EDITORS PICK cro

Phosphatidylserine dictates the assembly and dynamics of

caveolae in the plasma membrane
Received for publication, May 4, 2017, and in revised form, July 6, 2017 Published, Papers in Press, July 11, 2017, DOI 10.1074/jbc.M117.791400
Takashi Hirama, Raibatak Das, Yanbo Yang**1, Charles Ferguson, Amy Won, Christopher M. Yip,
Jason G. Kay, Sergio Grinstein**, Robert G. Parton, and Gregory D. Fairn**2
From the Keenan Research Centre for Biomedical Science, St. Michaels Hospital, Toronto, Ontario M5B 1W8, Canada, the

Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G1X8, Canada, the Department of Respiratory Medicine,
Saitama Medical University, Moroyama, Saitama 3500495, Japan, the Department of Integrative Biology, University of Colorado
Denver, Denver, Colorado 80204, the **Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, the

Institute for Molecular Bioscience and Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, Queensland
4072, Australia, The Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario
M5S 3E1, Canada, the Department of Oral Biology, School of Dental Medicine, the State University of New York at Buffalo,
Buffalo, New York 14214, and the Institute for Biomedical Engineering and Science Technology (iBEST), Ryerson University
and St. Michaels Hospital, Toronto, Ontario M5B 2K3, Canada
Edited by Dennis R. Voelker

Caveolae are bulb-shaped nanodomains of the plasma mem-
brane that are enriched in cholesterol and sphingolipids. They
have many physiological functions, including endocytic trans-
port, mechanosensing, and regulation of membrane and lipid
transport. Caveola formation relies on integral membrane pro-
teins termed caveolins (Cavs) and the cavin family of peripheral
proteins. Both protein families bind anionic phospholipids, but
the precise roles of these lipids are unknown. Here, we studied
the effects of phosphatidylserine (PtdSer), phosphatidylinositol
4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphos-
phate (PtdIns(4,5)P2) on caveolar formation and dynamics.
Using live-cell, single-particle tracking of GFP-labeled Cav1 and
ultrastructural analyses, we compared the effect of PtdSer dis-
ruption or phosphoinositide depletion with caveola disassembly
caused by cavin1 loss. We found that PtdSer plays a crucial role
in both caveola formation and stability. Sequestration or deple-
tion of PtdSer decreased the number of detectable Cav1-GFP
puncta and the number of caveolae visualized by electron
microscopy. Under PtdSer-limiting conditions, the co-localiza-
tion of Cav1 and cavin1 was diminished, and cavin1 degradation
was increased. Using rapamycin-recruitable phosphatases, we
also found that the acute depletion of PtdIns4P and PtdIns
(4,5)P2 has minimal impact on caveola assembly but results in
decreased lateral confinement. Finally, we show in a model of
phospholipid scrambling, a feature of apoptotic cells, that
caveola stability is acutely affected by the scrambling. We con-
clude that the predominant plasmalemmal anionic lipid PtdSer
This work was supported in part by Operating Grant MOP-133656 from the
Canadian Institutes of Health Research (to G. D. F.). The authors declare
that they have no conflicts of interest with the contents of this article.
This article was selected as one of our Editors Picks.
This article contains supplemental Figs. S1 and S2.
1
Supported in part by scholarships from the Li Ka Shing Knowledge Institute
and the Alzheimer Society of Canada.
2
Recipient of a New Investigator Award from Canadian Institutes of Health
Research and an Early Researcher Award from the Government of Ontario.
To whom correspondence should be addressed: Keenan Research Centre
for Biomedical Science, St. Michaels Hospital, 30 Bond St., Toronto, Ontario
M5B 1W8, Canada. Tel.: 416-864-6060 (Ext. 77330); E-mail: fairng@smh.ca.
is essential for proper Cav clustering, caveola formation, and
caveola dynamics and that membrane scrambling can perturb
caveolar stability.
Caveolae (little-caves) are bulb-shaped invaginations of the
plasma membrane (PM)3 enriched in cholesterol and sphingo-
lipids, with a diameter of 50 80 nm (1). A variety of physiolog-
ical roles has been attributed to caveolae, including as endocytic
carriers, mechanosensors, regulators of membrane stress, and
as regulators of lipid transport (2, 3). The primary protein com-
ponent of caveolae is the integral membrane protein caveolin
encoded by three homologous genes in mammalian cells: Cav1,
Cav2, and Cav3. Caveolin proteins are co-translationally
inserted into the endoplasmic reticulum (ER) (4) and are deliv-
ered to the PM via the secretory pathway (5). Newly synthesized
Cav1 molecules self-associate to form oligomers of 12 or 14
monomers (6, 7). These oligomers then traffic from the Golgi
apparatus to the PM via a vesicular carrier containing syntaxin
6, the ganglioside GM1, and glycosylphosphatidylinositol-
linked proteins (8). Once the Cav1 oligomers reach the PM,
they can assemble to form caveolae, higher order structures
containing 144 molecules of Cav1 protein (9). This basic
structure has been referred to as a quantal unit (9). However,
sub-quantal oligomeric structures of Cav1 have been described
in the PM (9), as well as caveolar clusters formed by multiple
caveolae in close, diffraction-limited proximity (10) or as larger
rosette structures (11).
From a structural standpoint, Cav1 contains a hairpin loop
structure, three palmitoylation sites, and a scaffolding domain
3
The abbreviations used are: PM, plasma membrane; PtdSer, phosphatidyl-
serine; PtdIns4P, phosphatidylinositol 4-phosphate; PtdIns(4,5)P2, phos-
phatidylinositol 4,5-bisphosphate; ER, endoplasmic reticulum; CFD, cumu-
lative frequency distribution; TIRF, total internal reflection fluorescence
microscope; PtdEtn, phosphatidylethanolamine; PJ, pseudojanin; BHK,
baby hamster kidney; DiI, 1,1-dioctadecyl-3,3,3,3-tetramethylindocar-
bocyanine; PH, pleckstrin homology; PLC, phospholipase C; EGFP,
enhanced GFP; CCD, charge-coupled device; MSS, moment scaling spec-
trum; FKBP, FK506-binding protein.

