Anderson et al., 1992; Razani et aI., 2000;
Gumbleton et al., 2000) as well as internalization of toxins and SV40 virus (Pelkmans
et al., 2001). This raft-dependent and clathrinlEpsl5-independent internalization
pathway is also taken by glycosyl phosphatidylinositol (GPI)- anchored proteins on
4
their way to conventional endosomes (Van Der Goot and Gntenberg, 2002; Sharma
et ai., 2002). Similarly, another ligand,
TGF-~
bound to its receptors is internalized
either by clathrin dependent or independent (raft dependent) pathways. However,
depending on the route of entry, the fate of internalized
TGF-~
receptors will be
different (Di Guglielmo et aI., 2003).
1.2.2.2 Macropinocytosis
In case of phagocytosis, the studies suggest formation of actin-rich phagocytic cup for
the internalization of large particles (Greenberg, 1995; Swanson and Baer, 1995), and
its extension for engulfment is probably driven by directed actin polymerization
against the plasma membrane. Similarly macropinocytosis too is an actin dependent
process but unlike phagocytosis and pinocytosis, it is a clathrin-independent endocytic
process (Johannes and Lamaze, 2002) that results in non-selective internalization of
extracellular solutes and nutrients by the formation of fluid-filled macropinosomes.
The macropinosomes are non-clathrin coated, irregular, large endocytic vesicles
ranging from
O.5-5.0~m,
formed when plasma membrane ruffles fuse together. In
addition to membrane ruffle fusion, role of certain other signaling molecules has been
implicated in the formation of macropinosomes including F-actin, integrins, AP I
adaptor complex, phosphatidyl Inositol 3-kinase and the Rho and Rab family GTPase,
Rah/Rab34.
1.3
INVOLVEMENT OF ENDOCYTOSIS IN VARIOUS CELLULAR
PROCESSES: THE SIGNIFICANCE
Endocytosis is a highly dynamic process responsible for many cellular functions. It is
an essential process for cell homeostasis since it controls many functions including
nutrient uptake, autophagy, transcytosis, signal transduction, growth factor and
hormone
responses,
antigen
presentation,
neurotransmission,
intercellular
communication, the entry of some pathogens such as viruses, toxins, and different
micro-organisms etc. (Gutierrez et al.• 2004; Klionsky. 2005; Mellman, 1996;
Miaczynca. 2004; Monks and Neville. 2004). In addition, of late, receptor mediated
endocytosis has been considerably exploited to design efficient site specific and target
oriented drug delivery system in order to achieve non-destructive delivery of various
bio-therapeutics into living cells.
1.3.1 Significance in pathophysiology
5
Endocytic process offers a highly selective mechanism of internalization, allowing
restricted entry of extracellular material in the intracellular environment. Thereby the
process restricts the intracellular entry of microbes and related agents. However,
certain pathogens like Salmonella typhimurium, Legionella pnelllnophila and
Mycobacterium tuberculosis have evolved with strategies to exploit the endocytic
pathway to gain entry in macrophage cells and manipulate them in a manner that
favours their own survival and growth inside the macrophage vacuoles (Aderem and
Underhill, 1999).
Salmonella typhimurium is a facultative intracellular pathogen. The macrophage
receptors that bind this pathogen are not yet known. However, its internalization
appears to be associated with an actin-dependent mechanism involving membrane
ruffling events that results in internalization of the bacterium into a compartment
resembling a macropinosome. Since the vacuole containing the pathogen is enormous
relative to the size of the bacterium, it has been called a "spacious phagosome"
(Alpllche-Aranda et al., 1994). Once internalized the spacious vacuole containing
virulent bacteria persists, and the bacteria multiply. There are conflicting reports
regarding maturation of spacious phagosomes; while some suggest acquisition of
lysosomal markers such as LAMP-I and cathepsin-L by spacious vacuole, others
suggest that the vacuole remains immature and does not fuse with the Iysosomes (Oh
et al., 1996; Rathman et al., 1997). Furthermore, Salmonella containing early
endosomes retain Rab 5 and Rab 18 and do not acquire late endosome characteristics.
In addition, Salmonella selectively depletes the vacuolar W-ATPases from the
membrane of host phagosome/early endosome to prevent endosomal acidification
(Hashim et al., 2000). Similarly, Mycobacterium species also has a capability of
surviving inside the host macrophages. The success of these bacteria as pathogens
depends on their ability to maintain infection inside the phagocytic vacuole of
macrophage. Once internalized, M tuberculosis resides in a membrane-bound vacuole
that resists lysosomal fusion (Hart et aI., 1972) and is only mildly acidified (StllrgillKoszycki et al., 1994). Phagosomes containing M avium also fail to acidifY below pH
6.5, and this appears to be due to the specific exclusion of the vesicular protonATPase (Stllrgill-Koszycki et al., 1994).
1.3.2 Endocytosis in target specific drug delivery
The rapid development in current pharmaceutical drug discovery has resulted in the
6
emergence of increasing numbers of novel therapeutic drugs for the treatment of a
variety of diseases. However, at present major problems associated with systemic drug
administration are (a) biodistribution of pharmaceuticals throughout the body; (b) lack
of drug-specific affinity towards a pathological site; (c) nonspecific toxicity and other
side effects resulting from high doses. An attractive strategy to enhance the
therapeutic index of drug is to specifically deliver it to the defined target cells and
keeping it away from healthy cells which are sensitive to the toxic effects of the drugs.
Furthermore, many therapeutic molecules such as anticancer drugs, proteins, and
peptides are generally excluded from transport from blood to brain, owing to the
negligible penneability of these drugs to the brain capillary endothelial wall, which
makes up the BBB (blood-brain barrier) in vivo. However, therapeutics may be
delivered to the brain with the use of strategy of coupling therapeutics to a BBB drug
transport vector. Brain capillary endothelial cells possess specific receptor-mediated
transport mechanisms that potentially can be exploited as a means to deliver
therapeutic molecules to the brain.
Polymer- and liposome-based delivery systems show potentials as specific and
target-oriented delivery systems (Langer, 1998; Maruyama et al., 1999; Vyas and
Sihorkar, 2000; Vyas et aI., 2001). Naturally existing proteins (such as transferrin)
have also received major attention in the area of drug targeting since these proteins are
biodegradable, nontoxic and nonimmunogenic. Moreover, they can achieve sitespecific targeting due to the high amounts of their receptors present on the cell
surface. The efficient cellular uptake of transferrin (Tr) pathway has shown potential
in the delivery of anticancer drugs, proteins and therapeutic genes into primarily
proliferating malignant cells that over-express transferrin receptors (TfRs) (Kratz and
Beyer, 1998; Singh, 1999; Vyas and Sihorkar, 2000; Kircheis et aI., 2002).
Transferrin is primarily iron-binding protein, but in human serum, Tf is only
about 30% saturated with iron, so there is a potential capacity for binding to other
metal ions that enter the body. Indeed, over 30 metal ions have been reported to bind
to Tf with either carbonate, oxalate, or other carboxylates as synergistic anions,
although Fe3+ has a higher affinity than any other metal ion for which the binding
constant has been determined (Aisen, 1998; Sun et al., 1999). Such binding may play
an important role in the transport and delivery of medical diagnostic radioisotopes
such as
67 Ga 3+
and IIIIn 3+ (Harris and Pecoraro, 1983; Ward and Taylor, 1988;
7
Harris etal., 1994} and therapeutic metal ions such as Bi 3+ (Sun et al., 2001; Zhang et
al., 2001), Ru 3+ (Kratz et al., 1994; Smith et aI., 1996) and Ti 4 + (Messori et al., 1999).
