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TITLE 1
Acto-myosin driven functional nanoclusters of GPI-anchored proteins are 2
generated by integrin receptor signaling 3
4
AUTHORS 5
Joseph Mathew Kalappurakkal1†, Anupama Ambika Anilkumar1,4†, Chandrima Patra1,§, 6
Thomas S. van Zanten1,§, Michael P. Sheetz 3, Satyajit Mayor1,2,*. 7
†,§ Equal contribution 8
AFFILIATIONS 9
1:National Centre for Biological Sciences, Tata Institute of Fundamental Research, 10
Bellary Road, Bangalore, India. 11
2: Institute for Stem Cell Biology and Regenerative Medicine, Bellary Road, Bangalore, 12
India. 13
3: Mechanobiology Institute, National University of Singapore, Singapore. 14
4: Present address: St Johns Research Institute, Bangalore, India 15
*Correspondence to: 16
Satyajit Mayor 17
e-mail: mayor@ncbs.res.in 18
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SUMMARY 26
GPI-anchored protein (GPI-AP) nanoclusters are generated by cortical acto-27
myosin activity. While our understanding of the physical principles behind this process is 28
emerging, the molecular machinery required for the generation of these nanoclusters is 29
unknown. Here, we show that ligand–mediated membrane receptor signaling triggers 30
nanocluster formation. Both soluble and surface-tethered RGD ligands bind the β1-31
integrin receptor and activate focal adhesion and src- kinases, resulting in RhoA 32
signaling. This cascade ultimately triggers actin-nucleation via specific formins, driving 33
nanoclustering of both GPI-APs and a model transmembrane protein with an actin-34
binding domain. Integrin signaling concurrently results in talin mediated activation of 35
vinculin. This is necessary for the coupling of the dynamic actin machinery to the inner 36
leaflet driving GPI-AP nanoclustering. Disruption of GPI-AP nanoclustering in either 37
GPI-anchor remodeling mutants or in cells that express vinculin mutants, provide 38
evidence that these nanoclusters are necessary for activating cell spreading, a hallmark 39
of integrin function. 40
41
INTRODUCTION 42
Sub-compartmentalization of the plasma membrane (PM) via the lateral segregation of 43
proteins and lipids into structural and signaling platforms is likely to play pivotal roles in 44
the spatio-temporal regulation of many signaling systems. Finely tuned signaling 45
systems such as T-cell receptor triggering at the immunological synapse (Gaus et al., 46
2005), B-cell receptor activation (Gupta and DeFranco, 2007; Mattila et al., 2013), and 47
cell-ECM adhesion (Gaus et al., 2006; Lingwood and Simons, 2010; Simons and 48
Toomre, 2000; van Zanten and Mayor, 2015), involve the generation of membrane 49
domains. Such membrane domains, enriched in cholesterol, sphingolipids and outer 50
leaflet lipid-tethered glycosylphosphatidylinositol (GPI)-anchored proteins, have often 51
been termed as membrane ‘rafts’ (Sezgin et al., 2017). 52
The mechanism whereby cells generate these domains remains controversial. The 53
size, scale and statistics of membrane heterogeneities in the cell are very different than 54
what is predicted from thermodynamically driven phase segregation observed in 55
artificial membranes or cell-free membrane preparations such as Giant Plasma 56
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Membrane Vesicles (GUVs) (Chiantia and London, 2012; Sezgin et al., 2012a, 2012b). 57
Many of the ‘raft’ components such as outer leaflet GPI-APs or inner leaflet Ras 58
molecules form nanoclusters (Plowman et al., 2005; Varma and Mayor, 1998). These 59
nanoscale clusters are hierarchically organized into larger scale optically resolvable 60
(mesoscale) domains where a significant fraction of the lipid-anchored proteins are 61
present as nanoclusters (Goswami et al., 2008; Tian et al., 2007; van Zanten et al., 62
2009). 63
In the ‘resting’ state, GPI-APs at the cell surface are distributed as monomers with 64
a small fraction of nanoclusters (20-40%) that is independent of total protein expression 65
levels (Sharma et al., 2004; van Zanten et al., 2009). Under conditions of activation 66
such as the binding of ligand to the integrin receptor, LFA-1, in immune cells, the 67
fraction of GPI-APs in nanoclusters increases (~ 80%), and appears in spatial proximity 68
to LFA-1 nanoclusters, regions designated as "hot-spots". Reduction of cholesterol 69
levels, a treatment that also prevents the formation of GPI-AP nanoclusters, drastically 70
inhibited the ligand binding capacity of these adhesion receptors (van Zanten et al., 71
2009). At the same time ligand-induced or crosslinking antibody-induced clustering of 72
GPI-APs is sufficient to drive downstream signaling responses in the cell (Harder et al., 73
1998; Stefanová et al., 1991; Suzuki et al., 2007). The regulation of nanoscale 74
clustering is, thus, likely to be an important determinant in high-fidelity signal 75
transduction processes that operate at the cell surface (Harding and Hancock, 2008; 76
Tian et al., 2007). 77
Extensive studies on the organization and dynamics of GPI-APs have indicated a 78
crucial role for a dynamic actin layer at the cortex juxtaposed to the inner leaflet of the 79
PM in the formation of such nanoclusters at the outer leaflet (Goswami et al., 2008; 80
Saha et al., 2015). In previous work, we had proposed that dynamic actin filaments 81
along with myosin motors form transient remodeling contractile platforms (asters) at the 82
inner leaflet (Gowrishankar et al., 2012). These ‘asters’ immobilize clusters of the long-83
acyl chain containing phosphatidylserine (PS) at the inner leaflet which interact with 84
long-acyl chain containing GPI-APs at the outer leaflet via a transbilayer coupling 85
interaction, thereby creating nanoclusters (Raghupathy et al., 2015). These and other 86
observations (Köster and Mayor, 2016; Rao and Mayor, 2014) indicate that the 87
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organization at the membrane might be better understood as an active actin-membrane 88
composite, wherein the constituents in the fluid membrane bilayer interact with the 89
dynamic actin cortex. In this context, membrane components can be classified into 90
three classes based on their ability to couple with and regulate this active machinery: 91
inert, passive and active (Gowrishankar et al., 2012). Inert molecules are those that are 92
unable to interact with the underlying actin (for example unsaturated lipids at the outer 93
leaflet or membrane proteins that lack any linkage to actin filaments), passive molecules 94
are those that bind (and unbind) actin filaments (such as the GPI-APs as well as trans-95
membrane proteins that possess actin-binding motifs at their cytoplasmic tails), and 96
active molecules which not only bind but also influence the actin cytoskeleton dynamics 97
at the membrane and in doing so could regulate local membrane organization. 98
Despite the emergence of a theoretical understanding of the active mechanics 99
behind the generation of the nanoscale assemblies and their distribution and dynamics, 100
the molecular machinery for their formation has been missing; the nucleators of actin, 101
triggers of myosin function, and the linkage between the actin and the PS lipid are 102
uncharacterized. In this manuscript we uncover the molecular machinery that governs 103
the formation of the dynamic actin-based membrane patterning system. 104
Here we show that integrin receptors behave as a prime example of an ‘active’ 105
molecule that regulates actin nucleators and myosin activity necessary to build the 106
hierarchical organization of clusters. We find that upon engagement with RGD-107
containing ligands, integrin receptors through their ability to activate the FAK and src 108
kinases and the resultant RhoA activation trigger formins necessary for the generation 109
of the dynamic actin filaments. RhoA also activates the ROCK pathway, required for 110
myosin activation. Importantly, we also identify vinculin, a ubiquitous protein that 111
associates with focal adhesions, as a molecule necessary for directing the generated 112
dynamic actin filaments to the inner-leaflet lipids and thereby generating GPI-AP-113
nanoclusters. Furthermore, using GPI-anchor remodeling mutants as well as vinculin 114
mutants, which fail to support nanocluster formation, we show that the nanoclusters 115
created by this active machinery are necessary for activating cell spreading, a hallmark 116
of integrin function. 117
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RESULTS 119
Integrin activation generates nanoclusters of the outer leaflet GPI-APs. 120
The integrin family of heterodimeric transmembrane receptors binds various 121
extracellular ligands that activate a multitude of structural and signaling molecules 122
(Hynes, 2002; Vicente-Manzanares et al., 2009). Integrins exhibit the hallmarks of an 123
‘active’ molecule that upon ligand engagement could alter the nanoscale organization of 124
cell surface molecules in its vicinity through its ability to regulate the cortical acto-125
myosin network. Earlier studies of the integrin LFA1 activation in fixed immune cells 126
have shown that upon binding to its ligand, ICAM-1, hot spots of GPI-AP clusters are 127
formed, localized to the site of integrin activation within 30 mins of activation (van 128
Zanten et al., 2009). This prompted us to test whether the activation of other integrins 129
had a similar effect on the nanoclustering of GPI-APs albeit in a different cellular 130
context. We used fluorescence emission anisotropy based microscopy to assess the 131
extent of homo-FRET between fluorescently-tagged GPI-APs (Ghosh et al., 2012). 132
Homo-FRET results in the lowering of anisotropy, providing a facile way to monitor nano 133
scale clustering in intact living cells (Sharma et al., 2004; Varma and Mayor, 1998). 134
Cells (CHO) stably expressing mEGFP or mYFP -tagged GPI (MYG-1) were de-135
adhered and re-plated under serum-free conditions either on glass coated with 1%BSA 136
or on glass coated with the extracellular matrix (ECM) protein fibronectin (FN) (Figure 137
1A). FN is capable of engaging with a specific subset of integrins (Humphries et al., 138
2006; Hynes, 2002) that promotes cell spreading (Figure1C), whereas the BSA surface 139
is relatively inert to cell spreading at a similar time point (Figure 1C). 140
Although the amount of EGFP-GPI expressed on the cell surface is comparable 141
(Figure1B-C; Total Intensity axis), the anisotropy (Figure 1B-C; Anisotropy axis) is 142
much lower in cells plated on FN compared to that measured on BSA coated glass. The 143
low (or high) anisotropy is discerned by ‘blue (or red)’ pixels in the heat map-encoded 144
anisotropy image in Figure 1B and used throughout this manuscript. 145
The observed decrease in anisotropy occurs in a FN-concentration dependent 146
manner saturating at ~10µg/ml FN solution concentration (Figure 1D). This decrease is 147
specific for FN since when plated on 0.01% Poly-L-Lysine (that permits integrin-148
independent adhesion; (Schlaepfer et al., 1994) or on Laminin, Collagen-1 or Vitronectin 149
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(that engages a different subset of integrins), there is no significant reduction in the 150
anisotropy of GFP-GPI (Figure S1A, B). 151
An increase in anisotropy upon photo-bleaching would indicate homo-FRET as a 152
cause for the lowering of anisotropy (Sharma et al., 2004). Photo-bleaching YFP-GPI 153
results in a net increase in anisotropy (Figure 1E, F) confirming our expectation. The 154
typical profile of a linear increase is consistent with the presence of nanoclusters of 155
YFP-GPI when cells are plated on FN (Sharma et al., 2004). The higher value of initial 156
anisotropy and the minimal change in the YFP-GPI anisotropy value in cells plated on 157
glass upon photo-bleaching, corroborates the low fraction of nanoclusters that are 158
formed under this condition (Figure1E, F). Additionally, the decrease in anisotropy 159
observed when cells are plated on FN is sensitive to the removal of cholesterol by the 160
cholesterol-sequestering agent methyl β-cyclodextrin that disrupts nanoscale 161
organization of GPI-APs [(Raghupathy et al., 2015); FN+mβCD; Figure 1B, C], 162
confirming the enhancement in nanoclustering of GPI-APs on this substrate. The 163
decrease in anisotropy occurs only for specific membrane constituents; there is a 164
decrease in anisotropy of an exogenously incorporated fluorescent GPI analogue (NBD-165
GPI) (Figure 1G, I; exo-GPI) whereas an ‘inert’ fluorescent short chain-containing 166
sphingomyelin analogue (C6-NBD-SM) incorporated into cells plated on FN does not 167
exhibit this decrease (Figure 1H, J; exo-scSM). 168
The αV-class (αVβ3) and β1-class (α5β1) integrins are the primary integrins that 169
