1

Cell-substrate adhesion drives Scar/WAVE 1

activation and phosphorylation, which controls 2

pseudopod lifetime 3

4

Shashi Prakash Singh+, Peter A. Thomason+, Sergio Lilla+, Matthias Schaks$, 5

Qing Tang%, Bruce L. Goode%, Laura M. Machesky+*, Klemens Rottner$ and 6

Robert H. Insall+* 7

8

+CRUK Beatson Institute, Switchback Road, Glasgow G61 1BD, UK; *Institute 9

of Cancer Sciences, University of Glasgow, Switchback Road, Glasgow G61 10

1BD, UK; $Zoological Institute, Technische Universität Braunschweig, 11

Spielmannstrasse 7, 38106 Braunschweig, Germany & Cell Biology, Helmholtz 12

Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, 13

Germany; %Brandeis University, 415 South Street, Waltham, MA 02453, USA. 14

15

Correspondence to Robert Insall 16

(R.Insall@beatson.gla.ac.uk; tel. +44/0 141 330 4005) 17

18

2

Abstract 19

The Scar/WAVE complex is the principal catalyst of pseudopod and 20

lamellipod formation. Here we show that Scar/WAVE’s proline-rich domain 21

is polyphosphorylated after the complex is activated. Treatments that stop 22

activation block phosphorylation in both Dictyostelium and mammalian cells. 23

This implies that phosphorylation modulates pseudopods after they have 24

been formed, rather than controlling whether a protrusion is initiated. 25

Unexpectedly, activation-dependent phosphorylation is not promoted by 26

chemotactic signalling, or by signal-dependent kinases such as ERKs, but is 27

greatly stimulated by cell:substrate adhesion. Scar/WAVE that has been 28

mutated to be either unphosphorylatable or phosphomimetic is activated 29

normally, and rescues the phenotype of scar- cells, demonstrating that 30

phosphorylation is dispensible for activation and actin regulation. However, 31

pseudopods and patches of Scar/WAVE complex recruitment last 32

substantially longer in unphosphorylatable mutants, altering cell polarisation 33

and the efficiency of migration. We conclude that pseudopod engagement 34

with substratum is more important than extracellular signals at regulating 35

Scar/WAVE’s activity, and that phosphorylation acts as a timer, restricting 36

pseudopod lifetime by promoting Scar/WAVE turnover. 37

3

Introduction 38

Scar/WAVE is the dominant source of actin protrusions at the edge of 39

migrating cells. In particular, lamellipods (in mammalian cells cultured in 40

2D) and pseudopods (in cells in 3D environments, or cells such as amoebas) 41

are driven by Scar/WAVE recruiting the Arp2/3 complex, which in turn 42

promotes an increase in the number of polymerizing actin filaments and 43

growth of actin structures (1). It works as part of a large, five-membered 44

complex, whose members have multiple names (2); in this paper they will be 45

referred to as Nap1, PIR121, Scar, Abi and Brk1 in Dictyostelium, and Nap1, 46

PIR121, WAVE2, Abi2 and HSPC300 in mammals. 47

48

The principal known activator of Scar/WAVE complex activation is the small 49

GTPase Rac. Inactive Rac is GDP-bound, but on stimulation becomes 50

temporarily GTP-bound. The GTP-bound, but not the GDP-bound form 51

binds to the complex (3), in particular through the A-site of PIR121, which 52

includes a Rac-binding DUF1394 domain (4). Interaction with GTP-bound 53

Rac is essential for the complex to be able to function (3, 5). However, though 54

it is clear that Rac is essential, it is not the only regulator (1). Various 55

experiments have found that Rac activation occurs later than the onset of 56

actin-based protrusion (6), and signal-induced actin polymerization can occur 57

earlier than Rac activation (7). In Dictyostelium, Scar/WAVE behaviour is 58

much more locally variable than Rac activity (8), so the Rac cannot simply be 59

driving the changes in Scar/WAVE. To understand pseudopod dynamics, it 60

will therefore be vital to enumerate different modes of Scar/WAVE regulation. 61

One potential form of regulation – phosphorylation - has been described in a 62

number of papers, and is also found in untargetted and high-throughput 63

screens. A typical narrative is that Scar/WAVE is phosphorylated in response 64

4

to external signalling, through kinases such as (particularly) the global signal 65