Besides transferrin receptors, folate receptors have also proved to be an excellent
target for anticancer therapies because they are over-expressed in most types of
human epithelial cancers including ovarian, choriocarcinomas, choroid plexus,
ependymomas, lung, kidney, breast, brain and colon (Ross et al., 1994; Weitman et
al., 1992a,b; Campbell et al., 1991). Furthermore, folate receptors are generally
absent in most normal human tissues with the exception of placenta, lung, thyroid,
and kidney. Low and co-coworkers have demonstrated that molecules can target the
high-affinity folate receptor (hFR) when conjugated to folic acid (Lu et aI, 2004;
Leamon and Low, 2001, 1994, 1991). As compared to antibodies or other ligands,
folic acid is presumed to be non-immunogenic due to its small size, has good
solubility, and is relatively specific for a variety of tumors. Few of the folate
conjugates that have been reported to be delivered to cancer cells over-expressing the
folate receptor are - (a) Folate conjugates of radiopharmaceutical agents (Ke et al.,
2004); (b) MRI contrast agents (Kanda et aI., 2000); (c) Low molecular weight
chemotherapeutic agents (Ladino et al., 1997); (d) Antisense oligonucleotides (Li et
al., 1998); (e) Proteins and protein toxins (Ward et aI., 2000); (I) Immunotherapeutic
agents (Kranz e/ al., 1998); (g) Liposomes with entrapped drugs (Lee and Low, 1995)
and (h) Plasmids (Douglas et al., 1996)
Of late researchers have started focusing on use of nanoparticles in targeted drug
delivery. Researchers at Harvard Medical School, US, Massachusetts Institute of
Technology (MIT), US, and Gwangju Institute of Science and Technology, Korea
were interested in developing a system for targeted delivery - intracellular uptake and controlled release of an active chemotherapy drug over an extended period of
time directly inside the cancer cells. To achieve this combination of effects, drugcontaining nanoparticles (-150 nm) using the polylactic-co-glycolic acid (PLGA)
controlled release polymer system have been developed. To the surface of these
nanoparticles RNA molecules, called aptamers have been attached. These Aptamers
can bind to the surface of prostate cancer cells. The resulting particles attach only to
prostrate cancer cells but not to normal cells, are taken up by the malignant cells via
receptor-mediated pathway and release the active drug over an extended period.
Aptamer-decorated nanoparticles were more toxic to cancer cells in in vitro tests than
8
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drug-containing nanoparticles without aptamers. In an animal experiment, five out of
seven mice showed complete tumour reduction (Farokhzad et al., 2006).
1.4
ENDOCYTIC PATHWAY - AN OUTLINE
The first question arising for understanding the pathway in depth is what are the
various steps of the pathway? - To a great extent this question has already been
answered and various steps involved in the uptake of a macromolecule from the
external medium to the cell interior are known (Figure 2). For example, the steps
involved in the internalization of a ligand from the external environment via a
receptor-mediated pathway can be listed as follows (Pastan and Willingham, 1985;
Alberts et aI., 1994; Lodish et al., 1999): (a) binding of the ligand to its specific
receptor present on the cell surface; (b) clustering of the ligand-receptor complex in
the c1athrin-coated pit and their internalization; (c) uncoating of the vesicle and
formation of receptosome (endosome); (d) sorting of ligand and receptor; (e) delivery
of either ligand alone or ligand and receptor both to the lysosome; and (e) ultimately,
degradation of the lysosomal content by hydrolytic enzymes.
Similarly, phagocytic process can be divided into four discrete steps (Greenberg
and Silverstein, 1993; Evans et al., 1993; Greenberg, 1995; Swanson and Baer,
1995): (a) particle binding to the plasma membrane; (b) assembly of an actin-rich
phagocytic cup that surrounds the particle; (c) internalization of the particle; and (d)
degradation of phagosome contents, following trafficking along the endo-lysosomal
pathway. Studies in Dic/yostelium indicate that phagocytosis also relies in part on the
activity of molecular motors of the myosin superfamily. Disruption of myosin lB
gene results in cells which are 30% slower in particle uptake than the parent strain
(Jung and Hammer Ill, 1990). In mutants lacking the actin-associated protein coronin,
phagocytosis is even more strongly inhibited, as uptake levels are reduced by 60%
(Maniak et al., 1995). In cells challenged with particles, both myosinlB and coronin
are enriched at the cell surface extensions induced by particle attachment, these being
the phagocytic cups (Fukui et al., 1989; Maniak et al., 1995; Hacker et al., 1997).
1.4.1 Necessities for ligand internalization
After drawing a general outline of the pathway, the next question cropping up in our
minds would be regarding conditions required for a ligand to be internalized.
1.4.1.1 Temperature
9
A ligand can bind to its specific receptor in the coated pit region at 4°C, but can be
internalized only at 37°C. This was shown using EGF (epidermal growth factor) as a
ligand, when added to the cells at 4°C, the receptor-bound EGF had a random
distribution on the cell surface. However, when the temperature was raised to 37°C,
the complex rapidly got accumulated in the coated pits and then after every 20 sec,
fonnation of a receptosome carrying the contents of pit was observed. In another
study it has been suggested that cooling mammalian cells to I-4°C reduces the
receptor mobility about 4-folds when compared to 37"C. Internalization of the ligandreceptor complex is also reported to take place at IS-20°C, but the uptake is again
very slow. These observations about the effect of temperature on internalization of
receptors are summarized by Pastan and Willingham (1985).
1.4.1.2 Size of ligand
If ligand is small enough to get trapped in a vesicle ISO nm or less in diameter,
internalization would be by pinocytic pathway and if it is too big to be engulfed by a
vesicle < 250 nm in size, it would also be internalized by phagocytic pathway.
However, studies in Dictyostelium, point to presence of 600 nm primary pinosomes.
Pinosomes larger than 200 nm are termed macropinosomes in mammalian cells
(Swanson and Watts, 1995) and are thought to be formed by large actin-containing
membrane ruffles, which protrude from the cell surface, circularize, and liberate a
large fluid-filled vesicle (Dowrick et aI., 1993). Involvement of actin cytoskeleton in
both phagocytic and pinocytic process indicates that internalization of particles and
fluid, depend to a similar degree on polymerized actin (Hacker et al., 1997).
1.4.1.3 Concentration of ligand
For the smaller ligands «150 nm), its concentration is an important factor deciding
the route of entry inside the cell: either by pinocytosis or receptor-mediated
endocytosis. If the concentration of the receptor-specific ligand is low enough
(usually few nM) to saturate the receptors, then most of it would follow receptormediated route, but if the concentration is much higher, it can enter the cell by
pinocytosis. Pinocytosis amounts to indiscriminate uptake of extra-cellular fluid
(Alberts et al., 1994; Lodish et al., 1999).
1.4.2 Fate of ligand and its receptor
10
A typical eukaryotic cell has a variety of receptors distributed on the cell surface. In
absence of certain ligands, receptors such as for EGF are randomly distributed on the
cell surface, but for LDL (low density lipoprotein) or transferrin, they are clustered in
specialized depressions in the cell membrane termed as coated pits, the site at which a
ligand bound to its specific receptor accumulates and is next internalized within the
cell. In most cells, around 500-1000 coated pits, occupying about 1-2% of the cell
surface are found. The vesicles bearing these ligand-receptor complexes are the
receptosomes (endosomes) (Pastan and Willingham, 1985), also called primary or
early endosomes. These transport organelles carry the ligand-receptor complex from
the cell surface to the interior with ligand still bound to its receptor. Further sorting of
the ligand and the receptors is carried out in this organelle. There are various
possibilities for the fate of this complex: (i) the receptor is recycled back to the cell
surface and the ligand is delivered to the Iysosomes for degradation e.g., LDL
receptor (Ladish et al., 1999); (ii) both ligand and receptor are passed on to the
Iysosomes e.g., EGF and its receptor (Pastan and Willingham, 1985), Antigenantibody complex along with Fc receptor; and (iii) ligand and its receptor are recycled
back to the surface and reutilized. e.g., after releasing iron in endosomes, transferrin
and its receptor both are sent back to the plasma membrane for reutilization (Pastan
and Willingham, 1985; Alberts et al., 1994).
1.4.3 Rapid speed of endocytic process
Endocytosis is a very rapid process. To know the speed of uptake of a ligand, binding
experiments have been carried out at 4°C. After a significant number of ligand
molecules are bound to the cell surface, then warming the cells to 37°C and studying
them by morphological methods indicate that ligands appear in receptosomes in 1-3
min and within 10-15 min they tend to appear in perinuclear golgi region. However,
when the ligand is added to cells already maintained at 37°C, it appears in
receptosomes within 25 sec (Pastan and Willingham, 1985).