mediate fibroblast cell spreading on FN (Humphries et al., 2006; Leiss et al., 2008).We 170
utilized various function perturbing antibodies targeted against either the β1 or the αV 171
class of integrins to discern which of these integrin sub-types are involved in the FN 172
mediated generation of GPI-AP nanoclusters in human U2OS cells (Byron et al., 2009) . 173
We observed a loss in nanoclustering of GPI-APs (increase in GFP-GPI anisotropy) 174
when U2OS cells were pre-treated with the increasing concentrations of β1-blocking 175
antibody and subsequently plated on FN (Figure S1D-E). There was also a significant 176
decrease in the cell spread area as a function of β1-blocking antibody concentration 177
indicating that U2OS cells predominantly utilize the β1 integrin to spread on FN. At the 178
highest concentration, the anisotropy values obtained were comparable to those 179
obtained after treatment with mβCD (Figure S1D). There was no increase in GPI-AP 180
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anisotropy when U2OS cells were treated with antibodies that do not block spreading 181
[(neutral non-function perturbing β1 antibody (K20) or Transferrin-receptor antibody 182
(OKT9); Figure S1E-F) or αV-blocking antibody (17E6; data not shown)] and 183
subsequently plated on FN. Additionally, U2OS cells plated on the αVβ3 ligand 184
vitronectin (Charo et al., 1990) does not exhibit an increase in GPI-AP nanoclustering. 185
Together, these data indicate that the enhanced nanoclustering occurs when cells are 186
plated on FN, and this is mediated by the activated β1-class of integrins. 187
We next probed if an increase in affinity of the integrin for its ligand can alter the 188
nanoclustering of GPI-APs. To test this, we plated U2OS cells on low concentrations of 189
ligand (0.5µg/ml) in the presence of increasing amounts of Mn2+, an ion that potentiates 190
integrin activation (Dransfield et al., 1992; Mould, 2002; Takagi et al., 2002). Prior 191
treatment of cells with increasing amounts of Mn2+ resulted in a dose-dependent 192
decrease in anisotropy of GPI-APs in the presence of low FN (Figure S1G-H). On high 193
FN (10µg/ml; and higher), addition of Mn2+ did not result in a further decrease in 194
anisotropy (Figure S1G-H) indicating no further increase in GPI-AP nanoclustering. 195
Taken together, these data indicate that shifting the equilibrium towards a ligand-196
engaged integrin, either by increasing FN density or by activation through Mn2+ 197
promotes the generation of GPI-AP nanoclusters. 198
Localized nanoclustering in the vicinity of the activated integrin receptor 199
When plated on FN, the cells go through three major phases of behavior from a round 200
state in suspension to a fully-flattened circular morphology (Dubin-Thaler et al., 2004). 201
These are: Phase 0 (P0), the wetting phase mediated by the initial engagement of the 202
integrin with its ligand; Phase 1 (P1), the rapid expansion phase where sensing of the 203
mechanical rigidity and chemical suitability of the substrate and the establishment of a 204
large contact area takes place (Giannone et al., 2004); and finally, Phase 2 (P2), where 205
myosin II contractility based probing of the substrate via periodic protrusion/retraction of 206
the cell edge and continued asymptotic spreading to maximum area is attained. These 207
phases define critical checkpoints for progression from a suspended state to a fully 208
spread state. This is organized in a specific spatial and temporal order involving distinct 209
sets of protein modules in each phase (Wolfenson et al., 2015), allowing us to correlate 210
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the changes in nanoclustering of GPI-APs with these universal characteristics of a 211
spreading cell. 212
We examined the effect of cell spreading (CHO) on fibronectin-coated surfaces on 213
GPI-AP nanoclustering (Figure 2A-C; Supplementary Movie 1). At the level of a single 214
cell, the cell surface that first comes in contact with the FN-coated area appears to be 215
devoid of nanoclusters (red areas in Figure 2A) and starts to acquire nanoclusters 216
(‘blue’ pixels at the cell periphery) co-incidental with the P0-P1 transition phase in cell 217
spreading (Figure 2B and C; Pink-yellow transition zone, Figure S2B). Analysis of this 218
behavior over a large number of cells shows that there is a sudden and consistent 219
decrease in the steady-state anisotropy of GFP-GPI (δAnisotropy) that precedes the 220
peak in cell expansion (δArea; that occurs in P2 phase) by ~100-200 seconds (Figure 221
2C). 222
In the P0 phase, integrin engagement and clustering is an early step in the 223
formation of cell-ECM adhesions and is independent of force (Choi et al., 2008). To 224
probe if the effects of integrin activation on the promotion of GPI-AP nanoclustering are 225
force-dependent and localized to the sites of integrin activation, we employed a 226
supported lipid bilayer (SLB) system functionalized with a mobile lipid-attached cyclic-227
RGD ligand (Figure S2C); Arg-Gly-Asp (RGD) ligand is the sequence motif in FN that 228
mediates integrin-engagement (Ruoslahti, 1996). Here, the transiently immobilized 229
fluorescently-tagged ligands serve as reporters of the ligated integrins (Yu et al., 2011, 230
Figure S2C). This system facilitates the observation of local membrane organization 231
during the early stages of integrin mediated cell adhesion by enabling the simultaneous 232
tracking of the dynamics of the nascent integrin clusters and the nanoclustering of GFP-233
GPI in the membrane in the vicinity of this cluster. (Figure 2D; Figure S2H). Although 234
the engaged integrin is unable to exert significant traction on the fluid bilayer which 235
results in the inability of cells to fully spread and arrest in the P0 phase (Figure S2F), 236
there is enhanced nanoclustering of GPI-APs on cells engaged with lipid-attached 237
mobile RGD ligands compared to cells plated on glass (Figure S2D, E). In many cases 238
we observe a characteristic pattern where integrin cluster formation often precedes the 239
local decrease in anisotropy of GFP-GPI (Figure 2D; see more examples in Figure 240
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S2G, H). These results show that the activation of the FN binding integrin receptors 241
triggers a localized change in GPI-AP nanoclustering. 242
The time from the initial contact until initiation of cell spreading is inversely 243
correlated with the ligand density (Dubin-Thaler et al., 2004), suggesting that the 244
process is triggered by the integration of chemical signals via integrin receptor 245
engagement to its ligand. To test the possibility that the extent of GPI-AP nanoclustering 246
is also an integral response of a chemical signaling process, we treated cells with a 247
soluble cRGDfV peptide that has been shown to activate signaling molecules 248
downstream of integrin receptor binding (Huveneers et al., 2008; Zhang et al., 2014). 249
Strikingly, we also observe an increase in nanoclustering of GFP-GPI in cells plated on 250
glass and treated with the soluble cRGDfV in a cholesterol and dose-dependent manner 251
(Figure 2E, F) indicating that the increase in nanoclustering is triggered by a signaling 252
response initiated by integrin- RGD-binding. 253
GPI-AP nanoclustering is mediated via a RhoA signaling pathway downstream of 254
integrin activation. 255
RhoGTPases, tyrosine kinases, and various bona fide cytoskeletal modifying proteins 256
are involved in the steps of integrin-mediated cell spreading behavior (Vicente-257
Manzanares et al., 2009). To investigate the signaling pathway activated by integrins 258
that leads to GPI-APs nanoclustering, we employed a chemical and genetic perturbation 259
approach. Pre-treatment of cells with the src-family kinase (SFK) inhibitor, PP2 (Hanke 260
et al., 1996) and the focal adhesion kinase (FAK) inhibitor PF 573 228 (Slack-Davis et 261
al., 2007) results in a dramatic decrease in GPI-AP nanoclustering in cells plated on FN 262
(Figure 3A,B). Correspondingly, FAK null fibroblasts also fail to support FN-induced 263
GPI-AP nanoclustering (Figure S3C, D). 264
A downstream target of the SFK and FAK kinases during integrin mediated 265
signaling is the Rho family GTPase member, RhoA (Cox et al., 2001; Guilluy et al., 266
2011; Ren et al., 1999). Increasing concentrations of the cell permeable Rho inhibitor 267
C3 exoenzyme which specifically inhibit RhoA activity (Aktories et al., 1987; Braun et 268
al., 1989)), also inhibited GPI-AP nanoclustering in a dose dependent manner when 269
cells were plated on FN (Figure 3C, D). In contrast, addition of a cell-permeable RhoA 270
activator [CN03; (Flatau et al., 1997; Schmidt et al., 1997)] induced enhanced 271
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nanoclustering of GPI-APs even when cells were plated on plain glass, a condition 272
where there is minimal integrin activation (Figure S3E, F). Furthermore, the failure in 273
promoting nanoclustering upon FN engagement when the cells were treated with both 274
SFK-FAK inhibitors can be rescued by the ectopic addition of CN03 (Figure 3G, H). 275
These results indicate that RhoA operates downstream of SFK-FAK in the molecular 276
pathway that mediates the nanoclustering of GPI-APs. 277
Formin nucleators are necessary for GPI-AP nanoclustering 278
We next investigated the role of the actin-nucleators that are downstream targets of 279
integrin activation in mediating the nanoclustering of GPI-APs. Pre-treatment of cells 280
with SMIFH2, a small molecule inhibitor of the formin class of actin nucleators (Rizvi et 281
al., 2009) led to loss of FN-mediated nanoclustering of GPI-APs (Figure 3E, F), 282
whereas the Arp2/3 inhibitor CK666 (Nolen et al., 2009) had no effect on GPI-AP 283
nanoclustering (Figure 3E, F). Moreover, the acute loss of GPI-AP nanoclusters 284
observed when cells were treated with inhibitors of SFK and FAK could be rescued by 285
treatment of cells with a formin activator (IMM01; Lash et al., 2013) which in turn is 286
reversed by SMIFH2 treatment (Figure 3G, H), suggesting that formins are downstream 287
of SFK/FAK-RhoA in this pathway that mediates nanoclustering of GPI-APs. In addition, 288
treatment of cells plated on uncoated glass with the formin activator (IMM01) resulted in 289
an increase in nanoclustering of GPI-APs even in the absence of integrin ligand 290
engagement (Figure S3G, H). This suggests that formin activation is an important step 291
in the integrin mediated signaling response that drives enhanced nanoclustering of GPI-292
APs. 293
To investigate the identity of the specific formin that mediates the nanoclustering of 294
GPI-AP, we utilized specific RNAis to reduce the expression of two candidate formins, 295
mDia1 (DIAPH1) and FHOD1 in U2OS cells. Upon reduction of the levels of mDia1 and 296
FHOD1 between ~80 and ~40%, respectively (Figure S4A, B), there was a drastic 297
decrease in nanoclustering of GPI-APs. The loss of nanoclustering was more significant 298
when FHOD1 levels were reduced when compared to mDia1 (Figure S4C, D), 299
implicating FHOD1 as one of the major formin members involved in this process. Taken 300
together, these results indicate that actin filaments nucleated by specific formins are 301
involved in the FN-integrin induced nanoclustering of GPI-APs. 302
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Integrin activation triggers an acto-myosin-based clustering mechanism. 303
To test whether changes in nanoclustering of GPI-APs induced by integrin activation are 304
mediated by the upregulation of the cortical acto-myosin based machinery described 305
previously (Gowrishankar et al., 2012), we monitored the organization of a model 306
chimeric receptor composed of an extracellular reporter domain derived from folate 307
receptor [FR], a transmembrane segment [TM] and a cytosolic domain derived from the 308
actin binding domain of ezrin [Ez-AFBD], FRTM-Ez-AFBD (Figure 4A). This chimeric 309
protein also forms nanoscale clusters that are dependent on its ability to bind actin and 310
associate with a dynamic acto-myosin machinery (Gowrishankar et al., 2012). Steady-311
state anisotropy measurements of fluorescently labeled FRTM-Ez-AFBD expressing 312
cells showed a decrease in anisotropy when plated on FN (Figure 4B, C), similar to that 313
observed for the GPI-APs (Figure 1C). By contrast, cells expressing a mutated version 314
of the FRTM-Ez-AFBD that is incapable of binding actin (FRTM-Ez-AFBD*; Figure 4D) 315
did not display changes in the steady-state anisotropy upon engagement with FN 316
(Figure 4E, F). 317
A signature of the dynamic actin-filaments at the membrane surface is the 318