transducer ERK2. This has been described in cultured fibroblasts (9), MEFs 66

(10), and endothelial cells (11). The phosphorylation is typically found to 67

change the complex from an inactive to an activatable state, so Scar/WAVE 68

phosphorylation directly leads to actin polymerization. Tyrosine kinases, in 69

particular Abl, have been found to be similarly activating (12, 13). These 70

reports are curious, for a number of reasons. First, actin is a strongly acidic 71

protein, so phosphorylation of binding proteins typically weakens their 72

affinity for actin and actin-related proteins. Second, ERK2 has a tightly 73

defined consensus sequence; however, the proposed phosphorylation sites 74

(and confirmed by us below) do not fit this consensus. We have therefore 75

explored the biological functions of Scar/WAVE phosphorylation in detail. 76

A separate set of phosphorylations is present in the C-terminal WCA domain 77

of Scar/WAVE. It is not detectable by, for example, a change in banding 78

pattern on western blots, and difficult to see by mass spectrometry, so is far 79

less widely described. We (14) and others (15) have shown that this is 80

constitutive, and has a role in tuning the sensitivity of the Scar/WAVE 81

complex rather than activating it; both phosphomimetic and 82

unphosphorylatable mutants are active. 83

One key process in Scar/WAVE biology that is particularly poorly understood 84

is autoactivation. It is clear that pseudopods of migrating cells (which are 85

caused by Scar/WAVE) are controlled through positive feedback – new actin 86

polymerization occurs adjacent to recent pseudopods (8), leading to travelling 87

waves at the edges of cells (16), but the mechanism of this regulation is not 88

well understood (17, 18). It does, however, emphasize the importance of 89

understanding the full dynamics of Scar/WAVE – its recruitment and release 90

from pseudopods, and its synthesis and breakdown – rather than focussing 91

exclusively on its activation. 92

5

93

There is no compelling reason to connect Scar/WAVE phosphorylation to the 94

activation step. Phosphorylation could alter the activity of the complex after 95

it is activated, or alter properties like the rate of autoactivation or the stability 96

of Scar/WAVE once recruited (18). Indeed, in the present work, we find that 97

phosphorylation’s primary role is to control the lifetime of Scar/WAVE after it 98

is recruited to the edge of the cells, meaning that it correlates with pseudopod 99

size rather than the probability of pseudopod generation. We show that 100

phosphorylation occurs after the complex is activated, and at a number of 101

separate, dissimilar sites throughout Scar/WAVE’s polyproline domain. Thus 102

its role appears to be centred around biasing pseudopod behaviour, as 103

required by pseudopod-based models of cell migration, rather than in 104

initiating new pseudopods or actin polymerization. 105

106

6

Results 107

Detailed analysis of Scar/WAVE phosphorylation in vivo 108

A number of authors have observed Scar/WAVE phosphorylation (10, 11, 14, 109

19, 20). Overall, it is clear that there is constitutive phosphorylation in inactive 110

Scar/WAVE, which we have shown is localised in the extreme C-terminus 111

(14). There is also additional phosphorylation seen in (for example) growth 112

factor stimulated cells, which a number of authors have placed downstream 113

of ERK2 (10, 11, 19), implying it is part of the process by which signalling 114

causes pseudopods to form. To analyse the phosphorylation in detail, we 115

devised an optimised electrophoresis regime. Examined by western blot on 116

normal SDS-PAGE gels, Dictyostelium Scar runs as a single band with a 117

diffuse upper edge; quantitative analysis shows this band forms a single, 118

diffuse peak (Figure 1A). To improve separation of different phosphoforms, 119

we optimised PAGE systems. The best separation occurred on 10% 120

acrylamide gels containing low bis-acrylamide concentrations of 0.06% (low-121

bis gels) compared to the usual 0.3%. In such gels, Scar from migrating 122

Dictyostelium resolves into at least six distinct bands (Figure 1B) with clear 123