1.5
MOLECULAR PLAYERS OF ENDOCYTIC MECHANISM
The entire endocytic process has been under thorough investigation and tremendous
insight has been obtained about the cellular, molecular and genetic aspects of
endocytosis. Although these studies have been carried out only in a few types of cells,
but it is now clear that many cell types carry out similar kind of internalization
II
processes. The realization that cells internalize many molecules, like hormones,
growth factors, toxins, nutrients, various transport proteins, viruses, etc., has aroused a
similar series of questions regarding the route of uptake of molecules, the mechanism
of internalization, other molecules and factors involved in the process and many more
(Pastan and Willingham, 1985). Answers to many of these questions are available,
which have enabled the elucidation of the significant role that endocytosis plays in
various cellular processes (Figure I). However, there is still a need to decipher more
details about the endocytic pathway.
1.5.1 Endocytic machinery involved in internalization events
The ligand is internalized either by membrane ruffling or invagination. The bestcharacterized mechanism for a ligand to gain entry into the cell is via clathrin-coated
pits (Brodsky, 1988; Robinson, 1987) present on the cell membrane, as well as in the
Golgi region of the cells. Two types of coated vesicles, clathrin-coated and coatomercoated have been reported. The former is involved in the trans membrane receptor
transport, like LDL receptors and the latter in the non-selective vesicular transport of
the default pathway, which includes transport from ER to Golgi apparatus, from one
Golgi cisternae to another and from trans Golgi to plasma membrane (Kreis, 1992).
Most of the molecules required for both clathrin-mediated receptor endocytosis
from the cell surface as well as intracellular vesicular trafficking have been identified
and characterized. Apart from involvement of clathrin and its adaptor protein, known
to be involved in the receptor-mediated endocytosis involving coated pits, various
other proteins like, a coatomer protein, COPII, and ARFs are now known to play very
important role in forming coated vesicles (Rothman, 1994). They are believed to
mediate the non-selective vesicular transport. The coatomer protein, COPH and ARFs
have their respective roles at different steps in the endocytic pathway.
1.5.1.1 Clathrin and adaptor proteins (AP's)
Two oligomeric protein complexes, clathrin and adaptor proteins, are major
constituents of clathrin coats. Clathrin is a protein whose basic assembly unit is a
triskelion (Robinson, 1994), consisting of three copies each of clathrin heavy (HC)
and light chains (LC). This triskelion assembles into a lattice of hexagons and
pentagons fonning a cage-like structure that encloses the membrane vesicle (Robinson
et ai., 1996). In low ionic strength, mildly acidic, high ci+ buffers, purified clathrin
12
triskelia self-assemble into closed polyhedral cages resembling the coats on transport
vehicles. Removal of LC subunits from the triskelia does not prevent cage fonnation,
indicating that self assembly is an inherent property of HC (Ungewickell and
Ungewickell, 1991). However, compared to intact clathrin, LC free triskelia assemble
more readily in absence of Ca2+, suggesting that LC might inhibit spontaneous
cIathrin assembly, thereby ensuring assembly only at membrane sites destined for
vesiculation (Ungewickell and Ungewickelll991; Pishvaee and Payne, 1998).
For fonnation of a clathrin-coated pit, clathrin should be recruited to the plasma
membrane from cytoplasm where it assembles into a lattice. In vitro studies suggest
involvement of a Tyr-containing 'internalization signal', located in cytoplasmic
domain of membrane proteins, notably receptors, in the recruitment of clathrin, where
the signal works by binding to protein complexes called adaptors or APs, that are also
components of the coat, fonning an inner layer and attaching the clathrin to the
plasma membrane (Robinson et al., 1996) (Figure 3). These adaptor proteins act in
both clathrin assembly and cargo collection (Schmid, 1997; Pishvaee and Payne
1998). They are heterotetrameric complexes consisting of two heavy chains of -100
kDa (generally called alpha and beta adaptins), a medium-sized subunit of -50 kDa
(f.!) and a small subunit of -20 kDa (s) (Kirchhausen, 1999). All APs share structural
similarities. Adaptor AP-I is associated with Trans Golgi network (TGN) and AP-2
with plasma membrane. Studies on these adaptors have also suggested that there may
be a specific AP-2 receptor on plasma membrane and an AP-l receptor on TGN
membrane but the receptors are yet to be identified. Two targeting signals on two
adaptor complexes specific for binding to their respective receptors are found to be
present (Robinson et al., 1996). The heterotetrameric APs bind to clathrin in vitro and
stimulate cage assembly at physiological ionic conditions. Assembly activity is
primarily attributed to the
AP-~
subunit, which alone can induce clathrin to fonn
cages. In, addition APs recognize sorting sequences on transmembrane cargo proteins.
In this way, APs function as adaptors, linking coat fonnation to cargo incorporation.
According to current view, coated vesicle fonnation initiates with binding of APs to
appropriate membrane sites. In vitro binding studies indicate that AP-l binding to the
TGN is regulated by the GTPase ADP-ribosylation factor 1 (ARF-l) and GTP. In
contrast, AP-2 binding to plasma membrane in vitro is GTP and ARF-l independent.
Once membrane-associated, APs bind clathrin to nucleate the assembly of polyhedral
13
coat that drives membrane budding. Phosphorylation and phosphoinositide binding
are implicated in regulating aspects of AP function. (Pishvaee and Payne, 1998)
Like AP 2, AP 180 too is a major component of clathrin coats that binds to
clathrin directly and can stimulate clathrin cage assembly in vitro; limiting the size
distribution of the resulting cages (Marijn et al., 2001). However, exactly how AP
180 and AP 2 stimulate the clathrin coat assembly and vesicle budding is not known,
but their ability to interact with each other and clathrin suggests that they may
coordinate clathrin coat assembly through dynamic and complex interactions. It is
likely that their interaction with PIP 2 tethers the clathrin lattice to the plasma
membrane (Gi/looty and Stenmark, 2001).
Besides, APs, specialized adaptors participate in cargo sequestration. Examples
include the nonvisual arrestins involved in agonist-induced internalization of the G
protein coupled p-adrenargic receptors (P-AR). Arrestin binds clathrin and P-AR, but
does not promote clathrin assembly (Goodman et al., 1997). These characteristics
suggest that arrestins act in addition to APs during endocytic coat formation.
Apart from clathrin and its adaptors AP-I and -II, four more coat proteins which
are specifically involved in controlling the vesicle formation at the level of
intracellular transport vesicles are presently known. They are adaptor related AP-3
and -4 complex, and COP-I and -II, each mediating different transport steps. The AP3 complex mediates vesicle transport from TGN to the endosomalllysosomal
compartment (Simpson et al., 1996; Dell Angelica et al., 1997; Simpson et al., 1997;
Le Borgne et al., 1998). Furthermore, it is implicated in reconstitution of synaptic
vesicles from early endosomes, suggesting its probable role in secretory and endocytic
pathways (Faundez et al., 1998). The role of recently cloned AP-4 complex in the
vesicular trafficking still remains unknown (Dell Angelica et aI., 1999; Hist et al.,
1999; Rohn et al., 2000).
1.5.1.2 Coatomer protein (COP f), COP II and ARFs
COP I is a multi-molecular complex consisting of seven COPs viz., a, P,
p-,
y, 8,
E
and s-COP g, which Conn a complex generally called a coatomer (Waters et al., 1991;
Schekman and Orci, 1996), known to function in anterograde transport within the
Golgi and in retrograde transport from Golgi to ER. COP I is also implicated in
transport from phagosomal membrane. It interacts with membranes through the small
14
GTPase, AOP-ribosylation factor 1 (ARFI). ARF is thought to be the eighth
polypeptide of coatomer and is a GTP-binding protein. The recruitment of COP I and
ARFI on endosomal membrane depends on acidic lumenal pH. Previous studies
indicate that interiors of endocytic vesicles and Iysosomes are characteristically
maintained at a low pH and H+ -gradients are considered to be critical for normal
functioning of endocytosis (Yamashiro and Maxfield, 1988; Anderson and Orci,
1988). Vacuolar type ATPase has been proposed to be responsible for acidification of
endosomal bodies (Padh et al., 1991a; Padh et al., 1991b; Nolta et al., 1991).
Neutralization of endosomal pH inhibits ARF I and COP I binding, thereby inhibiting
the endocytic process (Aniento et al., 1996; Clague et al., 1994).
A study suggests that coatomer-coated pits are involved
111
the vesicular
trafficking in Golgi region. For fonnation of coatomer-coated pits and then coated
vesicles, certain guanine nucleotide-exchange factors (GEFs) are required, which
convert GOP-bound inactive fonn of ARFI to ARF-GTP (active form), which is then
recruited to the membrane. It has been reported that Golgi membrane-localized ARF 1
regulates the recruitment of COP I, AP-l and AP-3 onto membranes (Chavrier and
Gaud, 1999). The assembly of the coatomer coat then pulls the membrane into a bud,
which then pinches off as a coated vesicle (Orci et al., 1993; Serafini e/ al., 1991).
