IDENTIFYING REGULATORS OF LYSOSOME REFORMATION: INHIBITOR SCREEN IN
MAMMALIAN CELL CULTURE
by
Ian Liu
____________________________
Copyright © Ian Liu 2016
A Thesis Submitted to the Faculty of the
DEPARTMENT OF MOLECULAR AND CELLULAR BIOLOGY
In Partial Fulfillment of the Requirements
For the Degree of
MASTER OF SCIENCE
In the Graduate College
THE UNIVERSITY OF ARIZONA
2016
STATEMENT BY AUTHOR
The thesis titled Identifying Regulators of Lysosome Reformation: Inhibitor Screen in
Mammalian Cell Culture prepared by Ian Liu has been submitted in partial fulfillment of
requirements for a master’s degree at the University of Arizona and is deposited in the
University Library to be made available to borrowers under rules of the Library.
Brief quotations from this thesis are allowable without special permission, provided
that an accurate acknowledgement of the source is made. Requests for permission for
extended quotation from or reproduction of this manuscript in whole or in part may be
granted by the copyright holder.
SIGNED: Ian Liu
APPROVAL BY THESIS DIRECTOR
This thesis has been approved on the date shown below:
Hanna Fares Ph.D.
Professor of Molecular and Cellular Biology
May 2, 2016
Date
2
ACKNOWLEDGEMENTS
Hanna Fares
Hope Dang
Julie Huynh
Gloria Le
Fares Lab Group
Keck Imaging Center, esp. Brooke Beam
3
TABLE OF CONTENTS
Abstract ............................................................................................................................................5
Introduction ......................................................................................................................................6
Materials and Methods .....................................................................................................................8
Results ..............................................................................................................................................9
Discussion ......................................................................................................................................23
References ......................................................................................................................................25
4
ABSTRACT
Lysosomes are membrane-bound organelles that have diverse functions in eukaryotic cells.
Malfunctions in lysosomes result in a range of diseases known as Lysosomal Storage Disorders.
After fusing with late endosomes to form hybrid organelles, lysosomes bud off and are reformed
in a poorly characterized process known as lysosome formation or reformation. Only one
mammalian regulator of lysosome formation has been identified, the non-selective cation
channel TRPML1. In the highly similar process of Autophagic Lysosome Reformation (ALR),
three known regulators have also been identified, the vesicle-coating protein clathrin and two
phosphatidylinositol kinases that catalyze the formation of the membrane phospholipid PI(4,5)P2.
Here, we use an inhibitor screen coupled with a live imaging assay to identify the actin
microfilament as a novel regulator of lysosome formation.
5
INTRODUCTION
Lysosomes are membrane-bound organelles that serve as the primary degradative compartment
of eukaryotic cells. The degradative functions of lysosomes are key to a diverse range of cellular
processes including catabolism (MOORE AND VIARENGO 1987), autophagy (DUNN 1994),
signaling regulation (SETTEMBRE et al. 2013), quality control of protein production (ARIAS AND
CUERVO 2011), and protection from pathogens (LEVINE AND KROEMER 2008). Defects in
lysosome function are implicated in a wide range of lysosomal storage disorders (LSDs), which
at present lack any sort of long-term therapeutic cure (VELLODI 2005; BALLABIO AND
GIESELMANN 2009). Given these crucial functions of lysosomes, it is striking that little is known
about the molecular mechanisms that regulate how lysosomes are formed.
Lysosome formation is known to occur through a budding event from hybrid organelles
near the end of the endocytic pathway; these nascent lysosomes that bud then extend away from
the hybrid organelles (also referred to here as parent compartments) while maintaining a
membrane bridge that eventually breaks, releasing the nascent lysosomes. Nascent lysosomes
can fuse with each other to form mature lysosomes; these mature lysosomes can also fuse with
late endosomes, forming a hybrid organelle (PRYOR et al. 2000; BRIGHT et al. 2005).