14292 J. Biol. Chem. (2017) 292(34) 1429214307

2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

that facilitates interaction with the PM (12). The scaffolding
domain of Cav1 (amino acids 82101) contains three cationic
residues that bind to the negatively charged headgroups of
PtdSer and PtdIns(4,5)P2 via electrostatic interactions (13,
14).These in vitro studies revealed that PtdIns(4,5)P2 is the
preferred ligand on a per mole basis. However, when lipo-
somes contained physiologically appropriate amounts of
PtdIns(4,5)P2 (1%) or PtdSer (17%), the PtdSer-containing
liposomes were the preferred substrate (13). In the cellular
context at first glance, the strength of the interaction
between three cationic amino acids and the anionic phos-
pholipid would be rather modest. However, as caveolae have
been estimated to contain 144 Cav1 molecules (9, 15), the
number of basic residues per caveola is amplified to 432.
Thus, the potential strength of the electrostatic interaction
is considerable.
Although Cav1 is essential for the formation of caveolae,
additional data have demonstrated the requirement for periph-
eral membrane proteins termed cavins. The cavin family con-
sists of four members, cavin1 to cavin4, that are required to
stabilize the Cav1 proteins and to shape caveolae (16 19). The
cavins also have the ability to bind to PtdSer (16, 17, 20). Fur-
thermore, a recent study demonstrated that there are 50 cavin1
molecules per caveola (15, 21). Together these observations
suggest that PtdSer may play a critical role in the formation and
stabilization of caveolae. Consistent with this notion, electron
microscopic (EM) studies revealed an enrichment of a PtdSer-
binding probe in caveolae (22, 23).
Despite these inferences, the precise role of PtdSer and
PtdIns(4,5)P2 in the assembly and stability of caveola has not been
analyzed directly. In this study, we used several approaches to alter
PtdSer and phosphoinositide levels of the inner leaflet of the PM,
and we assessed the consequences of these manipulations on the
caveolar number, size, and dynamics.
Results
Characterization of Cav1-GFP membrane distribution
To investigate the role of PtdSer and PtdIns(4,5)P2 in caveola
formation, we established a HeLa cell line stably expressing
Cav1-GFP (HeLa/Cav1-GFP), which was imaged using a total
internal reflection fluorescence (TIRF) microscope (Fig. 1A).
Because the features resolved by light (fluorescence) micros-
copy cannot distinguish true caveolae from sub- or supra-
caveolar clusters of Cav1-GFP, we will refer to all distinct foci of
GFP fluorescence as puncta and to structures identified by the
detection algorithms as features. Accordingly, Western blot-
ting using an anti-caveolin antibody suggested that in the stable
HeLa/Cav1-GFP cells, the ectopically expressed Cav1-GFP
contributes only modestly to the total Cav1 pool. Moreover, the
immunoblot analysis shows that the Cav1-GFP fusion appears
to be quite stable, as no significant breakdown products or free
GFP were detected using an anti-GFP antibody (Fig. 1B).
We observed a heterogeneous size and intensity distribution
of Cav1-GFP foci that is consistent with multiple caveolin-en-
riched structures, such as caveolae, sub-caveolar oligomers,
and rosettes (Fig. 1A, inset). To quantify this distribution, we
utilized an automated spot detection algorithm that uses wave-
PtdSer is required for caveolae
let transforms to extract image features at multiple length
scales (24, 25). As seen in Fig. 1C, the algorithm is robust at
identifying Cav1-GFP puncta of varying sizes and intensities.
For each detected feature, we computed an integrated intensity,
defined as the product of its area and its average intensity.
Assuming random incorporation of Cav1-GFP into endoge-
nous structures, this integrated intensity is proportional to the
total caveolin content of a feature. The distribution of inte-
grated intensities spans approximately 2 orders of magnitude,
reflecting the heterogeneity of the detected features (Fig. 1D).
To confirm that the ectopically expressed Cav1-GFP was
incorporated into caveolae and did not perturb their formation,
HeLa/Cav1-GFP cells were compared with untransfected HeLa
cells immunostained with an anti-caveolin antibody (Fig. 1E).
The distribution of the Cav1-GFP is virtually indistinguishable
from that of immunostained endogenous Cav1 (Fig. 1F), dem-
onstrating that the Cav1-GFP cell line is an excellent proxy to
study the distribution of endogenous Cav1 in the parental cells.
Diffusional analysis of Cav1-GFP features in untreated cells
Previous studies have shown that caveolae are largely immo-
bile in the plane of the membrane in unstimulated cells (5, 9, 26,
27). To confirm that our HeLa/Cav1-GFP cells behaved simi-
larly, we used rapid time-lapse microscopy (25 acquisitions/s
for a total of 20 s) and subsequently analyzed the mobility and
trajectory of detected features using single-particle tracking
(SPT) analysis (Fig. 1, G and H) (28). In this analysis, the type of
motion and the diffusion coefficient are determined using a
moment-scaling spectrum via a MATLAB algorithm (Fig. 1, H
and J) (29). SPT analysis showed that the majority of the iden-
tified Cav1-GFP features displayed sub-diffusive motion
(81.4% of 5046 particles), whereas free motion included a
small portion of the Cav1-GFP structures during the 20 s of
acquisition (Fig. 1I), consistent with previous reports (5). The
sub-diffusive Cav1-GFP features also had reduced diffusion
coefficients compared with the free Cav1-GFP features, with a
median diffusion coefficient of 6.7 103 m2/s compared
with 1.7 102 m2/s (Fig. 1J).
In contrast, caveolae are known to bud on and off the PM.
Because SPT determinations are discontinuous, budding and
reinsertion into the membrane can be interpreted as increased
lateral diffusion. Dynamin activity is associated with caveolae
pinching off the PM (26, 27, 30, 31). To determine the impact of
budding off and on the plasma membrane, we sought to inhibit
dynamin. The treatment of cells with Dyngo-4aTM, a potent
inhibitor of dynamin (32), illustrated that the fraction of tracks
classified as sub-diffusive and free motions in cells treated with
Dyngo-4aTM were very close to that of untreated, with the
median diffusion coefficients of 3.0 103 and 1.2 102
m2/s, respectively. This indicates that dynamin had minimal
impact on the mobility of caveolae with this analysis (Fig. 1, I
and J).
As a final step in quantitatively characterizing Cav1 dynam-
ics, we down-regulated cavin1, a crucial regulator of caveolar
formation (16, 33). As confirmed in Fig. 1K, we could effectively
deplete cavin1 using siRNA. Depletion of cavin1 caused a
noticeable decrease in the number of Cav1 puncta (Fig. 1L).
This was accompanied by a loss of the integrated spot intensity
J. Biol. Chem. (2017) 292(34) 1429214307 14293
PtdSer is required for caveolae

Figure 1. Characterization of Cav1-GFP-expressing HeLa cell. A, TIRF image of the HeLa cell stably expressing Cav1-GFP (HeLa/Cav1-GFP). Insets: magnifi-
cations of the indicated area by a dashed square. Scale bar, 10 m unless stated. B, Western blot analysis of HeLa and HeLa/Cav1-GFP whole-cell lysates.
Anti-caveolin antibody detected both the endogenous caveolin (22 kDa) and Cav1-GFP fusion protein (49 kDa). C, detection of caveolin in TIRF images using
Spot Detector function in Icy. The raw TIRF image (left) and the detected caveolae by spot detector (center) were shown to be identical in the overlay (right). D,
relative and the CFD of the integrated spot intensities in detected features of HeLa/Cav1-GFP, n 36 cells. The shaded region in a 95% confidence band around
each CFD, and the circle in the middle is the median. The same calculation for 95% confidence was applied throughout all the analyses. E, immunostaining of
HeLa cells probed with the anti-caveolin antibody and imaged using TIRF. F, CFD of the integrated intensity of Cav1-GFP puncta in HeLa/Cav1-GFP cells and of
endogenous Cav1 immunostained in untransfected HeLa cells, n 27 cells. G, inverted TIRF image of HeLa/Cav1-GFP recorded for 20 s at a rate of 40 frames/s.
H, representative image of the tracking for HeLa/Cav1-GFP with the tracks color-coded as follows: blue, sub-diffusive-Brownian trajectories (shown as sub-
diffusive); cyan, pure-Brownian trajectories (displayed as free); red, linear trajectories (shown as directed); and yellow, unclassified trajectories (shown as
unclassified, i.e. trajectories that were isotropic but too short for analysis). I, classification of Cav1-GFP features in HeLa/Cav1-GFP cells untreated (control) and
treated with the dynamin inhibitor, Dyngo-4aTM (Dyngo). Cells were incubated with 30 M Dyngo-4aTM for 30 min prior to observation. J, distribution of
estimated diffusion coefficients for tracks classified as sub-diffusive and free using the MSS analysis. All above experiments were performed on 3 separate days.
K, Western blot analysis demonstrating the levels of cavin1 in HeLa/Cav1-GFP cells treated with non-targeting or cavin1-targeting siRNA (top). L, TIRF images
of HeLa/Cav1-GFP transfected with cavin1-targeting siRNA. Inset, magnifications of the indicated area by a dashed square. M, CFD of the product of spot size and
intensity on HeLa/Cav1-GFP transfected with control-siRNA and cavin1-siRNA, n 16 cells. N, ratio of diffuse to punctate fluorescence for HeLa/Cav1-GFP
transfected with non-targeting or cavin1-targeting siRNA, n 12 cells, mean S.E. *, p 0.05.