The mammalian COP II coat consisting of small GTPase hSarlp and the
heterodimeric protein complex hSec23/24p and hSecl3/31 p (Barlowe et ai, 1994;
Wieland and Harter, 1999), functions in anterograde transport from the ER to the
Golgi. A working model of mechanism by which COP II recruits cargo and shapes a
bud has been put forward by Schekman and Orci (1996), where Sari p is recruited
directly to the ER membrane by virtue of its functional interaction with Secl2p, an
ER resident protein. The activated species, Sarlp-GTP, then recruits the Sec23pSec24p complex to form a binary complex free to diffuse within the plane of the ER
membrane, sampling potential partners by collisional encounters. Favourable
interactions may transfer the Sec23p complex to a protein marked for transport.
However, unfavouarable interactions such as with ER resident protein could trigger
premature GTP hydrolysis or have no consequence. Sari p-GTP hydrolysis stimulated
by the GAP activity of Sec23p recycles Sari p-GOP to the cytosol for next round of
activation and cargo recruitment. Sec-23p activated cargo or adaptor molecules may
then be clustered by multivalent interaction with the Secl3p complex to form a
15
concentrated patch of COP II coat and proteins selected for transport, ultimately
resulting in a COP II coated vesicle (Schekman and Orci, 1996).
1.5.2 Post internalization events
1.5.2.1 Vesicle budding from the membrane
Even after coated pits internalize ligand-receptor complex, they still remain bound to
plasma membrane and later, budding of coated pit from the plasma membrane takes
place. The vesicle containing ligand-receptor complex is called a coated vesicle, as
coat proteins are still present on the vesicle (Figure 4). After internalization when
vesicle budding takes place, a protein dynamin, having defined enzymatic activities
and a component of clathrin-coated vesicle machinery comes into play. This protein is
required for coated pit to pinch off from the membrane as a coated vesicle. Oynamin
is a GTPase that forms a ring around the neck of a budding coated vesicle. It gets
recruited to the neck of the fonning vesicle by binding to PIP2• a lipid known to be
essential for endocytosis. GTP hydrolysis constricts the ring, leading to membrane
scission resulting in the release of a free vesicle (Schweitzer and Hinshaw, 1998).
Oynamin is thought to possibly recruit the lipid-modifying enzyme endophilin to help
in the scission activity (Ringstad et aI., 1999). Endophilin has acyl transferase
activity. It mediates conversion of lysophosphatidic acid (LPA) to phosphatidic acid
(PA) and hence acts jointly with dynamin to induce vesicle fission (Conner and
Schmid, 2003; Kozlov. 2001) Amphiphysin is another protein that is needed for
recruitment of dynamin to coated pits and functions as a linker between dynamin and
clathrin coats (Takei e/ al., 2005).
In case of non-specific intracellular vesicular transport that takes place in ER
and Golgi regions, ARF is involved in the budding of coated vesicle from their
respective organelle membrane. As previously mentioned ARF-GTP, which is
recruited onto the membrane, leads to the assembly of coatomer protein on to the
membrane, giving rise to the coat. This ARF simultaneously brings about disassembly of coat proteins from the membrane of vesicle, which till then is attached to
the membrane. When coatomer-coated vesicle docks with its target membrane, a
specific GTPase-activating protein (GAP) in target membrane triggers ARF to
hydrolyze its bound GTP to GOP. This causes budding of vesicle from one membrane
and helps in its fusion to another membrane (Alberts et aI., 1994; Rothman, 1994).
16
1.5.2.2 Formation of early endosomes
Coated vesicles lose their coat in order to fuse with their appropriate target membrane
in the cell. Clathrin-coated vesicles lose their coats, as soon as they pinch off from
donor membrane, whereas coatomer-coated vesicles lose coats only after they dock on
to their target membrane. After the clathrin coat is disassembled, the components are
recycled back to the cytoplasm. Both in vitro and in vivo evidence support a role for
the chaperone hsc 70 in uncoating (Schmid, 1997; Pishvaee 1998). Auxilin, a coated
vesicle-associated protein, is also required for coat disassembly in vitro. Together
with auxilin, hsc 70 disassembles LC-free cages in vitro, arguing against proposals
that hsc 70-mediated uncoating requires binding to LC. Synaptojanin is an Inositol 5phosphatase which dephosphorylates PIP2, converting it to phosphatidylinositol 4phosphate (PIP), thereby leading to disassembly of clathrin coats from the coated
vesicle (Gil/ooty and Stenmark, 2001).
Un coating of coated vesicle gives nse to early endosomes (receptosomes)
(Figure 4). This compartment is morphologically defined as consisting of vacuoles,
cisternae and tubules and hence is also called tubular endosome (Mellman, 1996). The
small GTPase Rab5, acting through its effector rabaptin-5 also regulates homotypic
fusion between early endosomes (Robinson et al., 1996; Bucci et aI., 1992) and
controls delivery of cargo into this compartment. In these tubulo-vesicular
compartments, also termed as 'CURL', the uncoupling of ligand from its specific
receptor takes place. They are also called sorting endosomes for their efficient role in
sorting of recycled and down-regulated receptors. What still remains unclear is the
sorting signal differentiating the receptor to be recycled from the receptor that is to be
sent to lysosome for degradation. However, although lysosomal targeting signals have
been identified, it is still not clear as to at which transport step do they operate.
Sorting motifs have been found in the cytoplasmic domains of IL-2 receptor
~
chain (Subtil et aI., 1998), in P-Selectin (Blagoveshchenskaya et aI., 1998) and EGFR,
but bear little resemblance to each other. Recent study shows that ubiquitination of
cytoplasmic domain might contribute to the lysosomal sorting Rocca et al., 2001).
Ubiquitination process of EGFR requires proteins Cbl and CIN85, where Cbl is a
RING-type ubiquitin ligase that polyubiquitinates EGFR and monoubiquitinates
CIN85 which is an adaptor protein. Monoubiquitination of CIN85 is essential for
targeting of the complex to Iysosomes. Another study suggests that high affinity
17
binding to CIN85 requires tyrosine phosphorylation and a novel poly-proline motif
within the Cbl protein Roy and Gisou van der Goot, 2003). Sorting of ubiquitinated
proteins towards degradative pathway seems to occur in specialized domains of early
endosomes. From these early endosome, a few endocytosed receptors (e.g., transferrin
receptors) are recycled back to plasma membrane and the transit is through a
recycling endosome thought to be a separate highly tubulated compartment located in
peri-Golgi region, juxtaposed to Golgi stack and TGN. These vesicles are less acidic
than early endosomes. Recycling can occur by a fast or slow route. The fast route is
directly from early endosomes back to the plasma membrane and the slow route
involves recycling from the recycling endosomes. Rab4 and Rab 11 are proposed to
involve in the former and latter recycling, respectively. Transport along recycling
pathway involves actin and unconventional myosin motors. There is evidence
implicating the unconventional myosin Myr4 in transport from early sorting
endosomes to recycling endosomes (Gruenberg and Emans, 1993).
Rab 4 effector RABIP4 is known to contain a FYVE (phenyl alanine-tyrosinevaline-glutamic acid) motif and localizes to the early endosomes (Conmont et aI.,
2001). Also, Rab 4 interacts with Rab 5 effector rabaptin 5 (prekens et aI., 1998).
These findings suggest that possibly Rab proteins are functionally coupled.
1.5.2.3 Beyond early endosomes
From early endosomes, cargo can proceed further along the endocytic pathway.
Presence of an intermediate vesicle between early and late endosome is suggested.
These vesicles termed as endosomal carrier vesicles (ECV s) could fuse with late
endosomes in a microtubule-dependent manner, but not with early endosomes or with
each other (Robinson, 1996; Gruenberg, 2001). Formation of ECVs requires
coatomer coat protein,
~-COP.
In mammalian cells ECVs, also known as multi
vesicular bodies (MVBs) are clearly distinct from early endosomes and do not contain
early endosome-specific proteins, like Rab 5 or recycling receptors. It is not clear
whether they mediate transport of cargo between two stable compartments
(Gruenberg, 2001).