A process similar to lysosome formation occurs during autophagy (LEVINE AND
KLIONSKY 2004). After maturation, autophagosomes fuse with lysosomes to form hybrid
organelles (NAIR AND KLIONSKY 2005). Since multiple lysosomes can fuse with autophagasomes
at once, the presence of a cellular mechanism to maintain homeostasis in the cell involves
Autophagic Lysosome Reformation (ALR), which is similar if not identical to lysosome
formation that we study (YU et al. 2010).
Previous research has shown that the protein Phosphatidylinositol-4-Phosphate 5-Kinase,
Type I, Beta (P5KT1B) is necessary for the initial bud to form during ALR in starved NRK cells
(RONG et al. 2012). In addition, the well-known vesicle-coating protein clathrin was also found
to be necessary for nascent lysosome budding during ALR. Similar to endocytosis at the plasma
membrane, P5KT1B activity was required for clathrin recruitment to initiate ALR (see Figure
15).
Two proteins are implicated in the scission of the membrane bridge connecting nascent
lysosomes to hybrid organelles. First, the protein Phosphatidylinositol-4-Phosphate 5-Kinase,
Type I, Alpha (P5KT1A) is required for scission of the bridge connecting nascent lysosomes to
hybrid organelles during ALR (see Figure 15) (RONG et al. 2012). P5KT1A is encoded by a
separate gene than P5KT1B, but both proteins have 68% sequence identity, and both catalyze the
formation of PI(4,5)P2 (VAN DEN BOUT AND DIVECHA 2009). Second, loss of the mammalian
integral channel membrane protein TRPML1, or of its channel activity, also blocks membrane
scission (see Figure 15) (MILLER et al. 2015). Indeed, the calcium chelator BAPTA-AM also
blocks membrane scission (MILLER et al. 2015).
In humans, TRPML1 is encoded by the gene MCOLN1; mutations in this gene cause the
disease Mucolipidosis type IV (TREUSCH et al. 2004). In Caenorhabditis elegans, the orthologue
of the mammalian TRPML1 protein is known as CUP-5 (FARES AND GREENWALD 2001).
Mutations in the cup-5 gene result in defects in lysosome formation that are similar to those seen
6
due to the loss of TRPML1 in mammalian cells (FARES AND GREENWALD 2001; HERSH et al.
2002). C. elegans rab-2 mutants show a similar defect as cup-5 mutants (LU et al. 2008).
Interestingly, Rac2, the mammalian homologue of worm RAB-2, has been shown to physically
associate with TRPML1, suggesting a possible role for mammalian Rac2 in lysosome formation
(SPOONER et al. 2013).
Lysosome formation bears some resemblance to another budding and scission event that
occurs earlier in the endocytic pathway, endocytic vesicle formation. Although there are notable
differences in compartment sizes and lipid content, this yields a launching point for testing
whether some proteins that function in endocytic vesicle formation also function in lysosome
formation. Here, using an inhibitor screen of candidate proteins, we track the dynamics of
nascent lysosome budding, elongation, and scission, and identify new regulators of lysosome
formation.
7
MATERIALS AND METHODS
Cell culture and transfection
Cell line LS42 (MCOLN1-/-; GFP-TRPML1) (MILLER et al. 2015) was grown in Dulbecco’s
Modified Eagle Medium (DMEM) with 2 mM Glutamax supplemented with 10% Fetal Bovine
Serum (FBS), penicillin-streptomycin (100 U/mL penicillin and 100 U/mL streptomycin), and
hygromycin (DMEM/FBS/PS/HYG) at 100 µg/mL at 37°C in a 95% air/5% carbon dioxide mix.
Imaging LifeAct-mCherry
LS42 cells were transfected with the linearized plasmid DNA expressing LifeAct-mCherry (from
Roberto Weigert) using the TransIT-X2 (Mirus) transfection reagent according to manufacturer
provided instructions. Live imaging was then carried out as described below.