14294 J. Biol. Chem. (2017) 292(34) 1429214307

 PtdSer is required for caveolae

Figure 2. Sequestering phosphatidylserine impairs caveolae. A, schematic of the mCherry chimera with the Lact-C2 domain in tandem, mCherry-2Lact-
C2. B, HeLa cells transiently transfected with the indicated fluorescent proteins were imaged using spinning-disc confocal microscopy. C, TIRF image of
HeLa/Cav1-GFP cells overexpressing the mCherry-2Lact-C2 with the dual-view system. D, CFD of integrated spot intensities in HeLa/Cav1-GFP transfected
with 2Lact-C2, n 19 cells. E, TIRF images of HeLa/Cav1-GFP cells transfected with or without 2Lact-C2 were analyzed as described under Experimental
procedures to estimate the ratio of diffuse to punctate GFP, n 12 cells, mean S.E. *, p 0.05. F, confocal images of HeLa cells stained with filipin (left panel)
following transient transfection with GFP, GFP-Lact-C2, or GFP-2Lact-C2 (GFP and filipin merged, right panel) as indicated. Scale bar, 10 m.

of Cav1-GFP structures as assessed by TIRF imaging and using
our quantitative image analyses (Fig. 1M). Under circumstances
that cause disassembly of caveolae, such as loss of cavin1 or hypo-
tonic stress, the Cav1-GFP signal should become more diffuse in
the plane of the membrane. To this end, we developed an image-
processing scheme to measure the relative distribution of Cav1-
GFP between the punctate features and the rest of the membrane
(supplemental Fig. S1). Using this analysis, we find that in cells
lacking cavin1 there is an 2-fold increase in the ratio of diffuse
GFP to punctate GFP (Fig. 1N). Based on previous studies (16, 34)
showing a loss of caveolae upon cavin1 down-regulation, we sus-
pect that the remaining features detected by the algorithm are
small clusters or oligomers of Cav1-GFP.
PtdSer is required for the formation and stability of caveolae
PtdSer is enriched in the PM and comprises 1520 mol % of
the inner leaflet phospholipid pool (35). To visualize the sub-
cellular distribution of PtdSer on the cytosolic leaflet of organ-
elles, we use the discoidin C2 domain of lactadherin (Lact-C2)
fused to either GFP or mCherry (36). In this regard, we
hypothesized that gross overexpression of a construct con-
taining two Lact-C2 domains might be able to bind and
sequester a significant fraction of the available PtdSer (37) in
the PM and thereby serve as a tool to examine PtdSer-depen-
dent pathways (Fig. 2A). To examine this possibility, we tran-
siently expressed mCherry-2Lact-C2 in HeLa cells and
found that it localized to the same compartments as the mono-
meric Lact-C2, without obvious alterations to the cell struc-
ture (data not shown). We next confirmed that the expres-
sion of the probe did not significantly alter the presence and
distribution of PtdIns(4,5)P2 as monitored by the PH-PLC
sensor (Fig. 2B). We conclude that any alterations in caveolar
structure and function associated with the overexpression of
the 2Lact-C2 would be due to PtdSer sequestration and not sig-
nificant alterations in PtdIns(4,5)P2.
The overexpression of tandem Lact-C2 decreased the abun-
dance of Cav1-GFP puncta visualized by TIRF microscopy (Fig.
2C). Additionally, the remaining puncta were smaller and had a
reduction in the integrated fluorescence intensity (Fig. 2D),
suggesting that they were clusters of Cav1-GFP. Because of the
nature of these experiments, we were unable to determine
whether the loss of the puncta was due to the prevention of
assembly or enhanced disassembly of the structures. Superfi-
cially, the majority of the GFP signal is associated with puncta.
However, analyzing the diffuse to puncta ratio revealed that a
significant fraction of the GFP signal in the tandem Lact-C2-
expressing cells is diffuse, with only 15% of the signal associ-
ated with features as characterized by the algorithm (Fig. 2E).
We interpret these results to mean that a substantial amount of
PtdSer is required for proper caveola formation.
As cholesterol is required for the formation of caveolae, one
explanation for our results is that the overexpression of the
tandem Lact-C2 depletes cholesterol from the plasma mem-
brane. To examine this possibility, HeLa cells were transiently
transfected with plasmids encoding soluble GFP, GFP-Lact-C2,
or GFP-2Lact-C2, fixed and stained with filipin to visualize
the cholesterol distribution. As shown in Fig. 2F, regardless of
the GFP protein expressed, the filipin readily stains the plasma
membrane and a portion of the endosomes. Thus, it is unlikely
that the absence of caveolae seen in the 2Lact-C2-expressing
cells is due to a depletion of plasmalemmal cholesterol. How-
ever, we cannot completely rule out a disruption in the nano-
scale organization of cholesterol.

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PtdSer is required for caveolae

Figure 3. Sequestering phosphatidylserine depletes cavin1 levels and caveolae. A, Western blot analysis with anti-cavin1 antibody depicting the levels of
cavin1 (44 kDa) and cavin1-GFP (71 kDa) in HeLa and HeLa/cavin1-GFP cells. B, confocal images of the bottom of the cells with cavin1-GFP stably expressing
HeLa cells (HeLa/cavin1-GFP), which were transiently transfected with mCherry (top) or mCherry-2Lact-C2 (bottom). Scale bar, 10 m unless indicated. C,
normalized integrated cavin1 spot intensity from B. The sum of the product of spot area and intensity for each cavin1 punctum at the cell base slice in mCherry
or mCherry-2Lact-C2-expressing cells were compared with that of untransfected cells. n 18 cells, mean S.D. D, comparison of the total cellular cavin1-GFP
content in mCherry and mCherry-2Lact-C2-expressing cells. Z-stacks were used to measure the total cavin1-GFP intensity in the entire cell and divided by
pixel area, which was then contrasted by that of untransfected cells. n 15 cells, mean S.D. n.s., not significant, **, p 0.01; ****, p 0.001. E, caveolae
abundance was quantified in BHK and HeLa cells transiently transfected with fluorescent probes tagged with or without 2Lact-C2. F, trajectories of caveolin
features in HeLa/Cav1-GFP cells overexpressing the 2Lact-C2. G, classification of 4815 Cav1-GFP features in HeLa/Cav1-GFP cells overexpressing the 2Lact-
C2. H, relative frequency of the diffusion coefficients for the sub-diffusive and free motion of Cav1-GFP.

Sequestering plasmalemmal PtdSer interferes with caveolar
association of cavin1
Both Cav and cavin proteins bind to PtdSer in vitro, and this
binding is postulated to be necessary for the function and
assembly of caveolae in vivo (13, 14, 17, 20). To complement the
experiments investigating the localization and dynamics of
Cav1-GFP, we generated a cavin1-GFP stably expressing HeLa
cells (HeLa/cavin1-GFP) to monitor cavin1 localization follow-
ing perturbation of PtdSer. Again, Western blotting using an
anti-cavin1 antibody indicated that the cavin1-GFP fusion pro-
tein accounted for a small fraction of the total cavin1 in HeLa/
cavin1-GFP cells (Fig. 3A). Consistent with previous findings,
HeLa/cavin1-GFP formed punctate structures similar to HeLa/
Cav1-GFP as reported previously (16). Next, these cells were
transiently transfected with mCherry or mCherry-2Lact-C2
(Fig. 3B) and imaged using confocal microscopy. As depicted in
Fig. 3B, sequestering of PtdSer significantly reduced the num-
ber of plasmalemmal cavin1 puncta in tandem Lact-C2-ex-
pressing cells. Furthermore, not only was the number of
cavin1-puncta in the PM decreased, but also the total cellu-
lar cavin1-GFP was decreased in cells overexpressing the
tandem Lact-C2 but not neighboring untransfected cells
(Fig. 3, C and D). Taken together, these results suggest that
PtdSer is required for caveola formation and that a pro-
longed absence of caveolae results in the displacement of
cavin1 from the PM and ultimately its degradation consis-
tent with previous findings (38).
Despite the significant reduction in Cav1-GFP puncta in cells
overexpressing the tandem Lact-C2, Cav1-GFP features were
still detectable through TIRF. To verify that sequestration of
PtdSer by the overexpression of tandem Lact-C2 precipitated a
loss of bona fide caveolae, we used electron microscopy (EM).
As illustrated in Fig. 3E, a decline in the density of caveolae was
observed in baby hamster kidney cells (BHK) and HeLa cells
upon overexpression of tandem Lact-C2. The remaining Cav1-
GFP features demonstrated an increase in free motion and dif-
fusion, with an attendant decrease in the sub-diffusive compo-
nents (81.0 to 60.5%) (Fig. 3, FH). Thus, sequestration of
plasmalemmal PtdSer significantly inhibited the formation of
caveolar puncta, and the remaining structures (Cav1-GFP olig-
omers or caveolae) were on average more mobile and less
confined.