The next compartment distinct from ECVs involved in endocytosis is the late
endosomal compartment, which contains main lipid and protein constituents not
found in ECV s. The relationship between early and late endosomes is uncertain. One
18
view is that early endosomes slowly move inward to become late endosomes, which
fuse with the vesicles coming from Trans Golgi region bearing hydrolytic enzymes.
The increasing acidification by those hydrolytic enzymes and continuous membrane
retrieval leads to their conversion to Iysosomes (Dunn and Maxfield, 1992; Hopkins,
1992). The other view suggests that early and late endosomes are separate stationary
compartments and transport between them occurs with the help of an intermediate
transport compartment (Dunn and Maxfield, 1992; Hopkins, 1992). However,
although both early and late endosomes do differ in their membrane protein
composition, in particular Rab proteins, it has been difficult to draw the line between
early and late endosomes, and even more difficult to demarcate the late endosomal
and lysosomal compartments.
Both late endosomes and Iysosomes have almost same acidic pH (-5.5), contain
lysosomal enzymes, and their limiting membrane is primarily composed of same
glycoproteins. However, both compartments can be differentiated by Rab proteins,
like Rab 7, Rab 9, mannose-6-phosphate receptor (M6PR) and also phosphorylated
hydrolase precursors present in late endosomes, but absent in Iysosomes (Gruenberg
and Maxfield. 1995). In addition, late endosomes are known to possess a specific lipid
marker Iysobisphosphatidic acid (LBPA) (Gruenberg, 2001), which allows retention
and binding of specific molecules in whorls of compartment. Although late
endosomes function to degrade many proteins and lipids, they are not able to digest
all the material and hence Iysosomes play the role of a digesting organelle becoming
the end point in dynamic networking of endocytic pathway. Late endosomes fuse with
Iysosomes to form a hybrid organelle after M6PRs are recycled back to TGN.
Lysosomes can be differentiated by their physical properties on gradients, their
electron-dense appearance (Gruenberg, 2001) and a marker for Iysosomes - a heavily
glycosylated lysosomal associated membrane protein (LAMP).
1.5.3 Targeting, docking and fusion
Transport of cargo from donor to the acceptor membrane in the whole endocytic
pathway requires proper targeting, docking and fusion of transport vesicles with their
respective target membrane, without compromising with the structural integrity of
cellular compartments. To achieve this, vesicle buds off from the intracellular donor
organelle, gets targeted to, docks and ultimately fuses with an acceptor organelle
(Chen and Scheller, 2001).
19
1.5.3.1 SNARE proteins
Transport vesicles have to be highly selective about the target membrane with which
they fuse. Hence, it is essential for a signal to be present on the vesicle, which can be
recognized by the receptor on the appropriate target membrane. According to a
hypothesis, specific SNARE (soluble NSF-attachment protein receptor, where NSF
stands for N-ethyl-maleimide-sensitive fusion) proteins present on the respective
membranes probably mediate this specific recognition. These proteins are a family of
compartmentally-specific and cytoplasmically-oriented integral membrane proteins,
which provide a core mechanism that specifically pairs membranes. The SNARE
hypothesis suggests a SNARE complex to be involved in the membrane fusion events
(Grnenberg and Maxfield. 1995), consisting of v-SNARE (present on the donor
vesicle), a t-SNARE (present on the target membrane) (Sollner et al.. 1993; Sollner
and Rothman. 1996), SNAP [(soluble NSF attachment protein) (Clary et al.• 1990)]
and NSF (Block et al.• 1988). The v- and t-SNAREs bind each other in a cognate
fashion (Bennet et al.• 1993; Pevsner et al.• 1994; Soggard et al.• 1994) and must
reside in the opposite membranes for (fonnation of a SNARE complex) fusion to
occur (Nichols et al.• 1997) (Figure 5). Accordingly, each type of transport vesicle
bears a specific v-SNARE that pairs up with a unique cognate t-SNARE on the target
membrane and this interaction docks the vesicle onto the target membrane, with
subsequent dissociation of SNARE complex by ATPase activity of NSF, hence
driving the membrane fusion (Chen and Scheller. 2001). As several different
membrane systems are found in the cell, a vesicle is, therefore, more likely to
encounter many potential target membranes, before its v-SNARE fmds a
complementary t-SNARE to fonn SNARE complex.
1.5.3.2 Rab proteins
Some studies suggest that the assembly of v-t-SNARE complex is governed by other
proteins also, including members of Rab GTPase family, which are present in
membrane of budding donor vesicles and check for the correct pairing between the vand t- SNAREs (Soggard et al.. 1994; Schimmiller et al.. 1998; Gerst. 1999). When a
vesicle encounters correct target membrane, and the membranes of both come
together, v- and t-SNAREs bind, causing the vesicles to remain bound long enough to
allow Rab protein to hydrolyze its bound GTP, locking the vesicle to target membrane
for the fusion to take place. Eukaryotic cells contain several types of Rab proteins,
20
each specific for a particular endocytic organelle. Also involved are the sec1 proteins
which prevent the binding/assembly of v-t SNARE complex (Pevsner et al., 1994;
Jahn, 2000). Many of the Rab proteins are known to be remarkably specific with
stage-specific endosome morphology/functions and are recognized as markers for
endosomes at definite stages of endocytic pathway. For example, Rab 4, 5, 20, 21 and
22 are associated with early endosomes and differentially regulate membrane
transport through this compartment (Simpson and Jones, 2005). These Rab proteins
form the largest reported sub-group of the Ras GTPase family with almost 60
members identified in the mammalian system (Novick. and Zerial, 1997).
Like ARFs, Rabs also cycle between their inactive GDP-bound and active GTPbound forms requiring GEFs to catalyze GDP/GTP exchange. SNARE proteins were
thought to function in docking, since their known role was to bring the donor as well
as target vesicle membrane together, but the role in docking is challenged by the
finding that SNARE-cleaving neurotoxins do not affect vesicle docking at synapse
(Chen and Scheller, 2001) and that SNARE-deficient flies have an increased, not
decreased, number of morphologically docked vesicles (Schulze et al., 1995). Recent
study indicates that the role of SNAREs is to mediate fusion and not the docking and
it has been proposed that the small GTPases of Rab family are crucial in the early
stage of vesicle targeting and tethering (Zerial and McBride, 2001). The fusion events
also use tethering and docking proteins called EEA I and Rabaptin 5 which help align
the vesicles and bring them close enough to effect the fusion.
1.5.3.3 Minimal fusion machinery
Docking and fusion are, however, two distinct and separate processes. The process of
docking ends when membranes of both the vesicles come together. Membrane fusion
is required for the vesicle to unload its cargo. A crucial factor in vesicular transport
hence is, the coordination between Rab-dependent membrane tethering, docking and
SNARE-dependent membrane fusion. Upon successful tethering and priming,
SNAREs engage in trans-interactions between v- and t-SNAREs, leading to fusion of
two membranes. A question arises - could SNARE proteins be the minimal
machinery that fuses paired lipid bilayers? Since it is known that SNARE proteins
must be intact during the last few milliseconds prior to completion of fusion (Bruns
and Jahn. 1995; Parsons et al.. 1995; Banerjee et al., 1996a; Banerjee et al., 1996b),
the interpretation is that yes they are, at the very least, a part of minimal fusion
21
machinery. But then, are they sufficient? Although SNAREs alone are sufficient to
drive fusion of synthetic liposomes of certain lipid compositions, it has been
suggested that at least in some cases, SNARE complex fonnation might induce a
hemifusion intennediate state, after which SNAREs become dispensable (Jahn and
Sudhof, 1999), indicating the involvement ofa second catalyst too along with SNARE
proteins. Could this second catalyst be Ca2+, since it is the final trigger for many
membrane transport steps e.g. endocrine exocytosis (Xu et al., 1998) and intracellular
membrane fusion (Bechers and Balch, 1989)? Whether divalent Ca2+ can trigger
similar molecular events at distinct membrane fusion steps remains unknown. For
intracellular membrane fusion to take place, also required are SNAPs and ATPase
NSF which function to dissociate the otherwise stable complex of v- and t-SNAREs,
utilizing the energy provided by the hydrolysis of ATP (Sollner et aI., 1993; Hayashi
et aI., 1995). Although this biochemical activity of a-SNAP and NSF has not been
disputed, recent study shows that their revised roles is to act as a chaperone to
reactivate SNAREs after one round of fusion, so as to make them available for the
next round of membrane fusion (Chen and Scheller, 2001).