Cell preparation for imaging assay
For LS42 imaging, 3 x 105 cells in 90 µl of DMEM/FBS/PS/HYG medium were mixed with 10
µl of MW 10,000 dextran-rhodamine dye (10 mg/ml; Sigma-Aldrich) on a 35 mm tissue-culture
imaging dish (InVitro Scientific). Plates were left for 1 hour in a 37°C incubator, then 2 mL of
medium were added and cells were left overnight. Prior to imaging, cells were washed twice
with 2 mL of pre-heated DMEM/FBS/PS/HYG medium sans phenol red and then left in 2 mL of
this medium for imaging.
Inhibition screen for live cell imaging
Inhibitors were first tested to determine the maximal drug concentration that did not yield
cell death (as indicated by dissociation from the imaging plate) after 1 hour. Immediately after
the second wash for live cell imaging (using medium lacking phenol red), 2 mL of no-dye
medium was aliquotted into a 15 mL conical tube. Inhibitor drugs were then mixed with the
aliquotted, 2 mL portions of medium. The remaining medium on the 35 mm imaging cell plates
was then aspirated and replaced by the medium + inhibitor mix. Plates were then left to incubate
at 37°C for 1 hour prior to imaging.
Live imaging microscopy
Imaging was performed at room temperature in 3 z-planes with a step size of 0.6 µm every 1-2
seconds for ~3 minutes. SlideBook 5.5 (Intelligent Imaging Innovations) was used to generate
sum intensity z-plane projections that were analyzed using SlideBook 5.0 software (Intelligent
Imaging Innovations). Confocal images were collected using an Intelligent Imaging Innovations (3i) System built on a Marianas (Zeiss, Germany) microscope base with a Z-piezo stage
(ASI PZ2150FT), Yokogawa CSU-X1M Spinning Disk, 488 nm laser, 561 nm laser, 100× Plan
APO Objective and a Photometrics Evolve 512 CCD.
Statistical analysis
Student’s t-test was used to compare measurements from two samples using a two-tailed
distribution (Tails=2) and a two-sample unequal variance (Type = 2).
8
RESULTS
Microscopy assay using LS42 (MCOLN1-/-; GFP-TRPML1) transgenic cell line
Using a modified version of the spinning-disk confocal microscopy assay developed by Miller et
al. (2014), cells were imaged after incubation with one of the inhibiting drugs, Cytochalasin D,
Latrunculin A, Jasplakinolide, Torin 1, Dynasore, NSC23766, Nocadazole, BAPTA-AM, CK666, or ML-S13 for 45 minutes. Untreated cells or cells treated with the vehicle DMSO were
also imaged as controls. The inhibitors function as follows:
Cytochalasin D and Latrunculin A are inhibitors of actin polymerization (CASELLA et al. 1981;
MORTON et al. 2000).
Jasplakinolide either inhibits actin polymerization or stabilizes actin fibers (BUBB et al. 1994).
Torin 1 is an inhibitor of mTOR (LIU et al. 2010).
Dynasore is an inhibitor of dynamin (MACIA et al. 2006).
NSC23766 is an inhibitor of Rac1 and Rac2 (GAO et al. 2004).
Nocodazole is an inhibitor tubulin polymerization (JORDAN et al. 1992).
BAPTA-AM chelates cellular calcium (SAOUDI et al. 2004).
CK-666 is an inhibitor of the Arp2/3 complex (HETRICK et al. 2013).
ML-S13 is an inhibitor of TRPML1 channel activity (CHENG et al. 2010).
Cells were pre-loaded with the dye Dextran-Rhodamine. Spinning disk microscopy was used to
characterize the dynamics of lysosome formation.
Percent No Scission (PNS) indicates potential effects on lysosome formation post-budding
Lysosome formation is known to have three distinct stages: budding of the nascent lysosome
from the parent compartment, elongation of the membrane bridge connecting the two
compartments, and scission of the nascent lysosome from its parent compartment (Figure 1).