separation on an intensity plot. Lambda phosphatase resolved the multiple 124

bands to a single one, shifting the multiple intensity peaks (solid line) to 125

single peak (dotted line) (Figure 1C), confirming that the multiple bands are 126

due to phosphorylation, and Scar is typically hyperphosphorylated to varying 127

degrees. 128

Similarly, WAVE2 from two mammalian lines (human MDA-MB-231 and 129

mouse NIH3T3) appears on Western blots as a broad band with a single 130

intensity peak (Figure 1D). As with Dictyostelium Scar, low-bis gels reveal 131

multiple phosphorylated WAVE2 bands (Figure 1E). Intensity plots show 132

7

multiple well-resolved peaks that are shifted down after phosphatase 133

treatment confirming the bands represent WAVE2 phosphoforms. 134

Identification of phosphorylation sites in vivo 135

Previous reports identified Scar/WAVE phosphosites using protein expressed 136

in vitro (11), or overexpressed fusion proteins (9). Because Scar/WAVE’s 137

biological roles occur exclusively as part of the five-membered Scar/WAVE 138

complex, and overexpressed proteins are unevenly and incompletely 139

incorporated into the complex, we analysed the phosphorylation of native 140

proteins. We purified the Dictyostelium Scar complex using nap1- cells stably 141

transfected with single copies of GFP-Nap1 (14), yielding normal expression 142

levels, and pulled down the complex using GFP-TRAP. Under these 143

conditions Scar is only purified if incorporated in a properly assembled 144

Scar/WAVE complex. Gel-purified Scar bands (Supp. Figure 1A &B) were 145

analysed by LC-MS/MS to identify phosphopeptides. Scar/WAVE is 146

composed of N-terminal SH, central B and polyproline, and C-terminal V, C 147

and A domains (21). We identified three phosphorylated tyrosines (Y88, 129, 148

210) in the SH domain, three phosphorylated serines (S287, 290, 301) in the 149

polyproline and three (S384, 388, 389) in the V domain (Figure 1F). These are 150

additional to the constitutive phosphorylations found by Ura et al. from 430-151

440 in the A domain. The number of SCAR forms discernible as bands in 152

western blots from migrating cells is therefore not a surprise – at least 16 sites 153

are phosphorylated in vivo in normal cells. 154

Conspicuously, none of the phosphorylated serines made up MAP kinase 155

consensus sites (Supplementary Table 1). This was surprising, as a number of 156

papers report that Scar/WAVE bands (phosphorylation) are caused by ERK2, 157

which has a strong preference for prolines 2aa N-terminal to and 1aa C-158

terminal to the target site (22). The sites in human WAVE2 do not fit the MAP 159

8

kinase consensus (Supplementary Table 1), with the exception of T346, which 160

is relatively sparsely phosphorylated (https://phosphosite.org). 161

162

To confirm that we had identified the full range of phosphorylations, we 163

tested which amino acids had to be mutated to block extra bands in westerns. 164

We expressed un-phosphorylatable (Y-F; S-A) and phosphomimetic (S-D) 165

mutants in scar- cells. Removing the five phosphorylated serines in the A 166

domain of Scar (14) by deleting the VCA domain (ScardVCA) did not reduce the 167

number of bands (Figure 1G). Mutation of Y88/129/210F in SHD of Scar did 168

not affect the band shifts (Figure 1H). Mutation of the three serines in the 169

polyproline domain (ScarS3A, Figure 1H) to alanines reduced the number of 170

bands but did not abolish them. Mutating two more nearby serines (giving 171

ScarS5A; S284,290,301,335,339) gave near-complete loss of Scar bands (Figure 172

1I). Additional mutation of S384/388/389A from the V domain, giving ScarS8A 173

(Figure 1J), yielded Scar that was nearly homogeneous, with a single very 174

faint band above the main Scar population. The residual heterogeneity was 175

lost in cells whose Scar had also lost the sites in the A domain (14), giving 176