decreased diffusion of GFP-tagged-actin filament binding domain of Utrophin (GFP-Utr) 319
as measured by fluorescence correlation spectroscopy (FCS) (Gowrishankar et al., 320
2012). When FCS traces were taken from regions in the periphery of the cell that were 321
devoid of stress-fibers (Figure S4E), we detected at least two diffusing species (Figure 322
S4F); one corresponding to the diffusion timescale of unbound GFP-Utr (0.3 ms < τ < 3 323
ms) and another slower component (τ>10ms) that corresponds to GFP-Utr bound to 324
actin filaments with an approximate filament length of ~200nm (Gowrishankar et al., 325
2012). Treatment of cells with the formin inhibitor SMIFH2, resulted in a loss of only the 326
slow diffusing component (Figure S4F, G). This coincides with the loss of the dynamic 327
pool of actin filaments that is likely to mediate the nanoclustering of membrane proteins 328
(Saha et al., 2015). The nanoclustering of the chimeric transmembrane receptor 329
(FRTM-Ez-AFBD) was also abrogated upon inhibition of formins as well as SFK/FAK on 330
FN (Figure 4G, H). 331
Next we tested the role of integrin-stimulated ROCK activation in nanoclustering of 332
GPI-APs. RhoA via ROCK can stimulate myosin light chain (MLC) phosphorylation 333
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directly or indirectly through the inhibition of MLC phosphatase. Inhibition of ROCK 334
using the Y-27632 inhibitor (Uehata et al., 1997) results in loss of nanoclusters of GPI-335
APs when cells are plated on FN (Figure S4H, I) as does treatment with the MLC 336
kinase (MLCK) inhibitor ML-7 (Figure S4H, I). 337
Together these data provide evidence that integrin signaling mediated by src and 338
FAK kinases through active RhoA regulates actin polymerization via formins. This 339
couples integrin ligation to the generation of nanoclusters. The role of myosin activity in 340
promoting nanoclustering indicates that signaling activates a dynamic acto-myosin 341
machinery to promote the nanoclustering of GPI-anchored and other actin-filament 342
binding domain (AFBD) containing proteins. 343
Talin and vinculin are necessary for the generation of GPI-AP nanoclusters. 344
To further understand the mechanism of GPI-AP nanocluster generation, we tested the 345
role of focal adhesion proteins, talin and vinculin, that form an integral part of the 346
mechano-chemical signal transduction machinery downstream of integrin engagement 347
(Vicente-Manzanares et al., 2009). Anisotropy measurements on vinculin knock out 348
(vin-/-) mouse embryonic fibroblasts (MEFs) (Janoštiak et al., 2014) transfected with 349
GFP-GPI, and freshly plated on FN showed a relatively high anisotropy value, which 350
was unaffected by treatment with mβCD (Figure 5A,B). This indicates that cholesterol-351
sensitive nanoclusters do not form without vinculin. To restore vinculin function, we 352
transiently transfected full-length mCherry-tagged vinculin (Thievessen et al., 2013a) 353
into vin-/- MEFs re-plated on FN and measured anisotropy of co-transfected GFP-GPI. 354
Re-introduction of vinculin restored cholesterol–dependent GPI-AP nanoclustering 355
ascertained by decreased GFP-GPI anisotropy that was sensitive to cholesterol removal 356
(Figure 5A, B). These data indicate that the loss of nanoclustering in vin-/-cells was 357
only due to the absence of vinculin, providing a convenient test bed to explore the role 358
of vinculin in GPI-AP nanoclustering. 359
Vinculin exists in an auto-inhibited state in cells, and is activated by several 360
interacting molecules, which bind to specific domains (Figure S5C). A well-361
characterized mode of activation of vinculin is through the binding of its head domain to 362
talin, opening up the tail domains for interaction with actin and lipids (Case et al., 2015; 363
Golji and Mofrad, 2010). We first addressed the role of talin in nanoclustering of GPI-364
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APs, using talin1 knockout MEFs. Since loss of talin1 leads to over expression of the 365
talin2 isoform (Zhang et al., 2008), we additionally depleted these cells of talin2 with 366
talin2-shRNA co-expressed with GFP (Figure S5A, B). We monitored the 367
nanoclustering of GPI-APs in these cells using a fluorescently-tagged oligomerization-368
defective aerolysin variant (A568-FLAER) previously characterized to report on the 369
native distribution of endogenous GPI-APs (Raghupathy et al., 2015). A568-FLAER 370
exhibited a higher fluorescence emission anisotropy in the talin2 shRNA expressing 371
cells compared to the talin-1 alone deficient cells (Figure 5C, D), consistent with a loss 372
of nanoclustering of GPI-anchored proteins in talin1-/- cells after talin2 depletion. 373
However, this increase was less than that observed for the loss of vinculin; the partial 374
loss of talin2 (as indicated by immunostaining for talin2) could serve as a confounding 375
factor in these experiments (Figure S5A, B). 376
Expression of Vin-A50I, which is incapable of binding talin (Case et al., 2015; 377
Figure S5C) in vin-/-MEFs, fails to support GPI-AP nanoclustering (Figure 5E, F). 378
However, a constitutively activated vinculin, Vin-A50I-CA, which does not require talin 379
for its activation (Case et al., 2015) restored GPI-AP nanoclustering in the vin-/- MEFs 380
(Figure 5E, F). Together these results suggest that GPI-AP nanoclustering normally 381
requires vinculin activation by talin. 382
Vinculin activation specifically links integrin signaling and GPI-AP nanoclustering 383
We next asked if vinculin is necessary for triggering the actomyosin-based clustering 384
machinery downstream of integrin activation. We examined the status of nanoclustering 385
of FRTM-Ez-AFBD in vin-/- cells (Figure 5G, H). Monitoring anisotropy of FRTM-Ez-386
AFBD and the FRTM-Ez-AFBD* mutant constructs indicated that the nanoclustering 387
mechanism is unaffected in the absence of vinculin; FRTM-Ez-AFB exhibited a lower 388
anisotropy value compared to the FRTM-Ez-AFBD* in vin-/-cells as well as in the 389
vinculin restored cells (Figure 5G, H). Furthermore, the restoration of GPI-AP 390
nanoclustering observed in vin-/- MEFs by the expression of vinculin is completely 391
disrupted upon pre-treatment with the src family inhibitor, PP2, or FAK inhibitor, PF 392
573228 or the formin inhibitor, SMIFH2 (Figure 6A, B). GPI-AP nanoclustering is not 393
restored by treatment of cells with the formin activator, IMM01 in the vin-/- cells. By 394
contrast, transmembrane protein clustering is brought about upon integrin activation in 395
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these cells, indicating that vin-/- cells are not defective in generating the acto-myosin 396
machinery responsible for clustering. Addition of an artificial linker (LactC2-Ez-AFBD) 397
(Raghupathy et al., 2015) is able to fully restore the nanoclustering of GPI-APs, in the 398
vin-/- cells. This indicates that vinculin activation is not necessary for the creation of the 399
acto-mysoin machinery but is rather involved in the pathway that links actin to the inner-400
leaflet lipids (Figure 5I, J). 401
Lipid and actin binding capacity of vinculin are necessary for GPI-AP 402
nanoclustering 403
Vinculin possesses a negatively charged lipid binding site in its tail domain and mutation 404
of this site results in a loss in its ability to bind to negatively charged lipids in the 405
membrane (Humphries et al., 2007). Importantly, expression of this Vin-Ld mutant that 406
lacks lipid-binding capacity (Figure S5C) in vin-/- MEFs failed to restore GPI-AP 407
nanoclustering (Figure S6A, B). To determine if the failure to bind lipids, keeps vinculin 408
in an inactive state, we generated Vin-Ld-CA*, that is constitutively activated 409
(Humphries et al., 2007) (Figure S5C). This mutant also failed to restore GPI-AP 410
nanoclustering in vin-/- MEFs (Figure S6A, B). These results indicate that the lipid 411
binding capacity of vinculin is necessary for it to catalyze GPI-AP nanocluster formation 412
and accounts for the mechanistic differences between the nanoclustering of GPI-APs 413
and those of transmembrane proteins with actin binding motifs. 414
To assess if the actin-binding capacity of vinculin is necessary to bring about GPI-415
AP nanoclustering, we expressed a mutant version of vinculin which has reduced 416
capacity to bind to actin (Case et al., 2015) Vin AB1 (Figure S5C). Even though this 417
mutant of vinculin localizes to focal adhesions, it failed to rescue nanoclustering of GPI-418
APs consistent with the role of the actin binding domain of vinculin in GPI-AP 419
nanoclustering (Figure 6E, F). 420
Cells defective in the GPI-AP nanoclusters formation exhibit aberrant integrin 421
function. 422
Cells that lack vinculin have defective integrin mediated responses; they lack the P1 423
phase of integrin mediated spreading and exhibit aberrant FAs (Figure S7 H-I; see also 424
(Thievessen et al., 2013)). We hypothesized that some of these defects may be a 425
consequence of the inability of cells to build functional nanoclusters. To test this, we 426
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utilized mutant cells that are deficient in two enzymes (PGAP2 and PGAP3) required for 427
the remodeling of the unsaturated GPI-anchor acyl chains to long, saturated chains. 428
This defect results in the inability of PGAP2/PGAP3 mutant cells to make GPI-AP 429
nanoclusters (Raghupathy et al., 2015), and inefficient GPI-AP incorporation into 430
detergent-resistant membranes (Maeda et al., 2007). 431
When freshly plated on fibronectin, these mutant cells failed to exhibit a decrease 432
in the anisotropy of GFP-GPI and this defect could be reversed by restoring the 433
activities of the PGAP2 and PGAP3 enzymes (Figure S7A, B). In comparison with 434
either wild type cells or mutant cells rescued with wild type copies of PGAP2 and 435
PGAP3, the PGAP2/PGAP3 mutant cells lack the P1 spreading phase when plated on 436
substrates coated with fibronectin (Figure 6A, B). These mutant cells also lack a 437
protrusive lamellipodia and possess fewer smaller adhesions (Figure S7F-G) and 438
exhibit bleb-based cell spreading (Supplementary Movie 2). They also do not exhibit a 439
rapid increase in cell area when spreading on FN, characteristic of the P1 phase 440
(Figure 7C; Supplementary Movie 3). This defect is not due to defects in integrin 441
activation in the mutant cells, since antibodies that bind to either active or in-active 442
conformations of the β1-integrins bind equivalently to the mutant and wild type cells 443
(Figure S7C). 444
Several proteins that either create or reside within lo-like regions on the cell 445
membrane have been implicated in the process of cells spreading and migration 446
(Moissoglu et al., 2014; Navarro-Lérida et al., 2012). Recently we have shown that GPI-447
AP nanoclusters are associated with specific regions of the membrane that have an lo-448
like character (Saha et al, manuscript under preparation). Therefore, we tested if the 449
lack of GPI-AP nanoclusters in the PGAP2/3 mutants could lead to a global disruption of 450
ordered domains. Using the polarity-sensitive membrane dye Laurdan (6-lauryl-2-451
dimethylamino-napthalene) as a reporter of membrane order (Owen et al., 2012), we 452
found that the PGAP2 and PGAP3 mutant cells have a lower generalized polarization 453
(GP) value (Figure 7 D-E), which is consistent with the loss of ordered lo domains on 454
the cell surface. The decrease in the GP value was similar to that observed when 455
membrane cholesterol was depleted using mβCD treatment, and is fully restored in the 456
PGAP-2/3 add-back cell line (Figure 7D-E). To further confirm that the loss of lo 457
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domains is due to a specific defect in GPI-AP nanoclustering and not due to global 458
alterations of cholesterol or phospholipid composition of the plasma membrane, we 459
compared the levels of filipin-labeled cholesterol and performed mass-spectrometric 460
measurements on blebs extracted from them. We do not find any significant difference 461
in the levels of either membrane cholesterol or phospholipid profile of the mutant cells 462
(Figure S7 D-E; Supplementary Table S3). This suggests that the lack of GPI-AP 463
nanoclustering specifically contributes to the loss of lo-domains at the cell surface and 464
could account for the observable cell spreading defects. 465
466
DISCUSSION 467
Our results using both chemical and genetic perturbation show how GPI-AP 468
nanoclustering is initiated via a signaling cascade triggered by β1-integrin receptors 469
upon binding to its bonafide ligands: fibronectin (FN) or the fibronectin-derived peptide 470