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Chronic reduction of PtdSer results in a loss of caveolae
In mammalian cells, PtdSer is synthesized in the ER by two
separate enzymes, PtdSer synthase (PSS) 1 and 2. PSS1 cata-
lyzes PtdSer biosynthesis from phosphatidylcholine, and PSS2
uses phosphatidylethanolamine as a substrate (PtdEtn) (35).
Mice deficient for both PSS1 and PSS2 are not viable (39). How-
ever, previous studies identified and isolated a mutant Chinese
hamster ovary (CHO) cell line, termed PSB-2, with greatly
reduced PtdSer synthase activity, resulting in an 80% reduc-
tion in PtdSer (40). The sustained reduction of PtdSer causes
phenotypic changes in the cell with the most notable being
elongation (Fig. 4A). Conveniently, the levels of PtdSer can be
largely restored in these cells by supplementing the medium
with PtdSer (40), which is accompanied by reacquisition of a
more rounded phenotype, similar to the parental CHO cells
(Fig. 4A). To confirm the requirement for PtdSer in the forma-
tion of caveolae, we compared the number of caveolae in PSB-2
and parental CHO cells by EM (Fig. 4B). EM examination of the
PSB-2 cells revealed that these cells had a substantially reduced
caveolae density: from 14.1 per cell profile in CHO cells to 2.9
per cell profile in the PSB-2 cells (Fig. 4C). This defect could be
rescued by supplementation of the growth medium with Ptd-
Ser, restoring the number of caveolae to 9.7 per cell profile (Fig.
4, B and C).
Next, we examined the distribution of cavin1 in PSB-2 cells
and the contribution for the caveolae formation. CHO cells and
PSB-2 cells with or without PtdSer supplementation were tran-
siently transfected with a plasmid encoding cavin1-GFP. After
18 h of transfection, the cells were fixed, permeabilized, and
immunostained for endogenous caveolin. Consistent with pre-
vious findings, cavin1-GFP and immunostained caveolin were
both punctate in appearance and displayed extensive overlap in
the parental CHO cells. Conversely, in PSB-2 cells, the cavin1-
GFP was largely diffuse in nature, although caveolin remained
punctate (Fig. 4, D and E). Intriguingly, and as anticipated, the
cavin1 in PtdSer-supplemented PSB-2 cells relocalized with the
caveolin puncta. Together, the results suggest that the restora-
tion of PtdSer levels promotes not only the re-association of
caveolin and cavin1 but more importantly the reassembly of
caveolae.
The PSB-2 cells have not been extensively used for cellular
experiments. One possibility is that the reduction in PtdSer
could result in a reduction of PtdIns(4,5)P2. To compare the
distribution of PtdIns(4,5)P2 in CHO and PSB-2 cells, we used
the GFP-PH-PLC probe. As depicted the Fig. 4F, the probe
decorates the plasma membrane in both the parental CHO cells
and the PtdSer-depleted PSB-2 cells. Additionally, staining of
the actin cytoskeleton with rhodamine-phalloidin revealed the
presence of polymerized actin (not depicted) thereby confirm-
ing the presence of plasmalemmal PtdIns(4,5)P2.
Cav1-GFP features are more dynamic in PtdSer-depleted cells
To determine the lateral mobility of Cav1-GFP in PSB-2 cells,
we generated CHO and PSB-2 cell lines stably expressing the
Cav1-GFP construct: CHO/Cav1-GFP and PSB-2/Cav1-GFP.
Both cell lines showed similar levels of expression of Cav1 and
Cav1-GFP by Western blotting (Fig. 5A). Again, to confirm that
PtdSer is required for caveolae
the generated stable transfectants behaved as parental cells, we
examined the number and appearance of Cav1-GFP puncta
expressed in CHO/Cav1-GFP and PSB-2/Cav1-GFP and com-
pared it with the immunostaining caveolin I, the parental cells.
As depicted in Fig. 5, B and C, the stable expression of low levels
of Cav1-GFP did not impact the appearance of caveolin. We
next sought to characterize the fate of Cav1-GFP in PSB-2 cells
by using TIRF microscopy. The analysis revealed a reduction in
the integrated spot intensity (Fig. 5D). Additionally, we find a
trend toward an increase in the fraction of diffuse Cav1-GFP
(Fig. 5E). As before, we analyzed the mobility of the remaining
Cav1-GFP puncta in PSB-2 cells using SPT. The identified fea-
tures in the PSB-2/Cav1-GFP cells show less sub-diffusive
motion 65.5% compared with 80.1% in CHO/Cav1-GFP (Fig. 5,
FH) and display enhanced rates of sub-diffusion compared
with the parental CHO cells (7.8 103 m2/s versus 6.1
103 m2/s, respectively) (Fig. 5I). Although the PSB2/Cav1-
GFP cells showed more free motion of Cav1 than the CHO/
Cav1-GFP, this does not appear to be a result of budding off and
on the PM. As shown in Fig. 5H, inhibition of dynamin did not
alter the classification of the Cav1-GFP tracks. This dynamin-
independent motion seems logical because few bona fide cave-
olae are seen in PSB-2 cells by EM (Fig. 4B). The results suggest
that Cav1 clusters or oligomers may have free, long-range
motion that may, in fact, help with the reassembly of caveolae
after disassembly caused by stress (i.e. membrane stretching).
Phosphoinositide depletion increases the fraction of freely
diffusible Cav1-GFP features
The acidic nature of PtdIns(4,5)P2, PtdIns4P, phosphatidyli-
nositol, and PtdSer combines to generate the negatively
charged surface of the inner leaflet of the PM (41). The relative
contribution of each of these lipids to the surface charge is
dictated by both their relative abundance and their valency (41,
42). Thus, although the abundance of PtdIns4P and PtdIns(4,5)P2
is low, they are major contributors to the surface charge by virtue
of their relatively high valence, and we sought alternative ways to
alter the levels of PtdIns(4,5)P2 and PtdIns4P acutely to evaluate
their roles in caveola formation.
Pseudojanin (PJ) is a synthetic construct encoding a rapa-
mycin-based heterodimerization domain (FKBP) and two
phosphatase domains as follows: one isolated from INPP5E,
a PtdIns(4,5)P2 5-phosphatase, and a second one from Sac, a
4-phosphatase (43). Recruitment of PJ to the PM by co-
expression of the Lyn11-FRB targeting protein followed
by addition of rapamycin simultaneously degrades both
PtdIns4P and PtdIns(4,5)P2 (43, 44). As a control for these
experiments, a PJ-dead construct was developed in which both
phosphatase domains are inactivated (43). Additionally, to bet-
ter elucidate the specific roles of PtdIns4P and PtdIns(4,5)P2, a
PJ-INPP5E construct, which contains an inactivated Sac1
domain (and therefore serves only as a 5-phosphatase), and a
PJ-Sac construct, which contains an inactivated INPP5E
domain (and functions only as a 4-phosphatase), have also been
engineered (Fig. 6A and supplemental Fig. 2A) (43). When
recruited to the membrane, PJ was able to dephosphorylate
PtdIns(4,5)P2 and PtdIns4P, causing a sizable decrease in mem-
brane surface charge (Fig. 6A and supplemental Fig. 2A). Deple-
J. Biol. Chem. (2017) 292(34) 1429214307 14297
PtdSer is required for caveolae

Figure 4. Caveolae formation is impaired in PtdSer-depleted cells. A, differential interference contrast (DIC) images of CHO and PSB-2 cells. PSB-2 cells were
incubated in delipidated medium supplemented with 30 M PtdSer (middle), 30 M PtdEtn (right), or without supplementation (left). Scale bar, 40 m. B,
representative electron micrographs of CHO and PSB-2 cells with and without PtdSer supplementation. The arrowheads indicate caveolae or cavicles, and the
asterisks indicates clathrin-coated pits. Scale bar, 500 nm. C, quantitation of caveolae per cell slice examined by electron microscopy. *, p 0.05. D, cavin1-GFP
transiently expressing CHO and PSB-2 cells with or without PtdSer supplementation were immunostained for caveolin. Scale bar, 10 m. Insets, the magnifi-
cations of the indicated area shown by a square. E, Manders correlation coefficient calculated caveolin signal co-localizing with the cavin1 signal, n 20 cells,
mean S.D. ****, p 0.0001. F, confocal images of CHO and PSB-2 cells transiently transfected with the mCherry-PH-PLC. Scale bar, 10 m.

