2 acto-myosin driven functional nanoclusters of gpi ... · 11.12.2017 · 26 summary 27...

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: [email protected] 18

19

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not certified by peer review) is the author/funder.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

https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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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that stimulates outward translocation. Proc. Natl. Acad. Sci. U. S. A. 108, 20585–20590. 858

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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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

(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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

(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

1284

1285

1286

1287

1288

1289

1290

1291


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

0 1 2 3 40.08

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https://doi.org/10.1101/232223

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https://doi.org/10.1101/232223

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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


https://doi.org/10.1101/232223

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


E

mR

uby-

GP

I

p <0.0001

p <0.0001

p <0.0001

p <0.0001

p <0.0001


https://doi.org/10.1101/232223

-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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

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


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


https://doi.org/10.1101/232223

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


Ani

sotro

py,r


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


https://doi.org/10.1101/232223

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

https://doi.org/10.1101/232223

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



https://doi.org/10.1101/232223

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


https://doi.org/10.1101/232223

2 acto-myosin driven functional nanoclusters of gpi ... · 11.12.2017 · 26 summary 27...

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