RGD (see model in Figure 7F). Ligand binding results in the activation of the src and 471
FAK kinases. Potentially this step may involve activation of additional molecules 472
including ILK and kindlin-kinases (Calderwood et al., 2013). Regardless, downstream of 473
the kinases are the RhoA GTPases (Ishizaki et al., 1996; Leung et al., 1996), which 474
directly activate formins, necessary for nanoclustering. The nucleator of branched actin 475
filaments, Arp2/3, was not required for this process. Knockdown of both FHOD1 and 476
mDia via RNAi-mediated depletion inhibited nanoclustering of GPI-APs, although the 477
effect of FHOD1 depletion was more drastic. RhoA via ROCK activates FHOD1 through 478
the phosphorylation of C-terminal serine/threonine residues in its DAD region thereby 479
relieving its auto inhibition (Takeya et al., 2008). FHOD1 is also recruited to nascent 480
sites of integrin ligand engagement (Changede et al., 2015; Iskratsch et al., 2013), 481
implicating formins in effecting the nanoclustering of GPI-APs during early stages of 482
integrin mediated signaling. Consistent with this, we found that GPI-AP nanoclusters 483
were also formed on the supported lipid bilayer system, in the vicinity of integrin 484
clusters. Together with previous observations that the integrin LFA1-binding to its ligand 485
ICAM-1 results in a local concentration of GPI-AP nanoclusters, (van Zanten et al., 486
2009), these results show that integrin signaling generates a localized nanoclustering 487
response. 488
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In parallel, this mechanism requires a way to control myosin function. Indeed, 489
RhoA activates ROCK that regulates myosin light chain kinase (MLCK), and 490
perturbation of ROCK (via Y27632) and MLCK (via ML-7), inhibited actin-based 491
nanoclustering of GPI-APs. However, we do not exclude the possibility of this being a 492
myosin II independent ROCK or MLCK effect. Ectopic activation of RhoA via the agonist 493
CNO3 was sufficient to generate the necessary machinery for creating the GPI-AP 494
nanoclusters, independent of receptor signaling and despite the inhibition of the 495
upstream signaling cascade. Once generated, this actin machinery was also sufficient 496
to cluster transmembrane proteins with actin-binding capacity, exemplified by the model 497
transmembrane protein, FRTM-Ez-AFBD. Transmembrane proteins with actin-binding 498
motifs directly associate with the dynamic acto-myosin machinery, whereas GPI-APs at 499
the outer leaflet, require transbilayer interactions with long acyl-chain containing lipids 500
such as PS at the inner leaflet (Raghupathy et al., 2015). This in turn might require an 501
entirely different mechanism to connect to the actin machinery in the cortex. 502
Vinculin is known to be activated downstream of integrin signaling through the 503
activation of talin, and has several binding partners such as actin, paxillin and negatively 504
charged lipids like PS and PIP2 (Niggli et al., 1986). The role of these two proteins in 505
supporting GPI-AP nanoclustering was verified by the depletion of talin and vinculin in 506
MEFs, wherein GPI-AP nanoclustering was disrupted. The observation that 507
nanoclusters of TM-ABDs in vin-/- null cells were formed without any alteration implies 508
that the integrin receptor recruits distinct molecular players to facilitate a link between 509
the dynamic actin machinery and membrane lipids. Our results indicate a role for 510
vinculin as a molecular player which may direct actin to the membrane or serve as a 511
linker in connecting the inner leaflet to actin. The failure of the lipid and actin binding 512
mutants to restore GPI-AP nanoclustering support the latter hypothesis. However, 513
vinculin was not found measurably enriched at the membrane outside of focal 514
adhesions when examined at time points where GPI-AP nanoclustering is restored in 515
vin-/- cells (Figure S6C, D), supporting the former. 516
These results provide a molecular mechanism for the control of an active actin-517
membrane composite, wherein the fluid membrane is inextricably coupled to the cortical 518
actin-substructure beneath (Köster and Mayor, 2016; Rao and Mayor, 2014). The 519
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functioning of this composite implies the existence of three types of membrane 520
components: inert, passive and active. While we have previously described inert and 521
passive components and their characteristics (Gowrishankar et al., 2012), here we 522
provide evidence for the functioning of an active element, exemplified by the integrin 523
receptor family. 524
The relevance of GPI-AP nanoclustering in membrane function has been difficult 525
to probe because of the use of drastic perturbations such as cholesterol removal (Kwik 526
et al., 2003) or alterations in specific phospholipid levels (Lipardi et al., 2000). The 527
identification of a molecular mechanism behind the generation of these nanoclusters, 528
and the key role of integrin signaling in cell spreading provides an opportune 529
physiological context. Pertinently, many of the key components of the nanoclustering 530
molecular machinery identified here such as src, FAK, RhoA, formin, myosin, talin and 531
vinculin, are in the pathway of integrin-mediated signaling, and also have multiple roles 532
in cell physiology. Therefore, the well-documented cell spreading defects and alteration 533
in focal adhesion patterns that are exhibited by perturbations of these players, may be 534
difficult to directly relate to nanoclustering defects. As a consequence, we explored the 535
role of nanoclustering in membrane function by studying defects in integrin-mediated 536
functions in the GPI-anchor remodeling mutants. These mutants lack the ability to make 537
GPI-AP nanoclusters but they support FA-formation as well as integrin-mediated 538
activation. However, they exhibit dramatic defects in cell spreading that are restored 539
upon restoration of the cell’s ability to support nanocluster formation, similar to those 540
observed in vin -/- cells. This implicates a functional role for GPI-AP nanoclustering in 541
integrin-mediated signaling. 542
Why does signaling via integrin-ligation target the local construction of GPI-AP 543
nanoclusters? An answer to this, is related to the fact that GPI-AP nanoclusters form lo 544
nanodomains (Raghupathy et al., 2015) which in turn generate larger meso scale lo 545
domains (Saha et al , manuscript in preparation). Here we show that the loss of GPI-AP 546
nanoclustering results in the failure to enhance the overall lo characteristics of the 547
membrane observed upon integrin-mediated signaling. Coupled with the observation 548
that large cross-linked patches of GPI-APs accumulate src family kinase members at 549
the inner leaflet (Harder et al., 1998; Stefanová et al., 1991b; Suzuki et al., 2007), these 550
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results suggest a function for the lo-like GPI-AP nanocluster rich-regions in effecting 551
integrin signaling responses. Lipid modifications such as palmitoylation enable 552
molecules to partition into locally generated lo micro-environments. These membrane 553
domains are also likely to be important for the signaling activity of SFK and FAK (Seong 554
et al., 2011). Rac1 is a palmitoylated Rho family GTPase that regulates leading edge 555
protrusion dynamics, and its activity is restricted to lo domains (Moissoglu et al., 2014; 556
Navarro-Lérida et al., 2012; del Pozo et al., 2004). Moreover, the GAP activity of the 557
p190RhoGAP is also localized to potentially lo domains (Sordella et al., 2003) and its 558
recruitment to lo-like regions has been shown to be necessary for the cell spreading 559
process (Arthur and Burridge, 2001), as well as for the localized inhibition of the Rho 560
GTPase and the regulation of FA size. Palmitoylation of the fyn kinase, implicated in 561
rigidity sensing, is also required for the P1-based cell spreading process (Kostic, 562
2006).Thus, it is likely that the lo microenvironment created by the mechanism proposed 563
here, could serve to localize a number of important components of the effector cascade 564
in integrin-based activity to the leading edge where such sensing takes place. 565
Since the activation of the small GTPase, RhoA, is a pivotal feature downstream of 566
many signaling receptors besides integrins, such as Cadherins, RTKs, GPCRs (Olson 567
and Nordheim, 2010), this will likely culminate in the activation of such an acto-myosin-568
based mechanism as described here. Vinculin is also a downstream effector of many 569
signaling based systems (Hazan et al., 1997), ensuring that these dynamic acto-myosin 570
filaments also generate GPI-AP nanoclusters. The resultant membrane domains that 571
ensue, will serve as allosteric modulators of the output of the signaling system that 572
generates it (Harding and Hancock, 2008). This will naturally allow the cell to integrate 573
information that is encoded primarily in the composition of its membrane bilayer. In 574
conclusion, our results suggest a generalizable picture of how lo nanodomains may be 575
created and deployed in the context of a number of different signaling systems. 576
577
578
579
580
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AUTHOR CONTRIBUTIONS 581
J.M.K., A.A.A., and S.M designed the study. J.M.K, A.A.A set up the EA-TIRFM 582
microscope, performed experiments, and analysed the data; C.P performed the lipid 583
ordering experiments and filipin-labelling experiments; J.M.K also standardized the 584
supported lipid bilayer experiments working in collaboration with M.P.S laboratory. 585
A.A.A also performed the mass spectrometric experiments; T.S.V.Z performed and 586
analysed the FCS measurements and analysed the data involving the cell spreading 587
experiments. J.M.K., A.A.A., and S.M. drafted the manuscript with input from all the 588
authors. 589
ACKNOWLEDGMENTS 590
We thank Cheng-han Yu for help in standardizing the use of the supported-lipid bilayer 591
system; Kabir Husain and Balaji for Matlab codes to analyze the anisotropy and bilayer 592
data; Taroh Kinoshita, Yusuke Maeda, Daniel Rosel, Clare M. Waterman, Lindsey 593
Case, Ana Pasapera for their generous gifts of various reagents (as indicated in the 594
Supplemental Information); Max Planck-NCBS Lipid centre; Bini Ramachandran for 595
mass spectrometry; and H. Krishnamurthy and Manoj Mathew at the Central Imaging 596
and Flow Facility (NCBS). We thank Madan Rao, and Subhasri Ghosh for inspiration 597
and SM lab members for their critical comments on the manuscript. J.M.K. 598
acknowledges pre-doctoral fellowship from NCBS-TIFR. A.A.A acknowledges N-PDF 599
fellowship from DST-SERB (Government of India). T.S.V.Z. acknowledges EMBO 600
fellowship (ALTF 1519-2013) and NCBS fellowship. S.M. acknowledges JC Bose 601
Fellowship from DST (Government of India), a grant from HFSP RGP0027/2012 and 602
Wellcome Trust-DBT Alliance Margadarshi fellowship. 603
604
605
606
607
608
609
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FIGURE LEGENDS 889
Figure 1. Activation of fibronectin binding integrins leads to enhanced 890
nanoclustering of GPI-APs. 891
(A) Cartoon illustrates the assay used in this study. Serum-starved (3-8 hrs) fibroblastic 892
cells were de-adhered and re-plated back under serum-free conditions either on 893
Fibronectin (FN)–coated or 1%BSA coated glass-bottom coverslips. The extent of 894
nanoclustering of fluorescent protein-tagged GPI-APs expressed in these cells was 895
monitored by measuring the fluorescence anisotropy using an Emission Anisotropy 896
TIRF microscope (EA-TIRFM). Inset is the schematic representation of GFP or YFP 897
tagged GPI-APs localized to the outer-leaflet of the plasma membrane.(B-C) Intensity 898
and steady state anisotropy images (B) and anisotropy versus intensity plot (C) of GFP-899
GPI in live cells plated on 1%BSA (red) or on 10µg/ml FN (blue) or plated on FN and 900
subsequently treated with 10mM of the cholesterol depleting agent methyl-β-901
cyclodextrin (mβCD) (green) and collected using EA-TIRFM. (D) Box plot representing 902
the mean anisotropy values of GFP-GPI in CHO cells, plated on glass-bottom coverslips 903
coated with 1% BSA (red) or FN (blue) at the indicated concentrations before (blue) or 904
after (green) treatment with mβCD. (E-F) Intensity and steady state anisotropy images 905
(E) at the initial time point (I/Io =1) and at 50% photobleaching (I/Io=0.5) and graph 906
demonstrating the change in the fluorescence anisotropy upon photo-bleaching of YFP-907
GPI (F) expressed in CHO cells plated on glass (red) or on 10µg/ml FN (blue) or plated 908