Individual lysosome formation events were characterized as lacking scission if no scission of the
membrane bridge was seen over the time course of imaging.
We first determined the percent of events that did not result in scission (PNS); a higher PNS is
suggestive of an inhibitor affecting lysosome formation. However, this PNS has some caveats.
First, since events can only be tracked after the budding of the nascent compartment, any
inhibitor blocking a process prior to budding cannot be evaluated. Second, it is important to note
that although scission was not observed during ‘no scission’ events, it does not mean that
scission did not occur at a later time after the period captured during imaging.
9
0.9
0.8
PNSRatio
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Figure 2: PNS under different conditions. Each bar shows ratio of 'no
scission' events divided by the total number of observed events (both
scission and no scission).
PNS shows that the negative controls, untreated or DMSO-treated cells, behaved normally, with
scission:no scission numbers of 16:2 and 17:1, respectively (Figure 2). These results demonstrate
that most budding events in wild type cells proceed normally, as had previously been shown
(MILLER et al. 2015). All of the inhibitors, with the exception of CK-666, NCS23766, and
Dynasore showed an increased PNS, suggestive of effects on lysosome formation (Figure 2).
We next analyzed the dynamics of lysosome formation, including time and distance to scission
of native lysosomes from parent compartments.
Lysosome formation dynamics in wild type cells
Previous studies had measured the dynamics of lysosome formation in wild type LS44
(MCOLN1-/-; GFP-TRPML1) cells: there was scission of the membrane bridge in 15 out of 15
events, with an average distance before scission of 0.45 +/- 0.26 µm and an average time to
scission of 16.8 +/- 4.7 seconds (MILLER et al. 2015). We first repeated these studies in the
10
independently derived LS42 (MCOLN1-/-; GFP-TRPML1) cells; the reason for using LS42 is
that GFP-TRPML1 levels in LS42 cells are higher than in LS44 cells, thus making it easier to
visualize GFP-TRPML1. In LS42 cells grown under normal conditions, there was scission in 14
out of 16 events with an average distance before scission of 0.681+/- 0.049 µm and an average
time to scission of 21.010 +/- 4.782 seconds in the events where there was scission (Figure 3 and
Table 1). Similarly, in LS42 cells exposed to the vehicle DMSO, there was scission in 17 out of
18 events; in the events that showed scission, the average distance before scission was 0.669 +/0.089 µm (P 0.903 relative to no DMSO) and the average time to scission was 7.278 +/- 1.365
seconds (P 0.015 relative to no DMSO) (Figure 4 and Table 1). Thus, DMSO does not affect the
distance before scission. DMSO does seem to reduce time to scission. We compared all our
inhibitors studies to the DMSO data since all inhibitors were dissolved in this reagent.
1.4
Distance(microns)
1.2
1
0.8
0.6
0.4
0.2
0
0
5
10
15
20
25
30
35
40
Time(s)
Figure 3: Lysosome formation in the absence of inhibitors. Each plot represents
an individual lysosome formation event. Scission (when seen) is indicated by a red
diamond.
Inhibitor
No drug
treatment
DMSO
ML-S13
Table 1. Dynamics of Scission
Avg. time to
P values (twoAvg. distance to
scission (sec)
tailed) of Avg.
scission (µm)
time to
scission
(compared to
DMSO)
21.010 +/- 4.782
.015
0.681+/- 0.049
7.278 +/- 1.365
34.351
---
0.669 +/- 0.089
0.94
P values (twotailed) of Avg.