1.5.4 Other proteins involved in endocytosis
1.5.4.1 Annexins
Annexins are calcium-binding proteins whose role has been implicated in
endocytosis, though the specific role still remains obscure. Annexins I and-II are
enriched on early endosomes, while IV and -VI are more widely distributed. Annexins
II and -VI remain associated with the membrane even in the absence of calcium,
whereas annexins I and -IV are strongly calcium-dependent for their association to the
membrane. It has been envisioned that annexins, by using a variety of membraneassociated mechanisms might fonn platfonns on the membrane of different
subcellular compartments, which would specifically interact with cytosolic
components, including cytoskeleton. When N-tenninal deletion mutants were
generated in annexin I, some of these mutants were found to co-fractionate with late
endosomes and ECV's instead of primarily with early endosomes. Since Ca2+ is
implicated in a number of membrane trafficking events, hence it is suggested that
annexins might be responsible for a few of those events (Robinson et al., 1996;
Gruenberg, 2001).
22
1.5.4.2 Epsin
It is
a curvature fonning molecule that is first recruited to the PtdIns
(phosphatidyltinositol)( 4,5) P2 rich sites of the plasma membrane where it induces
membrane budding and facilitates recruitment of clathrin triskclions. Epsin molecule
then associates with AP 2 complex and becomes displaced when AP 2 molecule
triggers clathrin polymerization (LeRoy and Wrana, 2005; Ford et ai., 2002).
1.5.4.3 EPS 15
EGFR-pathway substrate IS (EPS IS) is an interacting partner of epsin and is located
at the edges of fonning coated pits where it is in complex with AP 2. The EPS l5-AP
2 complex becomes disrupted upon clathrin assembly (Conner and Smith, 2003;
Ford. et ai., 2002).
1.5.5 Lipid regulators of membrane traffic
A number of proteins regulate the assembly of the clathrin lattice and subsequent
vesicle budding, including CALM (clathrin assembly lymphoid myeloid leukemia
protein), its brain homolog AP 180 and epsin. These three proteins contain a
conserved amino-tenninal ENTH (epsin N-tenninal homology) domain, which binds
to PIP2, a lipid known to be a major recruiter of the endocytic molecular machinery.
In addition to CALM and epsin, several components of the endocytic machinery have
been found to bind to PIP2. These include AP-2, synaptotagmin and j3-arrestin, which
recruit receptors and other transmembrane proteins into clathrin coated-pits
{kirchhausen, 1999; Gaidarov and Keen, 1999), and dynamin, which causes the
scission ofclathrin coated vesicles from the plasma membrane (Schmid et al., 1998).
1.6
KEY ISSUES IN ENDOCYTOSIS
Few questions not answered completely and requiring more studies are:
(a) How does the plasma membrane remain intact and the cell functional, in spite of
many receptosomes being fonned continuously?
(b) How is the membrane integrity maintained?
(c) How does the cell know which ligand is to be internalized, if there are no specific
receptors, or is the internalization process a non-specific one?
(d) What decides and how, which ligand has to be uncoupled from its receptor in
23
CURLs (compartment of uncoupling of receptor and ligand) and which Iigandreceptor complex is to be directed to the Iysosomes for degradation?
(e) Are early endosomes and CURLs two sidcs of a coin?
(f) Do the early endosomes fuse together i.e., mature to give rise to a late endosomal
body or else, are both early and late endosomes two different stationary
compartments?
There are several questions still waiting to be answered, the most central being what
happens to endocytic process during cell division?
1.6.1 Endocytosis during cell cycle
One of the major questions coming up in ones mind is: what happens to endocytic
process during cell division as it is an ongoing process and so is the exchange of
materials between a cell and its external environment? The cell cycle transition from
interphase to mitosis in eukaryotic cells is reported to be accompanied by dramatic
inhibition of endocytosis. There are profound changes in cellular architecture and
inhibition of processes, like protein secretion, pinocytosis and receptor-mediated
endocytosis (Raucher and Sheetz, 1999).
For endocytosis to occur, one of the necessities is formation of endocytic
vesicles, which require substantial force to bend the membrane by overcoming
membrane tension, which has been suggested as a regulator of endocytic rate for both
plant (Kell and Glaser, 1993) and animal cells (Sheetz and Dai, 1996). The overall
process of endocytosis hence may be mechano-chemical in that the chemical energy
in clathrin and dynactin complexes is transduced into membrane bending and fission
(Raucher and Sheetz, 1999). Low membrane tension in interphase allows bending of
the membrane and formation of endocytic vesicles, so that endocytosis proceeds.
However, during transition from interphase to mitosis, membrane tension increases,
thereby, dramatically preventing the invagination of the membrane and fonnation of
endocytic vesicles, hence inhibiting endocytosis. Earlier, it was reported that if the
membrane tension in mitotic cells is decreased to interphase levels by addition of
tension reducing agents, then endocytosis is restored to interphase levels (Raucher
and Sheetz, 1999).
Membrane tension is modulatcd by changes in plasma membrane phospholipid
and protein composition that accompanies mitosis. Indeed, transferrin receptors and
24
active Na+fK+-ATPase pump sites are internalized during prophase and metaphase
and subsequently recycled to the cell surface (Raucher and Sheetz, 1999) and resynthesis of plasma membrane sphingomyelin is greatly decreased in cells undergoing
mitosis (Kallen et al., 1994). One of the parameters having an obvious role in
determining membrane tension in mitotic cells is the membrane-cytoskeleton
interaction (Sheetz and Dai. 1996), thereby raising the possibility that membrane
tension in mitotic cells may be modulated by changes in cytoskeletal lattice
underlying the plasma membrane. Alternatively, drop in secretion during mitosis also
leads to increase in membrane tension, until both endocytosis and exocytosis rates
match. The rise in membrane tension causes the membrane to stretch by 0.1 %. Since
an area of membrane equal to the plasma membrane gets endocytosed in -100 min,
the decrease in exocytosis rate would result in a rise in membrane tension in 6 sec.
Rate of endocytosis is altered dramatically after stimulated secretion which results in
a dramatic increase in the membrane surface area (Raucher and Sheetz, 1999).
In order to decipher more details of this pathway and to get clearer answers, it is
essential to first study and understand the endocytic pathway in tenns of cellular,
molecular and genetic aspects, based on the clues obtained till now.
1.7
STUDY OF ENDOCYTIC PATHWAY
1.7.1 Isolation of endocytic organelles
Attempts to purifY the endosomal compartments have met with limited success. This
limitation can be attributed to the absence of good methods for isolating the endocytic
subcellular organelles in sufficient purity and quantity (Pastan and Willingham, 1985;
Alberts et al., 1994; Ladish et al., 1999).
1.7.1.1 Conventional methods
Previously traditional methods of organelle purification like differential and density
gradient centrifugations were used, which fractionate the organelles on the basis of
their shape, size and density (Upadhyay et al., 1993; Alberts et al., 1994; Ladish et
al., 1999).
1.7.1.1.1 Differential centrifugation
It is probably the most commonly used method for the isolation of organelles from
tissue homogenate. In this the sample is separated into two phases: a pellet consisting
25
of sedimented material and a supernatant. The method is based on the differences in
sedimentation rate of organelles of different size and density, where the tissue
homogenate is centrifugally divided into a number of fractions by increasing (stepwise) the applied centrifugal field, which in tum is chosen with respect to
sedimentation rate of a particular organelle to be pelleted. However, a limitation of
this method is the insufficient purity of organelle fraction. Homogeneous organelle
fractions may be achieved by further subjecting the fraction to density gradient
centrifugation (Upadhyay el aI., 1993).
1.7.1.1.2 Density gradient centrifugation
In this method, separation of sample components
IS
achieved by sedimentation
through a density gradient. Separation under the centrifugal field is therefore
dependent upon the buoyant densities of the particles. Sedimentation of coated
vesicles to equilibrium in sucrose gradients has been a powerful purification tool.
Though such centrifugation methods lead to homogenous organelle fractions, but the
yield is very less (Upadhyay el al., 1993). In addition, these methods lead to posthomogenization degradation too. Furthermore, the endosomes are heterogeneous and
lack buoyant density, thereby leading to failure in isolating them. This limitation in
fractionating subcellular organelles suggest the inability ofresearchers in carrying out
the biochemical characterization of endocytic vesicles only till newer approaches
based on selective density shift or selective tagging for rapid isolation were developed
(Pastan and Willingham, 1985).