14298 J. Biol. Chem. (2017) 292(34) 1429214307

 PtdSer is required for caveolae

Figure 5. Chronic PtdSer depletion alters caveolin mobility. A, Western blotting analysis to examine the relative abundance of Cav1 in CHO, PSB-2, and the
CHO and PSB-2 cells stably expressing Cav1-GFP (CHO/Cav1-GFP and PSB-2/Cav1-GFP). Anti-caveolin antibody detects both the endogenous caveolin (22 kDa)
and Cav1-GFP fusion protein (49 kDa). B, TIRF images of CHO/Cav1-GFP and PSB-2/Cav1-GFP. Scale bar, 10 m. C, TIRF images of the CHO and PSB-2 cells stained
with the anti-caveolin antibody. D, CFD of integrated spot intensities in CHO/Cav1-GFP and PSB-2/Cav1-GFP cells, n 16 cells. E, ratio of diffuse to punctate
fluorescence for CHO/Cav1-GFP and PSB-2/Cav1-GFP cells, n 16 cells, mean S.E. n.s., not significant. F is tracks for the CHO/Cav1-GFP, and G is for the
PSB-2/Cav1-GFP cells. H, breakdown and classification obtained single-particle tracks for CHO/Cav1-GFP and PSB-2/Cav1-GFP with or without Dyngo-4aTM
(Dyg). I, distribution of the diffusion coefficient for the sub-diffusive and free motion of caveolin in CHO (dashed line) and PSB-2 cells (solid line).

tion of the phosphoinositides did not alter the transbilayer dis-
tribution of PtdSer, as monitored by annexin-V staining
(supplemental Fig. 2A). Recruitment of the construct with only
an active INPP5E domain led to the rapid depletion of available
PtdIns(4,5)P2, as monitored by the GFP-PH-PLC. Similarly,
the targeting of the Sac1 domain caused a rapid decrease in the
available PtdIns4P, as monitored with GFP-2PH-Osh2 (45).
The judicious use of these constructs, therefore, allowed us to
degrade PtdIns4P and/or PtdIns(4,5)P2 acutely in a controllable
fashion.
To examine the specific roles of PtdIns4P and PtdIns(4,5)P2
in the formation of caveolae, HeLa/Cav1-GFP cells were tran-
siently transfected with PJ and Lyn11-FRB. The following day,
the cells were visualized using TIRF microscopy either before or
after the addition of rapamycin. The depletion of both PtdIns4P
and PtdIns(4,5)P2 caused a mild reduction in the number of
Cav1-GFP puncta and a slight increase in the ratio of diffuse to
puncta (Fig. 6, B and C). However, the remaining structures
were unchanged as monitored by the integrated intensity of the
Cav1-GFP foci (Fig. 6, B and D). In cells expressing PJ and PJ-
Dead, both demonstrated a small shift to the left in the inte-
grated spot intensity (Fig. 6D) that could be the result of pho-
tobleaching in these time-lapse video microscopy experiments.
However, the depletion of the phosphoinositides resulted in a
decrease in the percentage of sub-diffusive Cav1-GFP puncta
from 83.2 to 57.8%, and a corresponding increase in the number
of freely diffusible puncta from 8.6 to 35.0% (Fig. 6, E and F).
Additionally, recruitment of PJ led to an increase in the
median diffusion coefficients, 0.84 102 m2/s (sub-dif-
fusive) and 4.2 102 m2/s (free) compared with the PJ-
dead controls, 0.6 102 m2/s (sub-diffusive) and 1.6
102 m2/s (free).
Through the recruitment of the PJ-INPP5E and the PJ-sac
constructs, we examined the impact of acutely and sepa-
rately decreasing PtdIns(4,5)P2 and PtdIns4P, respectively,
on the stability and (im)mobility of caveolae. Recruitment of
the specific phosphatases caused essentially no disassembly
of caveolae (Fig. 6C). However, such separate recruitment
led to an increase in the population of freely diffusible Cav1-
GFP features, with an accompanying decrease in the sub-

J. Biol. Chem. (2017) 292(34) 1429214307 14299

PtdSer is required for caveolae

Figure 6. Acute depletion of phosphoinositides alters the confinement of caveolae. A, recruitment of PJ to the plasma membrane depletes PtdIns(4,5)P2.
Spinning disc confocal images of a cell expressing the fusion proteins Lyn11-FRB, FKBP-mCherry-PJ, and GFP-PH-PLC before and after addition of rapamycin
are illustrated. Scale bars, 10 m. B, images acquired using TIRF microscopy of a HeLa/Cav1-GFP cell expressing the Lyn11-FRB and FKBP-mCherry-PJ constructs
before and after the addition of rapamycin for 5 min. Scale bars, 10 m. C, quotient of the fraction of fluorescent intensity in discrete puncta versus intensity in
undifferentiated PM per HeLa/Cav1-GFP transfected with PJ-dead (control), PJ (PtdIns(4,5)P2 and PtdIns4P depletion), PJ-INPP5E (PtdIns(4,5)P2 depletion), and
PJ-sac (PtdIns4P depletion). Error bars indicated the mean S.E. from five PJ-dead, 13 PJ, 10 PJ-INPP5E, and 9 PJ-sac recruited cells. All experiments were
performed at least on 3 separate days. n.s., not significant. D, cumulative frequency of the product of spot size and intensity as in Fig. 1 for cells expressing
PJ-dead (left, control) or PJ (right) before and after addition of rapamycin for 5 min. n 12 for PJ and n 11 for PJ-dead. E and F are the trajectories in
HeLa/Cav1-GFP transfected with PJ-dead, PJ, PJ-INPP5E, and PJ-sac as well as treated with rapamycin for 5 min. E, tracking analysis for HeLa/Cav1-GFP cells
following the plasmalemmal recruitment of PJ and PJ-dead constructs as described above (see Fig. 1). Scale bar, 2 m. F, classification and breakdown obtained
for 2821, 3136, 3426, and 3833 single-particle tracks for PJ-dead, PJ, PJ-INPP5E, and PJ-sac, respectively. All experiments were performed at least three times.

diffusive component (Fig. 6F). The reduction in the fraction of
sub-diffusive features and the rise in freely mobile features corre-
late with the charge density of the individual phospholipids,
consistent with PtdIns4P and PtdIns(4,5)P2 interactions being
important for the confinement and immobilization of caveolae/
Cav1-GFP puncta.
Membrane lipid scrambling alters Cav1-GFP features
The results suggest that caveolae are very sensitive to altera-
tions in PtdSer and to a lesser extent phosphoinositides. One
of the primary functions of PtdSer is to promote blood coagu-
lation by platelets and to act as an eat-me signal on apoptotic
cells. In both these situations and others, PtdSer is scrambled in

14300 J. Biol. Chem. (2017) 292(34) 1429214307

 PtdSer is required for caveolae

Figure 7. Dibucaine-induced scrambling alters Cav1 features. A, HeLa cells were treated with 1 mM dibucaine for 10 min resulting in PtdSer scrambling as
shown by annexin-V staining. Scale bar, 10 m unless indicated. B, TIRF images of HeLa/Cav1-GFP before and after dibucaine treatment. The dashed line in white
indicates the cell margin. Insets, magnifications of the indicated area by a square in blue. C, CFD of integrated spot intensities in HeLa/Cav1-GFP before and after
addition of dibucaine. Identical cells before and after treatment were selected for the analysis, n 18 cells. D, ratio of diffuse to punctate fluorescence, n 16
cells, mean S.E. EG, representative trajectories of Cav1-GFP features in cells treated with only dibucaine (Dib) (E), only Dyngo-4aTM (Dyg) (F), or both (G). Cells
were incubated with 30 M Dyngo-4aTM before dibucaine treatment. Scale bar, 2 m. H, classification of Cav1-GFP features in HeLa/Cav1-GFP cells from E to G.
Please note: As a reference we have included the control experiments from Fig. 1. I and J, relative frequencies of tracks from E to G. The dashed line indicates the
untreated cells (control, Fig. 1J). I is the relative frequency of the diffusion coefficients for the sub-diffusive movement, and J is free motion of caveolin in
HeLa/Cav1-GFP. *, p 0.05.