distance to
scission
(compared to
DMSO)
.903
--11
BAPTA-AM
Cytochalasin D
Latrunculin A
Jasplakinolide
CK-666
NSC23766
Dynasore
Torin 1
21.750 +/- 5.425
39.813 +/- 21.994
14.354 +/- 3.449
19.745 +/- 4.544
7.4 +/- 3.543
9.735 +/- 2.078
18.951 +/- 0.079
15.899 +/- 4.015
0.123
0.278
0.089
0.148
0.979
0.333
0.011
0.058
0.66 +/- 0.072
0.630 +/- 0.035
0.928 +/- 0.135
0.723 +/- 0.036
0.935 +/- 0.295
0.584 +/- 0.028
0.741 +/- 0.079
0.668 +/- 0.056
0.940
0.692
0.134
0.582
0.547
0.380
0.550
0.994
1
0.9
0.8
Distance(microns)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
2
4
6
8
10
12
14
16
18
20
Time(s)
Figure 4: Lysosome formation in the presence of DMSO. Each plot represents an
individual lysosome formation event. Scission (when seen) is indicated by a red
diamond.
ML-S13 and BAPTA-AM confirm previous involvement of TRPML1 channel activity
Previous studies have shown that there is a defect in scission during lysosome formation in the
absence of TRPML1 (genetic null), in TRPML1 channel mutants, or in the presence of BAPTAAM that chelates cellular calcium (MILLER et al. 2015). Indeed, in LS42 cells exposed to the
TRPML1 channel inhibitor ML-S13, there was scission in one out of seven events; in the event
that showed scission, the distance before scission was 0.94 µm and the time to scission was
34.351 seconds (Figure 5 and Table 1). Similarly, in LS42 cells exposed to the calcium chelator
BAPTA-AM, there was scission in three out of five events; in the events that showed scission,
the average distance before scission was 0.66 +/- 0.072 µm (P 0.94 compared to DMSO) and the
average time to scission was 21.75 +/- 5.425 seconds (P 0.122 compared to DMSO) (Figure 6
and Table 1). Though the number of events recorded so far is relatively low, these results are
12
consistent with previous studies using cells that lacked TRPML1 or after addition of BAPTAAM (MILLER et al. 2015).
1.4
Distance(microns)
1.2
1
0.8
0.6
0.4
0.2
0
0
10
20
30
40
50
60
70
80
Time(s)
Figure 5: Lysosome formation in the presence of BAPTA-AM. Each plot
represents an individual lysosome formation event. Scission (when seen) is
indicated by a red diamond.
1.2
Distance(microns)
1
0.8
0.6
0.4
0.2
0
0
10
20
30
40
50
60
70
80
Time(s)
Figure 6: Lysosome formation in the presence of MLS-13. Each plot represents
an individual lysosome formation event. Scission (when seen) is indicated by a red
diamond.
13
Actin implicated in lysosome formation
14
Lysosome formation shows some similarities with endocytosis at the plasma membrane. Given
the involvement of actin in endocytosis, we determined the possible involvement of actin in
lysosome formation (KAKSONEN et al. 2006). We first showed that actin localizes to the parent
compartment, the membrane bridge, and the nascent lysosome during lysosome formation
(Figure 7). We therefore assayed three actin inhibitors, Cytochalasin D, Latrunculin A, and
Jasplakinolide. In LS42 cells exposed to Cytochalasin D, there was scission in 3 out of 15
events; in the events that showed scission, the average distance before scission was 0.630 +/0.035 µm (P 0.692 compared to DMSO) and the average time to scission was 39.813 +/- 21.994
seconds (P 0.278 compared to DMSO) (Figure 8 and Table 1). In LS42 cells exposed to
Latrunculin A, there was scission in 8 out of 11 events; in the events that showed scission, the
average distance before scission was 0.928 +/- 0.135 µm (P 0.134 compared to DMSO) and the
average time to scission was 14.354 +/- 3.449 seconds (P 0.089 compared to DMSO) (Figure 9
and Table 1). In LS42 cells exposed to Jasplakinolide, there was scission in 21 out of 27 events;
in the events that showed scission, the average distance before scission was 0.723 +/- 0.036 µm
(P 0.582 compared to DMSO) and the average time to scission was 19.745 +/- 4.544 seconds (P
0.0148 compared to DMSO) (Figure 10 and Table 1). Thus, Jasplakinolide has significant effects
15
on time to scission in the events where there was scission. These results indicate that actin
inhibition causes defects in the membrane scission step of lysosome formation.