on FN and subsequently treated with mβCD (green), plotted against the fluorescence 909
intensity value (I) normalized to its value before photobleaching (I0). Note that the 910
starting anisotropy value of YFP-GPI in cells plated on glass was higher than that on 911
FN. The anisotropy increased upon photobleaching to attain a final anisotropy value 912
corresponding the value obtained in cells after cholesterol depletion (green horizontal 913
bar). This value likely represents the anisotropy value of YFP-GPI-monomers (A∞). (G-914
J) Intensity and steady state anisotropy images (G, H) and anisotropy versus intensity 915
plot (I, J) of CHO-K1 cells labeled for 3 hours with NBD-GPI (I; GPI analogue, exoGPI) 916
or C6NBD-SM (J; short chain SM; scSM), de-adhered and plated for 1 h at 37°C on 917
glass (red) or 10µg/ml FN (blue) and imaged on EA-TIRFM. Note the fluorescence 918
anisotropy of C6-NBD-SM does not change, whereas exogenously incorporated NBD-919
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GPI exhibits a lower anisotropy when cells are plated on FN compared to glass alone. 920
Scale bar 10 µm. Error bars represent the SD. See also Figure S1 921
Figure 2. Activation of RGD binding integrins leads to enhanced nanoclustering 922
of GPI-APs in its local vicinity. 923
(A-C) CHO cells stably expressing GFP-GPI were treated as described in Figure 1A and 924
re-plated on FN (10µg/ml)-coated glass-bottom cover slips, to observe the temporal 925
evolution of cell spreading and nanoclustering of GFP-GPI as monitored by EA-TIRFM. 926
Images (A) show snapshots of GFP-GPI Intensity and steady state Anisotropy at the 927
indicated times, post settling on FN. Kymograph (3 pixel average) (B) of intensity (top) 928
and anisotropy (bottom) of a line drawn perpendicular to the cell edge (yellow line in A) 929
showing the three phases of cell spreading and correlative changes in the anisotropy of 930
GFP-GPI. Note that the rapid decrease in anisotropy (increase in the nanoclustering) 931
occurs at the transition between P0-P1 phase. Graph (C) shows the change in area 932
(δArea;red curve) correlated to changes in GFP-GPI anisotropy (δAnisotropy;blue 933
curve) as a function of time. Note that the peak change in fluorescence anisotropy (blue 934
curve) occurs about 100-200 seconds before the peak increase in cell spread area. 935
Data depicts mean change in whole cell anisotropy values and cell spread area 936
between two consecutive frames (15s) of 11 cells measured at the indicated spreading 937
time; Data has been aligned relative to the timing of the peak area change. (D,E) 938
Representative snapshot taken from region of interest (2 µm X 2 µm) over time of CHO 939
cells expressing GFP-GPI plated (right panel) on RGD-functionalized SLB taken 940
sequentially, as described in Figure S 2C. Note the correlation between RGD cluster 941
intensity as reported with DyLight 650 labeled neutravidin (RGD; top panel; see also 942
Figure S 2G) and the GFP-GPI steady state anisotropy (Anisotropy; middle panel) 943
imaged sequentially in an Emission anisotropy –equipped spinning disc confocal 944
microscope; GFP-GPI intensity (bottom panel), shows no correlated patterns. Scale bar, 945
5 µm for whole cell image and 1 µm for ROI. (E-F) Intensity and steady state anisotropy 946
images (E) and box plots with mean anisotropy (F) of GFP-GPI expressing CHO cells 947
plated on Glass for 2-days without (0 µM cRGD; blue) or with indicated concentrations 948
of soluble RGDfV (green) for 30 mins at 37 °C , or with 10mM mβCD for 45 min at 37 °C 949
and imaged using EA-TIRFM. Higher concentrations of cRGD (>100 µM) induced cell 950
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rounding and detachment. Correspondingly the cells also exhibited a higher anisotropy 951
value. Scale bar 10 µm. Error bars represent SD. See also Figure S2. 952
Figure 3. Inhibition of SFK/FAK, Rho GTPase and formins leads to loss of FN-953
triggered nanoclustering of GPI-APs. (A-F) Intensity and steady state anisotropy 954
images (A, C, E) and intensity versus anisotropy plots (B, D, F) of GFP-GPI expressing 955
CHO cells plated on FN-coated or non-coated (Red; Glass) glass-bottom dishes imaged 956
via EA-TIRFM. The emission anisotropy of GFP-GPI was determined in cells pre-957
treated with 20 µM src family tyrosine kinase inhibitor PP2 (+PP2; green in A-B), 10 µM 958
focal adhesion kinase inhibitor PF-573-228 (+PF-573228; magenta in A-B) or both 959
(+PP2+PF 573 228; black in A-B), pretreatment (2 h) with indicated concentrations of 960
the cell-permeable Rho inhibitor exoenzyme C3 transferase (+C3; red in C-D), 10µM 961
formin inhibitor (+SMIFH2;magenta in E-F), or 10µM formin agonist (+IMM01; green in 962
G-H). Note in all cases, treatment with inhibitors increased the emission anisotropy of 963
GFP-GPI consistent with a failure of FN-engagement to enhance nanoclustering, 964
whereas activation of formin via IMM01 agonist, decreases emission anisotropy of GFP-965
GPI on cells plated on glass (green in G-H), in the absence of FN. (G, H) Intensity and 966
steady state anisotropy images (G) and plot of anisotropy versus intensity (H) of GFP-967
GPI expressing CHO cells pre-treated and plated on 10µg/ml FN in presence of SFK-968
FAK inhibitor (20µM PP2+10µM PF-573 228) without (black in G-H) or with 10µg/ml 969
RhoA activator (+CN03; green in G-H) or with 10µM formin agonist (+IMM01; blue in G-970
H), or with RhoA activator CN03 and 10µM formin inhibitor (+SMIFH2;red in G-H) and 971
imaged via EA-TIRFM. Note that the RhoA or formin activators reverse the effects of 972
SFK-FAK inhibition. The RhoA activator reversal remains sensitive to inhibition via the 973
formin-inhibitor (SMIFH2) indicating formins operate downstream of RhoA activation. 974
Scale bar 10 µm. Error bars represent SD. See also Figure S3. 975
Figure 4. Integrin activation induces changes in dynamic actin activity at the 976
cytoplasmic leaflet. 977
(A) Schematic of model transmembrane protein with the extracellular region derived 978
from the folate binding protein (FBP), transmembrane segment of IgG-Fc receptor (IgG-979
FcR TM) and an actin binding domain derived from the ezrin protein (FR-TM-Ez-AFBD) 980
and (D) a mutated version of the same construct as in A, FR-TM-Ezrin-R579A (FR-TM-981
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Ez-AFBD) that renders the Ez-AFBD domain incapable of interacting with actin. B-H) 982
Intensity and steady state anisotropy images (B, E, G) and plots of anisotropy versus 983
intensity (C, F, H) of CHO cells stably expressing FRTM-Ez-AFBD (B, C, G, H) or 984
FRTM-Ez-AFBD* (E,F) labeled with fluorescent folic acid analogue, Pteroyl-lysyl-985
BodipyTM(PLB), for 3 hours at 37°C and re-plated either on FN-coated (blue in C, F, H) 986
or uncoated (red in C, F) glass-bottom coverslip dishes and imaged via EA-TIRFM. In 987
panel G and graph H, cells were re-plated after pre-treatment with only DMSO (blue in 988
G-H), or 20µM PP2 and 10µM PF-573228 (red in G-H), or 10µM SMIFH2 (green in G-989
H). The difference in values of anisotropy of PLB-labelled FRTM-Ez-AFBD in G, 990
compared to B is due to the use of a different EA-TIRFM imaging station, equipped with 991
different NA optics. Error bars represent SD. See also Figure S4. 992
Figure 5. Talin and vinculin are required for facilitating GPI-AP nanoclustering in 993
mouse embryonic fibroblasts (MEFs) 994
(A-J) Intensity and anisotropy images of vinculin deficient (vin-/-; A, B, E-J) or talin 1 995
deficient (Talin1-/-) MEFs (C-D) either transfected with GFP-GPI (A, B) or labeled with 996
Alexa-568-FLAER (FLAER; C-F, I-J) or PLB (pteroyl lysine conjugated to Bodipy-TMR; 997
G, H) and imaged via EA-TIRFM after re-plating the cells on FN-coated glass-bottom 998
dishes. Cells were also transfected with mcherry-vinculin (A, B), Talin2 shRNA (C, D), 999
GFP-vinculin (Vin-WT; E, F),the indicated vinculin variants (E-F), FREZ , FREZ* (G, H) 1000
or Lact C2 Ez (I, J) 12-16 hours prior to being taken for imaging. mCherry vinculin 1001
(mCh-Vin; red in B) Talin2 shRNA (black in D) and Lact C2 Ez (green in J) expressing 1002
cells were additionally treated with mβCD. Transfected cells are marked by dotted 1003
magenta lines. Scale bar 10 µm. Error bars represent SD. See also Figure S5. 1004
Figure 6. Vinculin facilitates GPI-AP nanoclustering in an integrin signaling 1005
sensitive manner. 1006
(A-D) Intensity and anisotropy images (A, C) and the intensity versus anisotropy plot (B, 1007
D) of GFP-GPI expressing MEFs in the presence (A,B) or absence (C, D) of vinculin 1008
treated with various inhibitors like SMIFH2, PP2, PF573228 and IMM01 that interferes 1009
with the integrin signaling pathway that generates dynamic actin. Note that an increase 1010
in anisotropy was observed when cells were treated with SMIFH2 (red (A, B) or PP2 1011
and PF573228 in the presence (maroon; A, B) or absence of vinculin (red; C, D) as 1012
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indicated and a decrease in anisotropy was observed on treatment with a cocktail of 1013
PP2, PF573228 and IMM01 (black) in the presence of vinculin (A, B), but an increase in 1014
anisotropy was observed on treating cells with a cocktail of PP2, PF573228 and IMM01 1015
(black) in the absence of vinculin (C, D). (E-F) Intensity and anisotropy images (E) and 1016
the intensity versus anisotropy plot (F) of mRuby-GPI expressing vin-/- MEFs transiently 1017
transfected with Vin WT (black), Vin AB1 (green) or Vin AB1 CA (red). An increase in 1018
anisotropy was observed when cells were transfected with Vin AB1 and Vin AB1 CA 1019
compared to Vin WT suggesting a role for vinculin’s actin binding domain in GPI-AP 1020
nanoclustering. Error bar represent SD. See also Figure S6. 1021
Figure 7. Activity generated GPI-AP nanoclusters are necessary for efficient cell 1022
spreading. (A-B) Phase contrast images (A) and corresponding area versus time plot 1023
(B) showing the cell spreading dynamics of wild type (blue), PGAP2/3 mutant (red) or 1024
PGAP2/3 add back in mutant (Rescue; green) CHO cells or WT cells treated with 10mM 1025
mβCD (orange). Each data point is an average of cell spread area of approximately 100 1026
cells per time point. Error bars represent the SD. (C) Plot of change, between two 1027
consecutive frames of 15s, in cell spread area (δArea) as a function of cell spreading 1028
time on FN for the wild type (blue curve), PGAP2&3 mutant (red curve) and rescue 1029
(green curve) in the absence or presence of 10mM mβCD for 30 mins in suspension 1030
and during subsequent plating on FN. The data from individual cells was combined by 1031
aligning the peak area change (characteristic of the P1 phase) that occurs during 1032
individual cell spreading process. Note that the PGAP2&3 mutants and mβCD-treated 1033
WT cells lack the rapid increase in cell area characteristic of the P1 phase, consistent 1034
with the inability of these cells to make a lamellipodia (Supplementary Movie 3). (D-E) 1035
Laurdan total intensity and GP images and (E) Box plot representing the mean 1036
generalized polarization (GP) values of WT cells (blue) or PGAP2&3 mutants (red) or 1037
PGAP2&3 add-back (Rescue;green) or WT cells treated with 10mM mβCD. The lower 1038
values of GP observed in the PGAP2&3 mutants indicate the lack of lo-domains similar 1039
to that observed when WT cells were treated with mβCD, a well-characterized 1040
perturbant of lo-domains. Error bar indicates SD. 1041
(F) Model for the integrin signaling triggered generation of nanoclustering of GPI-APs. 1042
Integrin ligation triggers a cascade of biochemical signaling events leading to the 1043
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activation of RhoA via SFK-FAK kinases. This culminates in the activation of the formin 1044
class of nucleators that generate dynamic actin filaments along with RhoA-ROCK 1045
induced myosin contraction. Vinculin that is activated by talin downstream of integrin 1046
activation, either directly (i) or through the activation of additional adapters (ii; X) links 1047
the dynamic actin filaments to PS lipids at the inner-leaflet. This in turn couples to the 1048
GPI-anchored proteins at the outer-leaflet via a trans-bilayer coupling mechanism. The 1049
nanoclusters of GPI-anchored proteins that result from this active mechanism contribute 1050
to proper integrin function. Scale Bar 250µm (A) 20µm (inset),10µm (D). Error bar 1051
represents SD. See also Figure S7. 1052
1053
STAR METHODS 1054
Detailed experimental conditions are provided in the Extended Experimental 1055
Procedures in the Supplemental Information. 1056
Plasmids, Cell Lines, Antibodies and Other Reagents 1057
CHO cells stably expressing EGFP-GPI, Human U2OS cells stably expressing mEGFP-1058