1.7.1.2 Newer approaches
In recent years, various endocytic compartments have been identified and
characterized in Dictyostelium discoidellm by sucrose density gradient method76 ,
gold-induced density shift method (Steck and Lavassa, 1994) and a novel approach
based on electromagnetic separation (Padh, 1995). The latter has proved to be
beneficial in tenns of both yield and purity of endocytic vesicles.
1.7.1.2.1 Density shift method
Plasma membranes from D. discoidellm have been isolated, using a novel method
based on the adsorptive capacity of freshly prepared gold particles which when added
to the cells bind to the cell surface. The membrane-bound gold particles hence
become denser in comparison to other organelles and so they pellet down through
26
,....--~--....
. -- ,. .
Bhav[.
62% sucrose. Such preparation has >50% yield
f
---;J'I.t
~
'","
) Y
lais\tlJ·inetnbr~re. and
2%
contaminants, like lysosomes or mitochondria (Steck and Lavassa, 1994).
Shifting of the density of endosomes containing ligands by enzymatic reaction
resulted in better isolation but the isolated endosomes were modified (Courtoy et al.,
1984a; Quintart et aI., 1984b). Also, isolation of lysosomes based on the density
difference is much easier because the difference in density from other vesicles is
larger (Waltiaux, et al., 1978). However, the preparation contains primary and
secondary Iysosomes and those unrelated to the specific ligands under study. Shifting
of the density of lysosomes by feeding cells with triton WR·1339 (Waltiaux et al.,
1963) or iron-sorbitol-citric acid complex (Arborgh et al., 1973) has also been utilized
for isolation (Sato et aI., 1986).
1.7,1.2,2 Magnetic based separation techniques
In recent years, magnetic based separation techniques have greatly proliferated in the
area of biology and biotechnology. Magnetic separation techniques enable separation
of target biologically active compounds and cells from a variety of materials including
raw extracts, cultivation media, blood, body fluids, environmental samples, etc.
(Sajairik & Sajairikova 1997, 1999). Magnetic affinity adsorbents have been used for
the separation of various proteins (enzymes, antibodies, antigens, receptors, histidinetagged
proteins)
(Birkmeyer
et
al.,
1987),
nucleic
acids
(DNA,
RNA,
oligonucleotides) (Morrissey et al., 1989; Boom et al., 1990), low-molecular weight
biologically active compounds (drugs) and xenobiotics, carcinogens, water soluble
dyes, heavy metals ions, radionuclides) (Sajairik & Saja,'Yikova 1997). Furthermore,
magnetic particles are also being used for the isolation and separation of a variety of
specific mammalian cells (Kronick and Gilpin, 1986; kemshead and Ugelstad., 1985),
bacteria, viruses, subcellular organelles and individual molecules.
Magnetic separation technologies are based on the presence of certain affinity
groups on the surface of the magnetic particles. A suspension of these particles is
mixed thoroughly with a preparation of the target molecules or cells. During the
incubation period, the target molecules bind to the affinity ligand. Using a strong
magnet, the magnetic particles and the molecules attached to them can be captured or
immobilized. Following several washes, the target molecules generally are eluted
from the matrix before being analyzed further.
27
In contrast to other purification methods, magnetic separation methods expose
the target molecules only to very low levels of mechanical stress. Although often
called as magnetic, most of the particles currently used in these approaches actually
are paramagnetic. When the magnetic components of those particles (usually iron
derivatives) are small enough, they align themselves within the magnetic field without
themselves becoming independently magnetic. This feature is critical for the
mechanism of action of these particles. Thus, the particles are attracted by the
magnetic field. As soon as the magnetic field is removed, however the particles lose
attraction towards each other. This ensures efficient separation and complete
resuspension of the material.
Based on the novel approach successful isolation of endocytic vesicles from
lower eukaryote Dictyostelium discoideum has been achieved by feeding the cells
with magnetic particles followed by post-homogenization separation of endocytic
compartments using electromagnetic column chromatography (Figure 6). The method
has proved to be better than the conventional centrifugation techniques in terms of
yield of pinosomes (organelles involved in the uptake of extracellular fluid) which is
usually >60%. Also, the preparations are very pure containing <2% contaminating
markers for other sub-cellular organelles (Padh, 1995). The same magnetic based
separation technique has been used in our laboratory to isolate and characterize
pinocytic vesicles from primary cultures of rat peritoneal macrophages. Analysis of
the purified fractions confirmed that they were remarkably free from other subcellular
contaminants like endoplasmic reticulum, mitochondria or lysosomes. In addition the
technique was easily adaptable to different pulse-and-chase regimens that allowed
accurate isolation of specific class(es) of en dosoma I vesicles.
1.7.2 Approaches used to study and characterize vesicular transport
1.7.2.1 Cell-free systems
Vesicular transport can be reconstituted in cell-free systems. This was first achieved
for the Golgi stack where they were isolated from cells and incubated with cytosol
and ATP with subsequent following of formation of vesicles and the transport of
protein between cisternae. Such cell-free systems have been used to study transport
from medial to Trans Golgi network and then to plasma membrane, from endosomes
to lysosomes, and from TGN to late endosomes (Alberts et aI., 1994).
28
1.7.2.2 Semi-intact cell systems
Vesicular transport can also be studied in cells whose plasma membrane has been
permeabilized to allow both micro- and macromolecules to leave and enter the cell
freely. Such systems are particularly useful in studying transport from extended
membrane systems, like ER and TGN. Semi-intact cell systems have been used to
isolate vesicles mediating transport between TGN to apical plasma membrane
(Alberts et 01., 1994).
1. 7.2.3 III l'itro fusions
The evidence to prove the involvement of SNARE proteins in lipid bilayer fusion was
provided by in vitro fusion of artificially designed lipid bilayers (liposomes) with
SNARE proteins. v-SNARE containing vesicles were mixed with t-SNARE
containing vesicles and fusion was monitored, using a well-characterized lipid-mixing
assay. This approach has helped in a better understanding of details of vesicular
transport (Weber et 01., 1998).
1.7.2.4 Genetic approaches
Yeast although not a good model to study endocytosis, has proved to be an important
model for genetic studies of the process. Genetic studies of mutant yeast cells
defective for secretion at high temperature have helped in identification of more than
25 genes involved in the secretory pathway. Biochemical studies of mammalian cellfree systems and mutational studies in yeast have independently helped in identifying
many of the genes and their protein products involved in vesicular transport, clearly
demonstrating how genetic and biochemical approaches complement each other
(Alberts et 01., 1994).
1.7.2.5 Kinetic studies
Uptake of ligand being one of the major functions of endocytosis, major part of
endosome characterization has focused on the kinetics of ligand uptake and factors
affecting it. The studies have been carried out using various radio-labeled and
fluorescent labeled ligands. Recently, use of fluorescent labeled nanoparticles has
been employed to study the characteristics of the pathway. The studies have allowed
the characterization of factors like time of ligand uptake and saturation of the
pathway, temperature, ionic strength, binding constants of ligand to receptor etc
29
(Padh et al., 1993). Furthennore, the studies have served as a tool to analyse
endocytic function in health and disease (Tamada et aI., 1989).
1.7.2.6 Microscopic tools
Microscopic analysis has been a significant player in describing the sIze and
morphology of the endocytic organelles. With the help of phase contrast,
fluorescence, confocal microscopy and electron microscopic techniques various
endocytic organelles have been visualized in various cell types. Microscopy has
allowed visualization and characterization of endosomes in both purified fractions as
well as in intact cells. Based on various microscopic techniques primary endosomes
have been characterized to be tubulo-vesicular and secondary endosomes to be multivesicular bodies. Microscopy has used the unique capacity of endosomes that allows
incorporation of specific ligands like colloidal iron and gold preparations for
transmission electron microscopic analysis and fluorescent ligands like FITC-dextran,
acridine orange, pyranine, latex beads etc. for analysis by fluorescence or confocal
microscopy (Padh et al., 1993; Nalta et al., 1993).
1.7.2.7 Immunological tools
Using immunoprecipitation, immunofluorescence microscopy and immunogold
electron microscopy V-H+ ATPase was located on the membranes of lysosomes and
phagosomes in Dictyastelium (Nalta et aI., 1993). Also, presence of vacuolar proton
pump rich organelles called acidosomes was established in Dictyastelium.