Figure 7: Actin Localization in LS42 cells. Large arrows indicate nascent lysosome.
Arrowheads indicate parent compartment. Small arrows indicate bridge connecting
nascent lysosome to parent compartment. Stars indicate that scission has occurred.
16
0.8
0.7
Distance(microns)
0.6
0.5
0.4
0.3
0.2
0.1
0
0
10
20
30
40
50
60
70
80
Time(s)
Figure 8: Lysosome formation in the presence of Cytochalasin D. Each plot
represents an individual lysosome formation event. Scission (when seen) is
indicated by a red diamond.
Distance(microns)
1.5
1
0.5
0
0
5
10
15
20
25
30
35
40
Time(s)
Figure 9: Lysosome formation in the presence of Latrunculin A. Each plot
represents an individual lysosome formation event. Scission (when seen) is indicated
LS42-Latrunculin(.1microliter)-3.30.16-Capture1-…
by a red diamond.
17
1.2
Distance(miicrons)
1
0.8
0.6
0.4
0.2
0
0
5
10
15
20
25
30
35
40
45
Time(s)
Figure 10: Lysosome formation in the presence of Jasplakinolide. Each plot
represents an individual lysosome formation event. Scission (when seen) is indicated
by a red diamond.
Arp2/3 regulation of lysosome formation
Given the potential involvement of actin in lysosome formation, we decided to assay the actin
nucleation complex, Arp2/3 (MULLINS et al. 1998). In LS42 cells exposed to the Arp2/3 inhibitor
CK-666, there was scission in two out of two events; in the events that showed scission, the
average distance before scission was 0.935 +/- 0.295 µm (P 0.547 compared to DMSO) and the
average time to scission was 7.4 +/- 3.543 seconds (P 0.979 compared to DMSO) (Figure 11 and
Table 1). We need to identify more events before we can make any conclusions about the
involvement of Arp2/3.
18
1.2
Distance(microns)
1
0.8
0.6
0.4
0.2
0
0
2
4
6
8
10
12
14
Time(s)
Figure11:LysosomeformationinthepresenceofCK-666.Each plotrepresentsan
LS42-CK-666(2microliters)-4.6.16-Capture3-Event
individuallysosomeformationevent.Scission(whenseen)isindicatedbyareddiamond.
1
Rac regulation of lysosome formation
Previous studies have shown that Rac2, a regulator of actin dynamics, associates with TRPML1
(SPOONER et al. 2013). Indeed, mutation of the Rac2 homologue in worms results in a defect in
lysosome formation (CHUN et al. 2008). The Rac inhibitor NSC23766 did not seem to affect the
scission step of lysosome formation. In LS42 cells exposed to NSC23766, there was scission in
14 out of 14 events; in the events that showed scission, the average distance before scission was
0.584 +/- 0.028 µm (P 0.380 compared to DMSO) and the average time to scission was 9.735 +/2.078 seconds (P 0.333 compared to DMSO) (Figure 12 and Table 1). Thus, Rac1/2 do not seem
to be involved in lysosome formation.
19
1
0.9
Distance(microns)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
5
10
15
20
25
30
35
40
Time(s)
Figure 12: Lysosome formation in the presence of NSC23766. Each plot
represents an individual lysosome formation event. Scission (when seen) is
indicated by a red diamond.
Microtubules and lysosome formation
Having assayed the actin cytoskeleton, we decided to assay microtubules. In LS42 cells exposed
to the microtubule depolymerizing drug Nocodazole: no lysosome formation events were
observed in the presence of this inhibitor, even at very low concentrations. Depolymerizing
microtubules may have an inhibitory effect on all membrane trafficking steps (APODACA 2001).