GPI, FR-TM-Ez-AFBD and FR-TM-Ez-AFBD* (RA mutant) cell line, vinculin and Talin 1 1059
deficient mouse embryonic fibroblasts (MEF) and PGAP2/3 double mutant cell lines (of 1060
CHO origin) stably expressing CD59 and DAF was maintained in culture media as 1061
indicated in the extended experimental procedures with the appropriate antibiotics. The 1062
constructs used in the study were procured from various sources as indicated in the 1063
extended experimental procedures. 1064
GPI analogue incorporation: GPI analogues are incorporated into cell membranes by 1065
γ−CD method as described (Koivusalo et al., 2007; Riya Ragupathy doctoral thesis 1066
(http://hdl.handle.net/10603/77067). 1067
Preparation of RGD functionalized Supported Lipid Bilayers (SLBs): 1068
Supported lipid bilayers functionalized with cRGD was prepared based on the published 1069
protocol (Yu et al., 2011). 1070
FCS: Fluorescence correlation spectroscopy (FCS) measurements were performed as 1071
described previously (Gowrishankar et al., 2012). 1072
1073
Mass Spectrometry experiments 1074
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Briefly, Cells were treated with cytochalasin D in serum free media followed by 1075
centrifugation; the collected pellet (membrane blebs) was subjected to lipid extraction 1076
using chloroform/methanol solvent system (Raghupathy et al., 2015). The lipid extract 1077
thus obtained was subjected to LC/MS by performing mass spectrometric analysis on Q 1078
Exactive instrument (Thermo Fisher scientific) (Ramachandran et al, Manuscript in 1079
preparation) 1080
Anisotropy measurements: 1081
Steady state Emission anisotropy-total internal reflection fluorescence microscopy (EA-1082
TIRFM) (Swaminathan et al., 2017). Homo-FRET based anisotropy measurements 1083
were carried out on a NikonTE2000 microscope with polarized laser excitation and fitted 1084
with an 100x 1.49 NA TIRF objective with a dual camera imaging arrangement as 1085
described earlier (Ghosh et al., 2012). Confocal based anisotropy measurements were 1086
acquired on a custom-designed NikonTiE microscope coupled to a Yokogawa CSU-22 1087
spinning disc unit (Yokogawa) as described (Ghosh et al., 2012). 1088
1089
Cell spreading assay : 1090
Briefly, serum-starved cells were de-adhered and plated on pre-cleaned, pre-coated 1091
tissue culture dishes under serum-free conditions. After 5 mins of initial cell adherence, 1092
the unbound cells were washed off and the dishes were transferred back to 37°C/5% 1093
CO2 incubator. The cells were imaged in phase contrast with a 20X objective at each of 1094
the indicated time points. For quantification of cell-spread area, the cells were marked 1095
manually and the mean cell area was extracted using the ROI manager tool in Fiji. 1096
Statistical Analysis: 1097
Unless otherwise indicated each experimental condition was performed as technical 1098
replicates (with at least two dishes per experiment) and data was quantified from >200 1099
(~0.2X0.2µm) region of interest (ROI) taken from 20-30 cells for the anisotropy 1100
experiments. Each experiment that has been reported here was performed at least 1101
twice with similar results. 1102
1103
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Statistical analysis was performed using Matlab (‘ranksum’) functions to determine 1104
significance using the Mann-Whitney U test. This is a nonparametric test that assumes 1105
unpaired samples that does not necessarily follow normal distributions. Significance 1106
levels are indicated as * (p ≤ 0.0001) or n.s (p > 0.0001) where applicable. For details 1107
of the sample size and p-values associated with each experimental condition, please 1108
refer to Table S4 and Table S5 in Supplemental Experimental Procedures. 1109
1110
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SUPPLEMENTARY FIGURE LEGENDS 1111
Figure S1: Increase in GPI-AP nanoclustering is dependent on the presence of 1112
RGD ligand and occurs in a β-1 integrin dependent manner, Related to Figure 1 1113
(A-B) Intensity and steady state anisotropy images (A) and intensity versus anisotropy 1114
plots (B) of GFP-GPI expressing human U2OS cells plated on glass coverslips coated 1115
either with 0.01% Poly-L-lysine (PLL; green) or Collagen-1 (orange) or 20µg/ml Laminin 1116
or 10µg/ml Vitronectin (yellow) or on 10µg/ml FN (FN; blue) and on FN and treated with 1117
10mM mβCD (mβCD ; red) and imaged on EA-TIRFM. Scale bar 10 µm. Note that the 1118
fluorescence anisotropy of GFP-GPI in cells plated on FN is highly depolarized 1119
compared to cells plated on integrin-inert substrate PLL or on substrates that activates 1120
other classes of integrins. (C-D) Treatment of cells with the β1 integrin function antibody 1121
alone results in defective cell spreading response (top panel, Brightfield image), 1122
implicating β1 integrin as the primary integrin utilized by U2OS cells to spread on FN. 1123
These antibodies localize to focal adhesions (C) when probed with appropriate 1124
fluorescent secondary antibodies in an immunofluorescence assay (bottom panel, αMs 1125
Alexa 488). (D) Box plot depicting mean anisotropy of GFP-GPI in U2OS cells plated on 1126
10µg/ml FN (No ab; black) or pre-treated in suspension with increasing amounts of the 1127
β1 integrin function blocking antibody 4B4 (2-40µg/ml; green) or with 40 µg/ml of a 1128
neutral (non-function perturbing) β1 integrin antibody K20 (blue) and subsequently 1129
plated on FN in the presence of the respective concentrations of the antibodies and 1130
imaged on EA-TIRFM. Scale bar 10 µm. Note that while blocking the function of β1 1131
integrin disrupts the increase in nanoclustering of GPI-APs seen on fibronectin, blocking 1132
the αV integrins does not result in the loss of GPI-AP nanolcusters (Data not shown). 1133
(E-F) Intensity and steady state anisotropy images (E) and intensity versus anisotropy 1134
plots (F) of GFP-GPI expressing U2OS cells plated on FN (No ab, blue) or pre-treated 1135
in suspension with 40 µg/ml of a neutral (non-function perturbing) β1 integrin antibody 1136
K20 (Green) or 40 µg/ml of an antibody against the transferrin receptor (OKT9, 1137
magenta) and subsequently plated on 10µg/ml FN in the presence (red) or absence of 1138
10mM mβCD and imaged on EA-TIRFM. Scale bar 10µm. 1139
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(G-H) Intensity and steady state anisotropy images (G) and (H) Box plot depicting the 1140
mean anisotropy of GFP-GPI in U2OS cells plated on glass blocked with 1%BSA (red) 1141
without Mn2+ or on 0.5µg/ml FN (green) with or without 2mM Mn2+or on 10µg/ml FN 1142
(green) with 2mM Mn2+ (blue) imaged on EA-TIRFM. Scale bar 10 µm. Note that shifting 1143
the equilibrium towards ligand-engaged integrin, either by increasing FN density or by 1144
the activation of integrin by Mn2+ promotes the generation of GPI-AP nanoclusters. Error 1145
bars represent SD. 1146
Figure S2: Activation of RGD binding integrins leads to enhanced nanoclustering 1147
of GPI-APs in its local vicinity, Related to Figure 2 (A) Plot of cell spread area (red 1148
curve) with corresponding change in the anisotropy of GFP-GPI (blue curve) as a 1149
function of spreading time (log time-X axis) of CHO cells expressing GFP-GPI taken 1150
using EA-TIRFM. (B) Notch-box plot of the mean anisotropy of cells in the indicated 1151
phases of cell spreading quantified by drawing ROIs in the intensity kymographs and 1152
extracting the corresponding values from the anisotropy kymographs. In the box plots, 1153
box includes the median and the 1st quartile to 3rd quartile. Whisker extends 1.5 SD (C) 1154
Schematic representation of the RGD-functionalized supported lipid bilayer (SLB) 1155
system. Lipids used to prepare the SLB were 1,2-dioleoyl-sn-glycero-3-phosphocholine 1156
(DOPC) doped with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) 1157
(16:0 Biotinyl Cap PE) (Bottom bilayer). Biotin-cRGD attached to DyLight 650 1158
Neutravidin was used as a linker to facilitate the attachment of GFP-GPI and integrin 1159
expressing cells onto the SLB. The DyLight 650 neutravidin signal serves as a marker 1160
for the α5(β1) integrin clusters since they co-localize to α5 integrin clusters formed on 1161
α5-GFP expressing CHO-B2 cells (right panel; merge). Scale bar 5 µm. 1162
(D) Intensity and anisotropy images and (E, F) graphs representing the anisotropy (E) 1163
and cell spread area (F) of GFP-GPI expressing cells plated on RGD functionalized 1164
supported lipid bilayers (blue) or glass (red) show that the anisotropy of cells plated on 1165
cRGD functionalized SLBs were lower than those of cells plated on plain glass under 1166
comparable cell spread area. Error bars are SD. (G) Representative montages of ROIs 1167
demonstrating correlations between RGD cluster intensity (more ‘yellow’ pixels denote 1168
higher cluster intensity) with corresponding GFP-GPI anisotropy maps (more ‘blue’ 1169
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pixels represent regions with more GPI nanoclusters) taken from cells expressing GFP-1170
GPI re-plated on RGD functionalized SLBs. Scale bar 500 nm. 1171
(H) Representative plots that demonstrate correlations between the RGD-cluster 1172
intensity (blue curve) and GFP-GPI anisotropy (green curve) and taken over time from 1173
ROIs sampled from 30 cells (20-30 clusters per cell) plated on RGD-functionalized fluid-1174
SLBs. Note that the increase (or decrease) in RGD cluster intensity coincides with a 1175
decrease (or increase) in GPI-AP anisotropy indicating a local increase (or decrease) in 1176
GPI-AP nanoclusters. Error bar represent SD. 1177
Figure S3: Effect of perturbations of downstream targets of integrin signaling on 1178
GPI-AP nanoclustering, Related to Figure 3. 1179
(A-B) Intensity and steady state anisotropy images (A) and intensity versus anisotropy 1180
plots (B) of GFP-GPI expressing CHO cells on glass (red closed circles) or cells on 1181
glass and treated with 10mM mβCD (red open circles) or plated on FN (blue closed 1182
circles) or plated on FN and subsequently treated with 10mM mβCD (blue open circles. 1183
Note that the anisotropy of cells on glass closely resemble those of cells treated with 1184
mβCD and therefore either of these conditions can be used to represents cells with a 1185
loss of nanolcusters of GPI-APs. (C-D) Intensity and steady-state anisotropy images (C) 1186
and intensity versus anisotropy plots (D) of GFP-GPI expressing FAK+/+ (blue or green) 1187
or FAK-/- (red or black) mouse embryonic fibroblasts (MEFs) cells plated on 10µg/ml FN 1188
in the presence (green or black) or absence (blue or red) of 10mM mβCD imaged on a 1189
EA-TIRFM. (E-F) Intensity and steady state anisotropy images (E) and intensity versus 1190
anisotropy plots (F) of GFP-GPI expressing CHO cells plated on glass(red) or pre-1191
treated and plated on glass in the presence of 10µg/ml RhoA activator (CN03) (green) 1192
or plated on FN (blue) and imaged on a TIRF microscope. Note that the fluorescence 1193
anisotropy of GFP-GPI in cells treated with the RhoA activator is lower compared to 1194
DMSO treated cells on glass and resembles cells plated on FN indicating an increase in 1195
GPI-AP nanoclustering independent of FN binding under this condition.(G-H) Intensity 1196
and steady state anisotropy images (G) and intensity versus anisotropy plots (H) of 1197
GFP-GPI expressing CHO cells plated on glass(red) or pre-treated and plated on glass 1198
in the presence of 10µM formin activator (IMM01) (black) or plated on FN (blue) and 1199
imaged on EA-TIRFM. Note that the fluorescence anisotropy of GFP-GPI in cells 1200
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treated with the formin activator is lower compared to DMSO treated cells on glass and 1201
resembles cells plated on FN indicating an increase in GPI-AP nanoclustering 1202
independent of FN binding under this condition. Error bars indicate SD. Scale bar 10µm. 1203
Figure S4: Integrin activation alters cortical acto-myosin activity, Related to 1204
Figure 4 1205
(A) Western blot analysis of siRNA medicated knockdown of formins in human U2OS 1206
cells transfected for 72 hours with 5nmoles SMART pool of either control scrambled 1207
siRNA (black) or siRNA against the formins FHOD1 (red) or mDia1 (DIAPH1; blue) and 1208
probed with antibodies against the same. (B) Quantification of the blots indicate a 1209
knockdown of ~45% FHOD1 protein levels and ~80% in the case of mDia. The data is 1210
normalized first to the corresponding intensities of β-actin in each well (loading control) 1211
and then to the levels of the protein in the control siRNA well. (C-D) Intensity and 1212
steady-state anisotropy images (C) and intensity versus anisotropy plot (D) of U2OS 1213