the plasma membrane, which will effectively reduce the con-
centration of PtdSer by 50%. Thus, it is conceivable that
plasma membrane lipid scrambling will impact the stability of
caveolae. To test this hypothesis, we made use of dibucaine, an
analgesic, to scramble the lipids across the PM (46, 47). To
validate the effects of dibucaine, we added Alexa-555-conju-
gated annexin V, which detects exofacial PtdSer (Fig. 7A).
Treatment of HeLa/Cav1-GFP cells with dibucaine decreased
the abundance of Cav1-GFP puncta (Fig. 7B). The puncta that
remained at the PM were smaller and had a reduced integrated
fluorescence intensity (Fig. 7C), meaning disassembled. Con-
comitant with the decrease of punctate spots, an increase in
diffuse GFP signal was observed in the TIRF field in dibucaine-
treated cells (Fig. 7D).
Next, we determined the increase in mobility of the Cav1-
GFP features that persisted after dibucaine treatment, using
TIRF microscopy and SPT. The fraction of features showing
sub-diffusive Brownian motion decreased from 82.1% in
control cells (Figs. 1I and 7H) to 40.6% after dibucaine treat-
ment (Fig. 7, E and H). Conversely, Cav1-GFP foci displaying
free motion increased from 13.3% in controls to 45.1% after
dibucaine (Fig. 7, E and H). Furthermore, after treatment
with dibucaine, the diffusion coefficient raised to a median
17.8 103 m2/s for the tracks classified as sub-diffusive
and to 18.1 102 m2/s for freely diffusing features. The
distribution of diffusion coefficients for both modes of
motion shifted toward higher values in response to dibu-
caine treatment (Fig. 7, I and J). Thus, scrambling PtdSer

J. Biol. Chem. (2017) 292(34) 1429214307 14301

PtdSer is required for caveolae
from the cytosolic leaflet increased the lateral motion of
caveolin.
Sequestration and depletion of PtdSer led to increased Cav1-
GFP feature motion (Figs. 3, H and I, and 5, H and I). To distin-
guish between budding and bona fide lateral diffusion, we again
used Dyngo-4aTM to distinguish internalization from the pla-
nar mobility of the remaining Cav1-GFP features (Fig. 7, FH);
please note for comparison we have reposted data from Fig. 1, I
(frequency of motion types) and J (frequency of diffusion coef-
ficients). The fraction of tracks classified as sub-diffusive in
cells treated with Dyngo-4aTM, dibucaine, or both averaged
82.1, 40.6, and 71.4%, with median diffusion coefficients of
3.4 103, 17.8 103, and 7.0 103 m2/s, respectively 8, H and I) compared with the cells treated with dibucaine (Fig.
(Fig. 7, H and I). Conversely, 13.3, 45.1, and 21.3% of the tracks
exhibited free motion, with median diffusion coefficients of
1.2 102, 18.1 102, and 5.1 102 m2/s, respectively ity or sub-diffusive nature of caveolae.
(Fig. 7, H and J). These results indicate that a significant pro-
portion of the Cav1-GFP features display enhanced mobility
following dibucaine treatment and that the mobility is due to
both the stimulation of a dynamin-dependent internalization
pathway and an increase in lateral diffusion. These findings on
PtdSer scrambling together with the data on PtdSer sequestra-
tion and degradation support an important role of this lipid in
caveola formation. However, it should be noted that phospho-
lipid scrambling also scrambles other lipids, including PtdEtn.
Whether PtdEtn is required for caveola assembly or function is
currently unknown.
Transient increase in membrane fluidity does not alter
caveolae
In addition to being a potent inducer of membrane scram-
bling, dibucaine can also fluidize membranes (48, 49). This
raises the possibility that the substantial alterations in caveolar
behavior and abundance could be due to a combination of
scrambling and increasing membrane fluidity. Typically, mam-
malian cells maintain membrane fluidity within a narrow range
by a process known as homeoviscous adaptation (50). However,
this adaptation to temperature changes or fatty acid supple-
mentation occurs over the course of hours and requires exten-
sive remodeling of the lipidome. Fortunately, it has been
reported that valproic acid, a branched chain weak organic acid,
can rapidly increase membrane fluidity within minutes of addi-
tion to cells (51). To monitor the changes in membrane fluidity,
we used polarized TIRF microscopy to access the degree of
orientational order of the lipophilic membrane dye 1,1-diocta-
decyl-3,3,3,3-tetramethylindocarbocyanine (DiI) to deter-
mine the order parameter P2, as described previously (52, 53).
As depicted in Fig. 8A, cells were imaged at 0 (p-polarization)
and 90 (s-polarization) and processed to generate the single
pixel level P2 image. In control cells, the orientation of the
absorption transition moment of the DiI is largely parallel to the
membrane surface resulting in negative P2 values (Fig. 8B) (53,
54). Having determined the average value in control cells, we
next confirmed that dibucaine and valproate increased the flu-
idity of the cells. Again, the DiI-containing cells were captured
using the polarized TIRF microscopy before and after incuba-
tion with the drugs for 10 min. Following the incubation with
dibucaine or valproate, the calculated P2 value remains nega-
14302 J. Biol. Chem. (2017) 292(34) 1429214307
tive, but the magnitude is less than that obtained in control
cells. This indicates that the orientation of the absorption tran-
sition moment has changed as a result of increasing membrane
fluidity. Collectively, the images and histograms demonstrate
that treatment with either 1 mM dibucaine or 40 mM valproate
for 10 min increased membrane fluidity (Fig. 8, CF).
Next, we examined the impact of the addition of valproate on
the Cav1-GFP features using TIRF and signal particle tracking.
In contrast to the addition of dibucaine (Fig. 7B), the addition of
valproate had little impact on the number or intensity of the
Cav1-GFP features (Fig. 8G). Likewise, the addition of valproate
did not alter the diffusive properties of Cav1-GFP features (Fig.
7, E, G and H). Collectively, these results suggest that transient
increases in membrane fluidity do not greatly impact the stabil-
Discussion
Phosphatidylserine is required to support the formation of
caveolae
In this paper, we have examined the role of PtdSer and
PtdIns(4,5)P2 in the formation and assembly of caveolae. PtdSer
is abundant in the inner leaflet of PM and enriched in caveolae
(23), and now using a variety of techniques, we have shown that
PtdSer is required for their assembly. Cells with reduced avail-
ability of PtdSer, via the overexpressing of the tandem Lact-C2,
showed a reduction in the number of caveolae observed by EM
as well as a decrease in the intensity of Cav1 puncta detected by
TIRF microscopy. Intriguingly, the silencing of cavin1 results in
a similar pattern of Cav1 redistribution in the PM. This result is
consistent with cavin1 being the PtdSer-sensitive component
needed for proper function or stabilization. The polybasic sites
of cavin1, HR1 and HR2 domains, were proposed to bind ani-
onic phospholipids, including PtdSer through charge-based
interactions (16, 17, 20, 55). In support of this notion, we find
that chronic depletion of the majority (80%) of PtdSer limits
the ability of cavin1 to associate with Cav1 and the PM. Addi-
tionally, sequestering the plasmalemmal PtdSer using transient
overexpression of the tandem Lact-C2 also displaced cavin1
from the PM and ultimately caused the disappearance of GFP-
cavin1. These results are consistent with a previous study that
demonstrated that upon release from the PM, cavin1 can be
ubiquitinated on its HR1 domain leading to rapid proteasomal
degradation (38). Together, our results show that PtdSer is
required for Cav1 and cavin1 to form stable complexes in vivo,
thereby explaining the enrichment of PtdSer in caveola (23, 56).
Non-caveolar Cav1-GFP clusters are mobile
Sequestering or limiting PtdSer prevents the assembly of
caveolae but does not cause the loss of Cav1. Instead, PtdSer
limitation generates Cav1-GFP clusters capable of diffusing in
the plasma membrane. Although PtdSer-limiting manipula-
tions are somewhat extreme, other physiological conditions do
occur that result in the presence of non-caveolar Cav1. Cav1 is
delivered to the PM via vesicular transport with Cav1 organized
into oligomers of 1214 monomers (6). Upon delivery to the
plasma membrane, individual clusters will have to diffuse and
collide with other clusters to form the larger caveolae.
 PtdSer is required for caveolae

Figure 8. Rapid increase in membrane fluidity does not alter caveolae. A, HeLa cells containing the fluorescence probe DiI was excited by parallel (s) and
perpendicular (p) polarized light, relative to the substrate surface using polarized TIRF microscopy. The inset and B correspond to the order parameter, P2. Cells
loaded with DiI and treated with dibucaine (C and D) or valproate (E and F) were also imaged, and their relative order parameter was calculated. Scale bar, 3 mm.
G, TIRF images of HeLa/Cav1-GFP following a 10-min incubation with valproate. H, representative trajectories of Cav1-GFP features in cells treated with
valproate; I, the classification of the tracks.