Dynamin and lysosome formation
Dynamin is required for endocytosis at the plasma membrane. We therefore reasoned that it may
also be required for lysosome formation. In LS42 cells exposed to the dynamin inhibitor
Dynasore, there was scission in 16 out of 18 events; in the events that showed scission, the
average distance before scission was 0.741 +/- 0.079 µm (P 0.550 compared to DMSO) and the
average time to scission was 18.951 +/- 0.079 seconds (P 0.011 compared to DMSO) (Figure 13
and Table 1). Thus, Dynamin is not required for lysosome formation, though it may exert some
subtle effects on the timing of scission.
20
1.4
Distance(microns)
1.2
1
0.8
0.6
0.4
0.2
0
0
5
10
15
20
25
30
35
40
Time(s)
Figure 13: Lysosome formation in the presence of Dynasore. Each plot represents
an individual lysosome formation event. Scission (when seen) is indicated by a red
diamond.
mTOR and lysosome formation
Previous studies have suggested that the lysosomally localized mTOR complex may be involved
in lysosome formation, though those studies did not assay the dynamics of lysosome formation
by live imaging (KRAJCOVIC et al. 2013). In LS42 cells exposed to the mTOR inhibitor Torin 1,
there was scission in 15 out of 20 events; in the events that showed scission, the average distance
before scission was 0.668 +/- 0.056 µm (P 0.994 compared to DMSO) and the average time to
scission was 15.899 +/- 4.015 seconds (P 0.058 compared to DMSO) (Figure 14 and Table 1).
Thus, by our living imaging assay, mTOR does not seem to function in lysosome formation.
21
Distance(microns)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
5
10
15
20
25
30
35
40
Time(s)
Figure 14: Lysosome formation in the presence of Torin 1. Each plot represents
an individual lysosome formation event. Scission (when seen) is indicated by a red
diamond.
22
DISCUSSION
Lysosome formation is a dynamic process that proceeds through the budding of nascent
lysosomes from parent compartments, movement of nascent lysosomes away from parent
compartments while remaining connected by a membrane bridge, and scission of the bridge to
release nascent lysosomes (Figure 15). Previous studies had identified clathrin and P5KT1B as
required for the first budding step, and TRPML1 and P5KT1A for the membrane scission
(Figure 15). These four proteins are not sufficient to provide a bio-mechanistic model of
lysosome formation.
We used drug inhibition combined with live imaging to probe the involvement of known
proteins in lysosome formation. Our analysis shows that actin is required for the scission of the
membrane bridge connecting parent compartments to nascent lysosomes. The rapidlypolymerizing, microfilament protein actin has been implicated in a wide array of cellular
processes (POLLARD AND COOPER 1986). These include the critical processes of cell motility
(OLSON AND NORDHEIM 2010), transcription (PERCIPALLE AND VISA 2006), and more relevantly,
the early stages of clathrin-mediated endocytosis (YARAR et al. 2005). Similarly to clathrinmediated endocytosis, we hypothesize that actin functions in pushing the nascent lysosome away
from the parent compartment, either through polymerization or with the help of a myosin: this
push provides a directional movement that is required for the scission of the membrane bridge
(Figure 15).
In addition to actin we assayed several other proteins that we thought were good
candidate regulators of lysosome formation. We had sufficient data to draw more reliable
conclusions for three inhibitors. Based on these data, it does not seem that Rac1/2, Dynamin, or
Torin 1 are required for lysosome formation. The main caveat of this analysis is that we used the
inhibitors at very low concentrations. Indeed, in our future studies, we will use higher
concentrations of inhibitors and will assay cells immediately after administering these inhibitors.
The combination of these two approaches will yield more reliable data on the requirements of
some of these proteins during lysosome formation. In addition, we will be using shRNAmediated knockdown of other candidate genes to identify additional regulators of lysosome
formation, a crucial yet poorly understood process.
23
Figure15:Proposedmodeloflysosomeformation.ItisnotclearatwhichstepP5KT1A
functions.
24
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