Furthennore, the endocytic pathways of different cell types have been extensively
characterized by pulse feeding of fluorescent ligands or immunostaining for the
respective
proteins.
In
addition,
western
blots,
immunoassays
and
immunoprecipitation techniques have been extensively used to characterize
endosomes and related organelles.
1.8
NEED FOR STUDY OF RECEPTOR LINKED ENDOSOMES
Various ligand-receptor systems have been investigated for targeted or cellular drug
delivery. These include blood carbohydrate (lectin) receptors, transferrin receptors, Fc
receptors,
complement receptors, interleukin receptors, lipoprotein receptors,
scavenger receptors, receptors/epitopes expressed on tumor cells, folate receptors and
cell adhesion receptors. In addition, G-protein-coupled receptors are a major target for
the development of new marketable drugs (Gupta et aI., 2006). Thus, the role of
30
receptors as molecular target has opened new opportunities for cellular or intracellular
targeting using carrier systems appended with targeting ligands, utilizing the receptormediated endocytosis pathway.
Although in recent years, researchers have focused on exploiting receptormediated endocytosis (RME) to design a specific and target oriented drug delivery
system to deliver therapeutic agents like antibodies, genes, enzymes, peptides and
antisense-oligonucleotides and various other chemical drugs into living cells, the
success rate has been very low. The low targeting efficiency can be attributed to the
characteristics of RME. There are various issues that need to be looked upon while
targeting a drug to a specific site using RME, the prime concern being escape of the
internalized drug from en do-lysosomal pathway in order to avoid degradation by
lysosomal hydrolases. The failure of gene therapy can be majorly attributed to the
DNA ending up in lysosomes instead of being targeted to the nucleus. Recent
approaches have considered this limitation of receptor mediated endocytosis. Studies
are now focused to find certain endosomal escape signals so that the targeted
molecule instead of ending up in lysosome ends up in cytosol. One of the studies has
proposed a possible mechanism for the delivery of araC (anticancer drug) via folatereceptor (FR) targeted cationic lipid based pH-sensitive liposomes, where at first the
folate derivatized Ii po somes are taken into the cells by binding to the folate receptor
on plasma membrane followed by internalization via RME. This is followed by the
acidification of endosomes, which results in the protonation of the anionic lipid
component and generation of a net positive surface charge on the liposomes. Finally,
the electrostatic interactions between the liposomal and the endosomal membranes
result in bilayer fusion and the cytosolic delivery of the araC. Similar approaches can
lead to development of RME into a successful delivery system. Consequently, the
need of the present research is to focus on gaining deeper understanding of the
receptor mediated endocytosis pathway, thereby justifying the need to isolate and
characterize the receptor-linked endosomes along with identifYing the molecular
players involved with the same.
31
1.9
OBJECTIVES OF THE STUDY
After understanding the extensive and significant role played by endocytosis in
different cellular processes, it becomes imperative to isolate and characterize the
organelles involved in pathway. Highly acknowledged role of receptor mediated
endocytosis in developing the pathway to a target specific delivery system also
encourages one to isolate the endocytic compartments and search for certain
endosome escape signals so as to bypass the endo-lysosomal pathway in order to
achieve efficient and target specific delivery of the various potent therapeutics
designed for a variety of diseases. The need therefore is to develop a method for
isolation of subcellular organelles specifically involved in receptor mediated pathway
and ascertain the efficiency of the method in terms of high purity and reasonable yield
of the organelles. Also, endocytic vesicles isolated till date from various cell types
have been basically characterized kinetically, morphologically and immunologically.
However, biochemical characterization of endosomes might be an important clue in
the search of endosomal escape signals.
In order to manipulate the receptor mediated endocytosis pathway to achieve a
successful target specific delivery system, the need is to isolate the receptor-linked
endosomes and achieve their biochemical characterization. The prime focus of the
present study, therefore, became isolation and characterization of receptor containing
endocytic organelles from rat (Rattus novergicus) peritoneal macrophages by an
innovative technique based on electromagnetic fractionation. The technique has
already proved to be a better method over conventional centrifugation techniques for
sub-cellular organelle isolation from Dictyostelium and pinosome isolation from
primary cultures of rat peritoneal macrophages.
To achieve the prime aim, the work is divided into three steps as follows:
L
Preparation of receptor specific
fluorescent
labeled nano-sized super-
paramagnetic ligand for electromagnetic purification of endosomes containing
internalized receptor-ligand complex.
2.
Electromagnetic purification of Fc receptor-mediated endosomes from rat
peritoneal macrophages using the receptor specific ligand.
3.
Characterization of purified endosomal fraction: to establish the method's
32
validity for isolation of pure endosomal fraction the following confirmations will
be performed with the isolated endosomal fraction.
Microscopy
~
~
Fluorescence
IlllCroSCOpy
Yield
Protein
quantitation
~
~
~
Biochemical
analysis
Protein
profiling
Lipid
analysis
~
Electron
mIcroscopy
SDS-PAGE
Fluorescence Specific Fold
analysis
activity purity
~
Succinate dehydrogenase
for mitochondria
+
~
~
Immunoblotting
Marker
Lipid
enzyme
Phospholipid HPTLC
analysis extraction estimation
~
~
Acid phosphatase RNA for endoplasmic
reticulum and ribosomes
for lysosomes
V-H+ ATPase
for endosomes
33
I
Developmental process
Transcytosis
Nutrient uptake
Antigen processing
1-----1 and presentation
Biosynthesis of
structural components
Autophagy
Pathogen uptake
I
Hormonal action
Drug delivery
Neurotransmission
Figure1: Role of endocytosis in cellular processes
34
•
Ligand
Y
Recepter
•
(
"\ .
(l)
~coated
,escle
: ••
Early
-
pinesome
i
0~·
oJ
• •
Lale
Pinocytosis
~eC'lcJng
endosorre
J;Exocytosls
•••
•
p nose Ole
!-fydrdcse
1
.
Receptor ~edoateJd
,endoc yto,,,
~~
~,.J
'.."
Ecrly
Receplosorne~
•••• ./-
•••
Lofe
Recepfosorne
.0
: • +"
I
Figure 2: A cell depicting all three types of endocytosis viz.
pinocytosis, phagocytosis and receptor mediated pathway. [A cell
takes up extracellular material by all three routes. Final destination
for the material taken up by any pathway is Iysosomes. Whether
molecuales taken op by a different routes end up in the same
lysosome for digestion is still a question mark] (Neekhra and Padh,
2004)
Ligand
Recepta
r---- Plasma men"brane
HOOC
Adaplin
Figure 3: Internalization signal for endocytosis [The receptors
endocytosed with in the c1athrin-coated pits are thought to have a
'Tyr' containing peptide signal. The Adaptins are proteins which
recognized this internalization signal and link with the receptors and
clathrin to form a clathrin coated vesicle.] (Neekhra and Padh, 2004)
35
•
•
!
Hydlolose
Ugond
Receplol
Adoplor
~ Clothrin
1
~
eCYdng
enOOlome
/'
Receptor mediated
endocytosis
•••
••
Figure: 4 Clathrin- mediated endocytic pathway [Clathrincoated pits and vesicles provide an efficient pathway for the uptake
of an extracellular ligand. A ligand enters the intracellular
environment bound to its receptor by accumulating in the coated
pits, the complex further being captured in membrane vesicles which
then lose the coat to form early endosomes. Ligands like LDL
dissociate from its receptor in the acidic environment of early
endosomes and the receptor is recycled back to the plasma
membrane via recycling endosomes.] (Neekhra and Padh, 2004)
36
--------------------------------------------------------------
Donor Organelle
'="ACoat
protein
k
~
v-SNARE
Transport
Vesicle
Target organelle
Figure 5: Postulated role of SNARE proteins [A specific v-SNARE
present on the vesicle pairs up with a unique cognate t-SNARE on
the target membrane to guide the selective vesicular transport]
(Neekhra and Padh, 2004)
37
o Magnetic
Organelle with
ligand
o Magnetic
Organelle without
ligand
Column
Electromagnet
Electromagnet
Figure 6: A schematic diagram for electromagnetic separation
of endocytic vesicles containing magnetic ligand (Neekhra and
Padh,2004)
38