cells expressing GFP-GPI plated on 10µg/ml FN after treatment with 5nmol of either 1214
control siRNA (blue) or FHOD1 siRNA (red) or mDia1 siRNA (green) for 72 hours and 1215
imaged in EA-TIRFM. Scale bar 10µm. Error bars indicate SD. (E-G) Confocal images 1216
(E) and Average (circles) and standard deviation (shaded) autocorrelation decays (F) of 1217
GFP-tagged Utrophin actin filament binding domain (GFP-Utr) in cells plated on FN 1218
(blue) or pre-treated and plated on FN in the presence of 10µM formin inhibitor SMIFH2 1219
(red). FCS data was collected from regions in the cell periphery devoid of stable actin 1220
filaments as indicated (blue or red circles). Note the loss of the slow diffusion timescales 1221
corresponding to 10ms or longer for cells treated with the formin inhibitor. (G) Timescale 1222
and fraction of the slow moving population associated with moving actin filaments. (H-I) 1223
Intensity and steady-state images (H) and intensity versus anisotropy plot (I) of CHO 1224
cells stably expressing GFP-GPI plated on 10µg/ml FN after treatment with DMSO 1225
(control; blue) or 20µM MLCK inhibitor ML7 (yellow) or20µMROCK inhibitor Y-27632 1226
(green) or 20µM of both ROCK and MLCK inhibitors (ML+Y;black) or mβCD (orange) 1227
taken on EA-TIRFM.Scale bar 10 µm.Error bars indicate SD. 1228
1229
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Figure S5: Characterization of vinculin deficient and talin 1 deficient MEFs and 1230
schematic of vinculin mutations used in this study, Related to Figure 5. 1231
(A-B) Intensity images (A) and corresponding histogram (B) quantifying endogenous 1232
talin levels in talin 1-/- cell line in the presence or absence of GFP-tagged talin 2 shRNA 1233
using a pan talin antibody. The data shows a significant decrease in talin levels in cells 1234
treated with talin 2 shRNA (C) Schematic depicting the vinculin variants used in this 1235
study, and the typical characteristics of each molecule. Error bars represent SD. 1236
Figure S6: Characterization of the effects of vinculin mutants on GPI-AP 1237
nanoclustering, Related to Figure 6. 1238
(A-B) Intensity and anisotropy images (A) and intensity versus anisotropy plot (B) of 1239
vinculin deficient (vin-/-; MEFs transfected with the indicated vinculin variants and 1240
labeled with Alexa-568-FLAER 12-16 hours post transfection and imaged in EA-TIRFM 1241
after replating the cells on FN coated glass bottom dishes. Transfected cells are marked 1242
by dotted magenta lines. (C-D) vin-/- MEFs were transfected with GFP-tagged Vinculin 1243
and co-transfected with a membrane marker (RFP-tHRas) and imaged on a confocal 1244
microscope (C). Plot (D) shows the line intensity profiles of Vin-WT and RFP-tHRas 1245
suggesting the absence of Vin-WT at the plasma membrane post 12-16 hours of 1246
transfection. White line in (C) depicts the region of line scan measurement. Scale bar 10 1247
(A), 5 (C)µm. Error bar represent SD. 1248
Figure S7: Functional significance of GPI-AP nanocluster formation, Related to 1249
Figure 7 1250
(A-B) Intensity and steady-state anisotropy images (A) and intensity versus anisotropy 1251
plot (B) of GFP-GPI expressed in wild type (WT; blue), PGAP2/3 double mutant (red) 1252
and PGAP2/3 add back (Rescue; green) re-plated on 10µg/ml FN. Scale bar 10µm.Note 1253
that an increase in anisotropy in mutant cells was observed corresponding to a loss of 1254
nanoclustering of GFP-GPIs. (C) Quantification of the relative amount of active β-1 1255
integrins (marked by HUTS4 antibody) and inactive β-1 integrins (marked by 4B4 1256
antibody) normalized to the levels of neutral antibody (K20) of WT cells (blue bar), 1257
PGAP2&3 mutants (red bar), rescue cells (green bar). Data is also additionally 1258
normalized to the levels in the WT scenario. (D-E) MS/MS Mass spectrometric analysis 1259
(D) and filipin-staining of WT cells (blue bar) or PGAP2&3 mutants (red bar) or rescue 1260
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(green bar) or WT cells treated with 10mM mβCD (control in E) quantifying the levels of 1261
the indicated lipid species (in D) and free cholesterol (in E). Data represents the mean 1262
+/- SD. 1263
(F) TIRFM images of WT (blue), PGAP2&3 double mutant (red) and Rescue cells 1264
(green), de-adhered and allowed to spread on FN coated dishes for 60 mins and 1265
subsequently fixed, permeabilised and stained for paxillin (Left panel) to mark focal 1266
adhesions, or labeled with Phalloidin to mark actin filaments (Middle panel) or a merge 1267
of both (Right panel. Scale bar 10µm. (G) Frequency histogram of the binned focal 1268
adhesion sizes (marked by paxillin) of WT (Blue bars), PGAP2/3 double mutant (Red 1269
bar) and Rescue line (Green bars) Note that the mutant cells have larger adhesions and 1270
lack the smaller nascent adhesions that are usually found at the cell 1271
periphery/lamellipodia (Red arrow heads in F). (H-I) Phase contrast images (H) and 1272
histogram (I) quantifying the extent of spreading of vin-/- MEFs or vin-/- MEFs 1273
transiently transfected with the indicated Vin constructs. Cell area was quantified at 0 1274
and 30 mins after seeding on 10µg/ml FN-coated glass-bottom dishes. Inset depicts the 1275
cell spreading versus time profile of vin-/- cells on FN. The transfected cells are marked 1276
by a dotted magenta line. Scale bar 50 µm. Error bars represent SD. 1277
1278
1279
1280
1281
1282
1283
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1285
1286
1287
1288
1289
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1292
Supplementary Movie Legends: 1293
Supplementary Movie 1: Cell spreading dynamics of GFP-GPI expressing CHO cells 1294
on 10µg/ml FN; Imaged after every 15 seconds in 100X EA-TIRFM mode at 37oC. Left 1295
panel: Total Intensity image; Right Panel: GFP-GPI anisotropy image with the 1296
corresponding LUT bar. Scale bar 10µm. Notice that the cells initially blebs and rapidly 1297
acquire GPI-AP nanoclusters (blue pixels) before the cell extends out a prominent 1298
lamellipodia and begins spreading rapidly. 1299
Supplementary Movie 2: 20X Phase contrast time series images of WT or PGAP2&3 1300
double mutant CHO cells spreading on FN. Notice that the PGAP2&3 double mutants 1301
spread slowly and extend by producing blebs (and lack a prominent lamellipodia). Scale 1302
bar 50µm. 1303
Supplementary Movie 3: Cell spreading dynamics of GFP-GPI (Cell membrane 1304
marker) expressing WT (Left Panel), PGAP2&3 mutant (Middle Panel) or Rescue (Right 1305
Panel) CHO cells spreading on 10µg/ml FN;imaged every 15 seconds in 100X TIRF at 1306
37oC. Notice that the PGAP2&3 mutant cells exhibit defects in cell spreading due to 1307
their inability to produce a prominent lamellipodia. Scale bar 10µm.1308
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0 1 2 3 40.08
0.10
0.12
0.14
0.08
0.10
0.12
0.14
exo-scSM
exo-GPI
Figure 1
FN+mβCD
0.17
0.15
0.21
0.20
0.16
0.140.10
G H
GFP
-GP
IA
niso
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Ani
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Total Intensity, a.u x104
NB
DA
niso
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B
I
Ani
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J
YFP-GPI
D
C
F
exo-GPI
0.06
exo-scSM
0.19
0.17
0.210.190.18
0.16
0.22
YFP
-GP
IA
niso
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FNGlassI/I0 = 0.5I/I0 = 1I/I0 = 0.5I/I0 = 1
A
BSA-coated glass FN-coated glass
ii.De-adhered
i.Serum-starved ‘resting’ cells growing on petridishes
iv.Re-plated under serum-free conditions and imaged in EA-TIRFM
Outerleaflet
Innerleaflet
GFP
GPI
anc
hor
Outerleaflet
Innerleaflet
YFP
GPI
anc
hor
E
Normalized Intensity I/Io
YFP-GPI
1% BSA 10μg/ml FN
0 2 4 6 8 10
0.16
0.18
0.20
0.22GFP-GPI
Total Intensity, a.u x104
Glass FNGlass FN
0.2 0.4 0.6 0.8 1.00.18
0.20
0.22
0.24+mβCD
Photobleaching
0.14
0.16
0.18
0.20
0.22
1%B
SA
* *
*
0 0.1
0.5 1 5 10 20 50
+mβC
D
FN conc. (μg/ml)
GFP-GPI
iii.Cells in suspension for recovery
p <0.0001
p <0.0001
p <0.0001
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted December 11, 2017. ; https://doi.org/10.1101/232223doi: bioRxiv preprint
0.16
0.17
0.18
0.19
0.20
0 0.1 1 10
+mβC
D
c(RGDfV) conc. (μM)
100
T+20sec T+40sec T+60sec T+80secT0sec x104
x103
Figure 2
0.230.210.190.170.15
Aniso
tropy
, r
GFP
-GP
IA
niso
tropy
E F
GFP-GPI Anisotropy BR
GD
D
A
1μm
150s
x-position (in μm)
150
300
4500.511.52
2 4 6 8 10 12 14 16 18
150
300
450 0.10
0.15
0.20
0.25
Tim
e (in
sec
s)
P0P1
P2GFP
-GP
IA
niso
tropy
GP
I-GP
IA
niso
tropy
1.51.31.10.90.7
0.320.280.240.200.16
5.04.03.02.01.0
x104
0 μM cRGD +10μM cRGD +mβCD
C
0.26
0.24
0.22
0.20
0.18
0.16
0.14
0.12
0.10
15
135
195
240
420
time
(sec
onds
)
time (seconds)
δAre
a (μ
m2 )
δAni
sotro
py
-200 -100 0 100 200 300 400
0
20
40
60
80
-0.04
-0.02
0.00P0
P1
P2
*
p <0.0001
*
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted December 11, 2017. ; https://doi.org/10.1101/232223doi: bioRxiv preprint
0.20
0.10
0.15
0.05
Figure 3A B
0.22
0.18
0.10
0.14
0.06
GFP
-GP
IA
niso
tropy
C
E
H
0.20
0.10
0.15
0.05
D
F
G
PF573228 -- ++- +
+-
PP2 G
FP-G
PI
Ani
sotro
py
- +C3 exo
0.18
0.16
0.10
0.14
0.06
- +SMIFH2 - +CK666
GFP
-GP
IA
niso
tropy
PP2P573228
IMM01CN03
++--
GFP
-GP
IA
niso
tropy
+++-
++-+
SMIFH2 - - -
+++-+
0 2 4 6 8 10
0.10
0.12
0.14
0.16
Glass
0 2 4 6 80.10
0.12
0.14
0.16
Total Intensity, a.u x104
+1μg/ml C3 -
Ani
sotro
py,r
0 2 4 6 8 10
0.10
0.11
0.12
0 2 4 6 8 10 12
0.10
0.12
0.14
+mβCD+3μg/ml C3
0.22
0.160.18
0.12
0.20
0.14
Glass
Glass
p <0.0001
p <0.0001
p <0.0001
p <0.0001
0.140.150.160.170.180.19
0
1.0
2.0
3.0
+mβC
D
C3 exo conc. (μg/ml)
p <0.001
*p <0.0001
*
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted December 11, 2017. ; https://doi.org/10.1101/232223doi: bioRxiv preprint
0.28
0.24
0.20
0.16
0.12
PLB
Ani
sotro
py
Ani
sotro
py,rPLB
Ani
sotro
py
FN
B C
E F
Figure 4
0.13
0.11
0.09
0.07
0.05
PLB
Ani
sotro
pyFR
TM-E
z-A
FBD
HG
0.28
0.24
0.20
0.16
0.12
FRTM
-Ez-
AFB
DFR
TM-E
z-A
FBD
*
Ezrin-AFBD
FBP
IgG
-FcR
TM
Outerleaflet
Innerleaflet
Ezrin-AFBD
FBP
IgG
-FcR
TM
Outerleaflet
Innerleaflet
*
A
D
0 1 2 3 4
0.18
0.20
0.22
0.24
0 2 4 6 8 10
0.18
0.20
0.22
0.24
PF573228 ----+
++
PP2
SMIFH2 - -
1 2 3 4 50.06
0.08
0.10
0
Total Intensity, a.u x104
Glass
FNGlass
p <0.0001
p <0.0001
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted December 11, 2017. ; https://doi.org/10.1101/232223doi: bioRxiv preprint
A
5 10 15
0.08
0.12
0.16
0.20
0.200.160.12
0.24
GFP
-GP
IA
niso
tropyVi
n-/-
E F
+Vin +mβCD+mβCDVin-/-
Ani
sotro
py
+Vcl
0.18
0.14
0.10
0.06
FLA
ER
Ani
sotro
py
+mβCD
0 0.5 1.0 1.5 2.00.08
0.10
0.12
0.14
Talin1-/- +Talin2 shR +Talin2 shRTalin1-/-+mβCD
B
C D
Figure 5
+Vcl
FR-EZ FR-EZ* FR-EZ FR-EZ* H
0.14
0.16
0.18
5 10
Total Intensity, a.u x 104
FLA
ER
Ani
sotro
py
+Vin-WT
G
+Vin +Vin0.20
0.16
0.12
0.10Ani
sotro
pyP
LB
0.04
0.08
0.12
0.16
+Vin-A50I +Vin-A50I-CAVin-/-
Vin-
/-
I
Vin-
/-
0.18
0.14
0.10
0.06
FLA
ER
Ani
sotro
pyVi
n-/-
+mβCDVin-/- +Lact C2 Ez +Lact C2 Ez
J
0 5 10 150.04
0.06
0.08
0.10
0.12
0.14
0 5 100.14
0.16
0.18
0.20
0
p <0.0001
p <0.0001
p <0.0001
p <0.0001
p <0.0001
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted December 11, 2017. ; https://doi.org/10.1101/232223doi: bioRxiv preprint
0.08
0.12
0.16
0.20+ Vcl-WT
0 5 10 15
0.08
0.12
0.16
0.20
Vin-/-mch-VclSMIFH2
PP2
IMM01
Figure 6
0.200.160.12
0.24
0.200.16
0.12
0.24
GFP
-GP
IA
niso
tropy
GFP
-GP
IA
niso
tropy
10µm
10µm
+Vin WT
Vin-/-
0 5 10
0.08
0.16
0.24
A B
C D
F
PF573
--+- ++-- ++-- + +---
0.10
0.15
0.20
0.25
Vin-/- Vin AB1 CAVin WT
----
----
+---
-++-
-+++
SMIFH2PP2
IMM01PF573
0.08
0.16
0.24
---
Anis
otro
py,r
Vin AB1
0 2 40.14
0.16
0.18
0.20
0.22
Ani
sotro
py
Total Intensity, a.u x 104
E
mR
uby-
GP
I
p <0.0001
p <0.0001
p <0.0001
p <0.0001
p <0.0001
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted December 11, 2017. ; https://doi.org/10.1101/232223doi: bioRxiv preprint
-200 -100 0 100 200 300 400
0
20
40
60
80
Cel
l spr
ead
area
x 10
3 (μm
2 )
0 15 30 45 60 75 900
0.2
0.6
1.0
1.4
1.8
2.2 WT PGAP2/3 mutant Rescue
90 m
ins
WT
Rescue
+mβCD
PGAP2/3 mutant
A BFigure 7
C
time (seconds)
δare
a (μ
m2 )
time (minutes)
F
D
0.600.500.40
0.70
0.200.30
Laur
dan
GP
valu
e
WT +mβCD
Laur
dan
GP
valu
e
PGAP2/3 mutant Rescue
0.600.500.40
0.70
0.200.30
E
GP
Valu
e
0.3
0.4
0.5
0.6
0.7
WT
PGAP2&3
mutantRes
cue
WT
+mβCD
P0
P1
P2
SFK
FAK
Tailin
Vinculin
GTP
RhoA
SFK
FAK
Formins
Fibronectin
Integrin-activation
Actin filaments
RGD
inner-leaflet
outer-leaflet
RhoA
GDP
FibronectinRGD
Formins
FibronectinRGD
nanoclustersmonomers
GPI-APs
inner-leaflet
outer-leaflet
FibronectinRGD
nanoclustersmonomers
inner-leaflet
outer-leaflet
GPI-APs
VhVt
Talin
inner-leaflet
outer-leaflet
ROCK
Myosin
p
p
p
p p
p
p
p
p
ppp
p
p
p p
p
Myosin
MLCK
MLCP
Vinculin
Actin filaments
PS
GPI-APmonomers
Integrin receptor
Integrin-activationIntegrin-activation
i iiX X X
1.