Growing evidence suggests that non-caveolar Cav1 may also
have biological significance. For instance, select prostate can-
cers contain non-caveolar Cav1 due to the absence of cavin1
(16, 57). Non-caveolar Cav1 has been described to impact a
variety of traits necessary for the progression of cancer. In pros-
tate cancer cells, Cav1 influences JAK/STAT and Src signaling
with high levels of Cav1 correlating with worse outcomes (58).
Importantly, in these cells exposure to radiation or the chemo-
therapeutic agents, gemcitabine and 5-fluorouracil, results in
enhanced levels of Cav1 (58). This up-regulation could be quite
problematic as Cav1 is thought to promote the dissemination of
cancer via metastatic migration through the lymphatic system
and to protect cells from apoptosis (59). Although a previous
study has demonstrated that caveolae can provide resistance to
apoptosis in androgen-independent prostate cancer cells (60),
the biological signals associated with non-caveolar Cav1 are
incompletely understood. In this regard, non-caveolar Cav1 has
been shown to influence the expression of important mediators
of metastasis and lymphangiogenesis such as matrix metallo-
protease 9 and vascular endothelial growth factor, respectively

J. Biol. Chem. (2017) 292(34) 1429214307 14303

PtdSer is required for caveolae
(59, 61). Thus, it is tempting to speculate that improved under-
standing of the biophysical properties of non-caveolar Cav1 will
help understand its role in cancer metastasis.
Anionic lipids in caveolae formation and immobilization in the
PM
Examination of the role of the plasmalemmal phosphoi-
nositides PtdIns4P and PtdIns(4,5)P2 in caveolae formation
required more acute depletion of these lipids. Thus, we used the
controllable enzymatic degradation of the phosphoinositides
rather than the inhibition of their synthesis. The acute deple-
tion of PtdIns(4,5)P2 had minimal impact on the total number
of observable caveolae. Because of the robustness of this detec-
tion algorithm to identify puncta in the TIRF images, depletion
of phosphoinositides had minimal impact on the number of
puncta. However, integrated spot intensity for the population
of foci was only minimally decreased after the exhaustion of
PtdIns4P and PtdIns(4,5)P2 and to a slightly lesser extent
PtdIns(4,5)P2 alone. Consistent with this finding, rapid deple-
tion of phosphoinositides cause only a minor increase in the
fraction of diffuse Cav1-GFP. The depletion of PtdIns(4,5)P2
and PtdIns4P did lead to changes in the percent of sub-diffusive
versus freely diffusible caveolae. It is possible that this observa-
tion is related more to the impact of phosphoinositide deple-
tion of actin dynamics. Furthermore, although the requirement
for PtdIns(4,5)P2 in caveola formation is less obvious than Ptd-
Ser, it is possible that the caveolar pool of PtdIns(4,5)P2 is not
readily exchangeable with the rest of the plasma membrane.
In summary, through the use of a variety of techniques to
manipulate the levels or availability of plasmalemmal PtdSer,
we have dissected the role for the lipid in supporting the forma-
tion of caveola and Cav1-cavin1 association. PtdSer appears to
be essential to support caveola formation, although Cav1 mol-
ecules still cluster when it is greatly diminished.
Experimental procedures
Reagents and solutions
Rabbit polyclonal antibodies to caveolin (catalog no. 610060)
were purchased from BD Biosciences, and rabbit polyclonal
antibodies to cavin1 (ab48824) were from Abcam. Goat poly-
clonal anti-GFP antibodies were purchased from Rockland Inc.
(catalog no. 600-101-215). Mouse monoclonal anti-GAPDH
antibodies were obtained from Millipore (MAB374). Cy3-con-
jugated IgG for a secondary antibody was obtained from Jack-
son ImmunoResearch Laboratories. Enhanced chemilumines-
cence reagents were obtained from GE Healthcare, and BCA
protein assay reagent was from Pierce. Fluorescently conju-
gated annexin-V and puromycin were purchased from Thermo
Fisher Scientific. Dyngo-4aTM was also acquired from Abcam.
Oligonucleotides for PCR and siRNA-mediated gene silencing
were purchased from Integrated DNA Technologies and Dhar-
macon, respectively. All other reagents were from Sigma unless
stated.
Plasmids
Construction of the plasmids encoding Lact-C2 (36), PH-PLC
(62), and EGFP-PTRF (cavin1) (16) has been described previously.
14304 J. Biol. Chem. (2017) 292(34) 1429214307
The mCherry-tandem Lact-C2 construct was constructed as fol-
lows. First, Lact-C2 was amplified from bovine Lact-C2 using the
forward primer (5-GCAGACCTGCAGTCTGCACTGAACCC-
CTAGGCCTG-3) and the reverse primer (5-GCAGACGGTA-
CCCTAACAGCCCAGCAGCTCC-3) and then introduced into
the pEGFP-C1 vector (Clontech) at the PstI and KpnI sites. Next,
the other Lact-C2 fragment was amplified on Lact-C2 using the
forward primer (5-GCAGACTCCGGATGCACTGAACCCCT-
AGGCCTG-3) and the reverse primer (5-GCAGACCTCGAG-
AACAGCCCAGCAGCTCC-3) and then inserted into the
pEGFP-Lact-C2 made in the first step at BspEI and XhoI sites. This
construct consisted of two Lact-C2 cDNAs separated by the 30-bp
linker. Finally, the EGFP-tandem Lact-C2 was digested with re-
striction enzymes BspEI and KpnI, and subsequently, the tandem
Lact-C2 cassette was cloned into the mCherry-C1 vector with the
same restriction sites.
The pMSCV-Cav1-GFP plasmid was constructed by first
performing PCR using the forward primer (5-GCGAGAT-
CTATGTCTGGGGGCAAATACGTAG-3) and the reverse
primer (5-GCGGAATTCTTACTTGTACAGCTCGTCCATG-
3) and subcloning using BglII and EcoRI restriction sites built
into the primers and cloned into the pMSCV-puro vector
(Clontech). The dual phosphatase PJ and its related constructs
were the gifts from Robin Irvine (Addgene plasmids catalog no.
37999-38002) and have been described elsewhere (43).
Cell lines and transfection
HeLa, BHK, and CHO cells were obtained from the Ameri-
can Type Culture Collection. The PSB-2 cell line was the kind
gift of Dr. Osuma Kuge (Kyushu University, Japan) and was
maintained as described previously (63). To establish lines sta-
bly expressing Cav1-GFP, cells were transfected with pMSCV-
Cav1-GFP using FuGENE6 (Promega) and maintained for 3
weeks in DMEM/F-12 containing 1 g/ml puromycin (Life
Technologies, Inc.) for HeLa and 20 g/ml for CHO and PSB-2
cells. The HeLa cells stably expressing cavin1-GFP (HeLa/
cavin1-GFP) were established by FuGENE 6 transfection with
cavin1-GFP into cells, which were incubated for 3 weeks in
DMEM/F-12 containing 800 g/ml G418 (Wisent Bio-Prod-
ucts). The fluorescent cells were then collected using the
InfluxTM cell sorter (BD Biosciences). HeLa, HeLa/Cav1-GFP,
HeLa/cavin1-GFP, CHO, and CHO/Cav1-GFP were incubated
in DMEM/F-12 (Wisent Inc.) with 5% fetal bovine serum (FBS).
PSB-2 and PSB2/Cav1-GFP were maintained in DMEM/F-12
with 5% charcoal-stripped FBS (Wisent Inc.). Where indicated
a suspension of PtdSer (from porcine brain; Avanti Polar Lip-
ids) or PtdEtn (Avanti Polar Lipids) was prepared as described
(40) and added to the growth medium.
Transient transfection of plasmids was performed using
FuGENE 6, according to the manufacturers instructions. For
single-plasmid transfection, 1.0 g of cDNA and 1.5 l of
FuGENE 6 were used per coverslip. Transfection of siRNA tar-
geting cavin1 (SMARTpool, Dharmacon) and the scrambled
oligomer (control) was done using HiPerFect transfection
reagent (Qiagen). The sequences of siRNA used were CCU-
ACUAGAAGGACGUGAA, CCAUCUCUACUAAGCGAAA,
GCGAGAAACUGAAGACGUC, and CGGGACAAGUU-
GCGCAAAU. 100 l of serum-free DMEM/F-12 containing