4.
2.
3.
p <0.0001
p <0.0001* *
*
WT+mβCD
WTPGAP2/3 mutantRescue WT+mβCD
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted December 11, 2017. ; https://doi.org/10.1101/232223doi: bioRxiv preprint
2 4 60.14
0.16
0.18
0.20
0.22
FN+mβCD
2 4 60.14
0.16
0.18
0.20
0.22
Figure S1A
E
GFP
-GP
IA
niso
tropy 0.22
0.140.12
0.16
0.24
0.20
+OKT9 +K20No ab
GFP
-GP
IA
niso
tropy 0.24
0.20
0.12
0.16
0.28
+ 4B4
DC
Ani
sotro
py,r
1%BSA 0.5 μg/ml FN
0mM Mn2+ 0mM Mn2+ 2mM Mn2+
0.220.20
0.160.18
0.14
0.24
GFP
-GP
IA
niso
tropy
HG
B
F
Total Intensity, a.u X104
10 μg/ml FN
2mM Mn2+
Total Intensity, a.u X104
αTfR K20 4B4
Brig
htfie
ld
+αTfR +K20 +4B4
αMs
Ale
xa 4
88VitronectinPLL LamininCollagen-1
+ mβCDp <0.0001
p <0.0001
0.14
0.16
0.18
0.20
0.22
FN conc. (μg/ml)
No
Mn2+
+0.5
mM
Mn2+
+2m
M M
n2+
+10m
M M
n2+
+2m
M M
n2+
No
Mn2+
No
Mn2+
0 0.5 10
0.12
0.14
0.16
0.18
0.20
0.22
+ m
βCD
No
ab
4B4 ab conc. (μg/ml)
402 5 10 20 30 40K
20
p <0.0001*
*
n.sn.s
n.s
*
n.s
p <0.0001
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted December 11, 2017. ; https://doi.org/10.1101/232223doi: bioRxiv preprint
Figure S2A
Glass RGD-SLB0
50
100
150
GlassRGD-SLBC
ell a
rea,
in μ
m2
0.35
0.30
0.25
0.20
0.15
GP
I Int
ensi
tyA
niso
tropy
Neu
travi
din
0.15
0.20
0.25
0.30
GlassRGD-SLB
p<0.0001
*
p=0.1030
F
Glass RGD-SLBD
RG
D in
tens
ity, a
.u
Anis
otro
py,r
E G
DyLight650-Neu
Merge
α5-integrin GFP
0.35
0.25
0.15
H
GP
I-anisotropy,r
0.24
0.23
0.22
0.23
0.21
0.19
0.21
0.19
0.17
0.18
0.16
0.14
0.22
0.20
0.18
0.25
0.23
0.210 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100
1.15
1.05
0.05
1.8
1.4
1.0
1.45
1.35
1.25
2.2
1.8
1.4
2.5
2.0
1.5
1.4
1.0
0.6
GP
I-ani
sotro
py, r
cyclic-RGDLigated Integrin
Neutravidin DyLight 650
Plasma membrane
GFP-GPI
Supported lipidbilayer doped with
Biotinylated PE lipid
Glass
500nm
time (seconds)
RGD GPI-anisotropy
0.14
0.16
0.18
0.20
0.22
0.24
0.26 P0 P1 P2
Anis
otro
py,r
C
B1 10 100
10
100
1000
0.16
0.19
0.22
0.25
time (seconds)
Are
a (µ
m2 ) Anisotropy
* *
*
n.s
p<0.0001
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted December 11, 2017. ; https://doi.org/10.1101/232223doi: bioRxiv preprint
0 3 6 9 12
0.10
0.12
0.14
0 2 4 6 8 10 12
0.14
0.16
0.18
0.20
Figure S3G
FP-G
PI
Ani
sotro
py
0.10
0.14
0.18
0.22
FAK +/+ FAK -/-
0.16
0.12
0.08
0.200.24
GFP
-GP
IA
niso
tropy
Ani
sotro
py,r
Ani
sotro
py,r
C D
E F
DMSO +IMM01
Total Intensity, a.u x104
GFP
-GP
IA
niso
tropy
G H
0.22
0.18
0.10
0.14
0.06
DMSO +CN03
Glass
+mβCD +mβCD
FNDMSO
0 2 4
0.10
0.12
0.14
0.16
FNDMSO
0 2 4 6 8 10
0.20
0.22
0.24
Glass FNA B
Glass
p <0.0001
p <0.0001
p <0.0001
p <0.0001
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted December 11, 2017. ; https://doi.org/10.1101/232223doi: bioRxiv preprint
0 2 4 60.14
0.16
0.18
+mβCD
10-3
10-2
10-1
0.0
0.2
0.4
0.6
0.8
1.0
time
(s)
fraction bound Utr-G
FP
+mβCD
αmDia1C
ontro
l m
Dia
1
FHO
D1
GFP
-GP
IA
niso
tropy
αactin
αactin
αFHOD1
DC
A
F
0.00.20.40.60.81.0
Knoc
kdow
n ef
ficie
ncy
(Fra
ctio
n re
lativ
e to
con
trol)
Contro
l
FHOD1
mDia1
I
GFP
-GP
IA
niso
tropy
Total Intensity, a.u x104
Ani
sotro
py,r
Total Intensity, a.u x104
Ani
sotro
py,r
Figure S4
H
0.16
0.14
0.10
0.20
DMSO
+ SMIFH2
E
Utr-
GFP
72 hrs siRNA
siRNA
0.20
0.10
0.15
0.25
ML7Y27632
--
+-
-+
++
FHOD1 mDia1 Control
siRNA
10-5 10-4 10-3 10-2 10-1 1000.0
0.3
0.6
0.9
timelag (s)
Nor
mal
ized
G00
(-)
SMIFH2DMSO
B
G
0 2 40.14
0.16
0.18
0.20
8
p <0.0001
p <0.0001
* *p <0.0001
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted December 11, 2017. ; https://doi.org/10.1101/232223doi: bioRxiv preprint
Figure S5
pan-
Talin
ab
Talin 1-/-
0.2
0.6
1.0
Nor
mal
ised
Inte
nsity
Control Talin 2 shR
A
+Talin2 shR
B
CVin-WT
Head TailNeck1 1066
Head TailNeck1 1066
N773A/E775A
Head1 1066821
Head TailNeck1 1066
A50I
Head TailNeck1 1066A50I N773A/E775A
Head TailNeck1 1066K952Q/K956Q/R963Q/K966Q/R1060Q/R1061Q
Head TailNeck1 1066D974A/K975A/R976A/R978A
Head TailNeck1 1066K952Q/K956Q/R963Q/K966Q/R1060Q/R1061Q D974A/K975A/R976A/R978A
Vin-CA
Vin head
Vin A50I
Vin A50I-CA
Vin Ld
Vin CA*
Vin Ld CA*
wild type
constitutively active
head domain
talin binding mutant
constitutively active talin binding mutant
lipid binding mutant
constitutively active
constitutively active lipid binding mutant
Head TailNeck1 1066N773A/E775A
constitutively active actin binding mutantI997A
Vin AB1 CA
Head TailNeck1 I997A
Vin AB1 actin binding mutant
1066
1066
Vin Ld
p <0.0001not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted December 11, 2017. ; https://doi.org/10.1101/232223doi: bioRxiv preprint
Figure S6
0.04
0.08
0.12
0.16
FLA
ER
Ani
sotro
py
+Vin-CA +Vin-head +Vin-Ld-CA* +Vin-LdA B
GFP-Vin-WTMembrane (RFP-tH) Merge
0 5 10 15 20 25 30 35 40
200
300
400
500
600 Vin-WT MembraneC D
0 5 10 150.04
0.06
0.08
0.10
0.12
0.14
Distance (x 102 nm)
Inte
nsity
a.u
p <0.0001
Ani
sotro
py
Total Intensity, a.u x 104
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted December 11, 2017. ; https://doi.org/10.1101/232223doi: bioRxiv preprint
0 3 6 9 12 150.14
0.16
0.18
0.20
Figure S7WT RescuePGAP2/3
mutantG
FP-G
PI
Ani
sotro
py
Ani
sotro
py,r
0.24
0.20
0.12Total Intensity, a.u x104
0.16
A B
I
D
H
<0.15
0.15-0
.3
0.3-0.
45
0.45-0
.6
0.6-0.
75>0
.750.0
0.1
0.2
0.3
0.4
Freq
uenc
y
Adhesion size(μm2)
PGAP2/3mutantWT
Rescue
WT
PG
AP
2/3
mut
ant
Res
cue
αPaxillin Phalloidin MergeF G
C
0.0
0.4
0.8
1.2
1.6
Active
Norm
alize
d le
vels
Inactive PS PC PE PI0
1020304050
pmol
es/μ
g pr
otei
nE
0 min 30 min
Vin-
/-+V
in-W
T+V
in-C
A
+Vin
-Ld
+Vin
-Ld-
CA
*
FN0 min 30 min
FN
Cel
l spr
ead
area
X 1
03 μm
2
0 min 30 min
Vin-/- Vin-WT Vin-CA Vin-Ld Vin-Ld-CA*
0.00.3
0.6
0.9
1.2
1.5
1.8
00.51.01.52.02.5
Filip
in in
tent
isy,
a.u x104
WTPGAP2&3 mutant
RescueWT +mβCD
p <0.0001
*
n.s
n.s
n.s
n.s n.s
n.s
**
*
n.s
**
**
p <0.0001 p <0.0001 p <0.0001
p <0.0001
p <0.0001
0 10 20 30 40 50 600
0.2
0.4
0.6
0.8
1.0
minutes
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted December 11, 2017. ; https://doi.org/10.1101/232223doi: bioRxiv preprint