siRNA (final 20 nM) mixed with 8 l of HiPerFect was incubated
at room temperature for 15 min. Cells expressing PJ and its
related constructs, PJ-INPP5E, PJ-sac, and PJ-dead, were
treated for 5 min with 1 M rapamycin before analysis. Unless
otherwise indicated, all treatments and assays were at 37 C.
Western blotting
Cells were lysed in RIPA buffer, 150 mM NaCl, 1.0% Triton
X-100, 0.5% sodium deoxycholate, 0.1% SDS, 20 M leupeptin,
and 20 mM Tris, pH 7.4, supplemented with protease inhibitor,
cOmpleteTM (Roche Applied Science). Protein was quantified
with the BCA Protein Assay Reagent Kit, using bovine serum
albumin as a standard. The lysate was denatured with Laemmli
Buffer and separated by electrophoresis on a 10% polyacryl-
amide gel. Proteins were transferred to a PVDF membrane and
the blots probed with the anti-caveolin, anti-GFP, anti-
GAPDH, or anti-cavin1 antibodies as indicated. The proteins
were visualized with horseradish peroxidase-conjugated sec-
ondary antibodies using the ECL system (GE Healthcare).
Immunostaining
Cells were fixed with 4% paraformaldehyde in PBS,
permeabilized blocked with 0.1% Triton X-100 and 5% skim
milk in PBS, and incubated with the anti-caveolin antibody
diluted 1:2000 in 5% skim milk containing PBS, for 60 min.
After extensive rinsing with PBS, cells were incubated for 1 h
with Cy3-coupled donkey anti-rabbit antibodies in PBS con-
taining 5% skim milk.
Microscopy
In the acquisition of all images or movies, coverslips with
cells were transferred to an 18-mm magnetic chamber (Cham-
lide CMB, Live Cell Instrument); medium was replaced with a
synthetic medium as indicated (see under Reagents and solu-
tions), and the chamber was placed on a microscope stage
maintained at 37 C. For acquiring differential interference
contrast microscopy, the cells were imaged using a Leica DM
IRB microscope, and images were captured by a cooled charge-
coupled device (CCD) camera (Cascade II; Photometrics) using
MetaMorph software (MDS Analytical Technologies). Fluores-
cence images were acquired by spinning-disc confocal systems
(Quorum Technologies). An Axiovert 200M microscope (Carl
Zeiss) with 63 objective lenses and a 1.5 magnifying lens
was equipped with diode-pumped solid-state lasers (440, 491,
561, 638, and 655 nm; Spectral Applied Research) and a motor-
ized XY stage (Applied Scientific Instrumentation). Images
were acquired using back-thinned, electron-multiplied cam-
eras (model C9100-13 ImagEM; Hamamatsu Photonics) driven
by Volocity software 4.3.2 or 6.2.1 (PerkinElmer Life Sciences).
TIRF microscopy was performed using an Olympus cell TIRF
microscope system. The instrument is based on an Olympus
IX81 inverted microscope, with a 150, 1.45 numerical aper-
ture (NA) oil immersion objectives, Hamamatsu C9100-13
electron multiplying (EM)-CCD, and 491- and 561-nm laser
excitation lines. Volocity 4.3.2 software (PerkinElmer Life Sci-
ences) was used to visualize Cav1-GFP within the plane of PM.
All experiments using TIRF microscopy were performed with
precision coverslips, glass thickness No. 1.5H (Paul Marienfeld
PtdSer is required for caveolae
GmbH & Co. KG, Germany). For simultaneous dual-channel
imaging, a Photometrics DualView (Tucson, AZ) adapter was
used. The dual view is an emission splitting system capable of
simultaneous acquisition of two separate emission spectra.
Polarized total internal reflection microscopy
Cells were treated with lipophilic membrane stain DiI. Fluo-
rescence microscopy was carried out on a custom-built combi-
natorial microscopy system. The rig is built around an Olympus
IX70 inverted microscope with four laser lines and two Evolve
512 EM-CCD cameras (Photometrics, Tucson). The system is
capable of performing polarized and traditional TIRF micros-
copy, atomic force microscopy, confocal microscopy, and
direct stochastic optical reconstruction microscopy. The
images were captured with a 60 1.45 NA oil-immersion
TIRF objective (Olympus Canada). The fluorescence probe
DiIC18(3) was excited by parallel (s) and perpendicular (p)
polarized light, relative to the substrate surface, through a half-
wave liquid crystal variable retarder LCC25 1111A (Thorlabs,
Newton, NJ). Both the EMCCD cameras and liquid crystal vari-
able retarder were controlled by Micro-Manager. Cells were
imaged before and after a 10-min treatment with dibucaine or
valproate, respectively. The P2 values were calculated using an
ImageJ macro that considers the evanescent electric field vector
amplitude, excitation incident angle, and fluorescence detected
dichroic rotation, see Ref. 53 for further details.
Electron microscopy
Cells were rinsed briefly in 100 mM cacodylate buffer, pH 7.4
(Electron Microscopy Sciences, PA), and then fixed for 1 h in
primary fixative 100 mM cacodylate buffer, pH 7.4, containing 1
mg/ml ruthenium red (Calbiochem) and 2.5% glutaraldehyde at
room temperature. Next, cells were washed with 100 mM caco-
dylate buffer and stained with 100 mM cacodylate buffer, pH
7.4, containing 1% osmium tetroxide and 1 mg/ml ruthenium
red for 3 h at room temperature. After that, cells were washed
with 100 mM cacodylate buffer, pH 7.4, and rinsed with double-
distilled water. Cells were observed in a transmission electron
microscope (JEOL 1011; JEOL Ltd.), and electron micrographs
were taken with a digital camera (Morada; Olympus) using
AnalySIS software (Olympus).
Post-acquisition analysis
Quantifying punctate versus diffuse fluorescenceTo restrict
our analysis to a region of interest, we first constructed a mem-
brane mask, algorithm for DualView images (Cav1-GFP
membrane label). For these images, we first split the image into
the two channels (supplemental Fig. 1A), averaged the mem-
brane channel images, and performed the following operations
on the averaged image: 1) threshold to a binary image; 2) apply
morphological closing with a disk-structuring element; 3) fill
any holes to create a membrane mask (supplemental Fig. 1B).
The membrane mask was combined with the spot detection
output to generate a mask for punctate Cav1-GFP (supplemen-
tal Fig. 1C), and with the inverse of the spot detector output to
generate a mask for diffuse Cav1-GFP (supplemental Fig. 1D).
Applying these masks to the Cav1-GFP generated correspond-
ing images of the membrane distribution of Cav1-GFP in either
J. Biol. Chem. (2017) 292(34) 1429214307 14305
PtdSer is required for caveolae
punctate (supplemental Fig. 1E) or diffuse partitions (supple-
mental Fig. 1F). We then summed the pixel intensity, for each
image and computed the ratio of total pixel intensities of the
two images. All image-processing operations were carried out
in MATLAB using custom code (Mathworks, Natick MA).
Particle trackingThe motion of individual particles was
defined by SPT. For analysis, raw images were cropped in
ImageJ (National Institutes of Health, Bethesda) (Fig. 1G), and
tracking was performed with MATLAB (MathWorks, Natick,
MA), detecting local intensity maxima and then fitting two-
dimensional Gaussian kernels approximating the point-spread
function of the microscope as described previously (28). The
detected particles throughout a time-lapse image sequence
were first linked between consecutive frames, and the resulting
track segments were then linked to generate complete trajecto-
ries by closing gaps and capturing merging and splitting events
(Fig. 1H).
Diffusion analysisMotion types and diffusion coefficients
were also derived with MATLAB using a moment scaling spec-
trum (MSS) analysis (64). The motion type (sub-diffusive, free,
or directed) was determined by the value of the slope of the MSS
(29).
Author contributionsG. D. F. conceived the study, coordinated the
experiments, and wrote the paper. T. H., Y. Y., C. F., and A. W. per-
formed experiments and analyzed data. R. D. generated Matlab code,
analyzed data, and generated figures. J. G. K. provided new materials
used in the manuscript. S. G., C. M. Y., and R. G. P. helped to edit the
paper and provided input into the experiments. All authors reviewed
the results and approved the final version of the manuscript.
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