ScarS13A (Figure 1I, J & K). Similarly, mutation of all 5 polyproline 177

serines(ScarS5D), or all 8 polyproline+V domain serines to aspartate (ScarS8D), 178

caused a substantial mobility shift and two distinct bands on low bis gel 179

(Figure 1K). The mobility shift of ScarS8D was also obvious on a normal-bis gel 180

(Supp. Figure 1C), but only a single band of ScarS8D was resolved. These two 181

forms are apparently not caused by phosphorylation; strong phosphatase 182

treatment, which also caused significant proteolysis, caused only partial loss 183

of the upper band (Figure 1L); the difference may be due to a small modifier 184

like ubiquitin, though we were unable to find ubiquitin itself by western blot 185

in any variant (ScarWT, ScarS8A or ScarS8D; Supp. Figure 2). 186

9

The mammalian Scar/WAVE2 has been also shown to be phosphorylated at 187

multiple positions (Figure 1M, Supp. Table 1). Similar to Scar, mutation in 188

WAVE2S8A/T1A (S293/296/298/308/343/351/429/442/T346A) removed all 189

phosphorylated bands (Figure 1N). This confirmed that the multiple bands in 190

WAVE2 are due to S/T phosphorylations. 191

In conclusion, the multiple bands seen in westerns are mostly caused by 192

differential phosphorylation at sites in the polyproline region, with minimal 193

contributions from the V and A domains. 194

195

Scar/WAVE is phosphorylated after activation 196

The Scar/WAVE complex must interact with the GTP-bound, active form of 197

Rac1 before it can catalyse Arp2/3 complex activation and pseudopod 198

generation (3, 23–28). We would not expect Rac interactions to be important to 199

the phosphorylation if, as described in several publications (3, 23, 26), the 200

phosphorylation is an upstream process regulating Scar/WAVE activation. 201

We therefore determined the effect of Rac1 binding on Scar/WAVE 202

phosphorylation. First we inhibited the Rac1 activity in both Dictyostelium 203

and B16F1 cells by EHT1864, an effective inhibitor of RacGEF activity (29, 30), 204

and assessed the band shifts of Scar/WAVE. EHT1864 treatment resulted in 205

complete loss of Pak-CRIB-mRFPmars2 from cell periphery and depolarized 206

Dictyostelium cells (Figure 2A, Suppl. Video 1), confirming that the inhibitor 207

reduced active Rac levels to almost nothing. Similarly, lamellipods of B16F1 208

cells collapsed after EHT1864 treatment; their F-actin at cell edges also 209

appeared reduced (red, Figure 2B). 210

To assess the effect of Rac1 inhibition on Scar phosphorylation, Dictyostelium 211

cells were incubated with 50µM EHT1864 for 2, 5 and 10 min, lysed, and 212

analysed by low-bis western. Inhibition of Rac1 resulted in a strong reduction 213

10

in Scar phosphorylation (Figure 2C). The intensity profile shows the 214

disappearance of peaks for polyphosphorylated Scar even more clearly 215

(Figure 2D). Quantification of the fraction of Scar in the upper bands shows 216

approximately 50% reduction (Figure 2E). This figure underestimates the total 217

change in phosphate, because the higher bands that are disproportionately 218

lost contain several phosphates. Similarly, inhibition of Rac1 in B16F1 cells by 219

EHT1864 reduced the intensity of the upper bands of WAVE2 within 2 h; the 220

change became even clearer after 6 h of treatment (Figure 2F). Intensity plots 221

of WAVE2 bands show strong reduction in the highest peak of WAVE2 (0h; 222

pink) after EHT1864 treatment (Figure 2G). Quantification of the ratios of the 223

upper (intense) and lowest bands also suggest a substantial loss of WAVE2 224

phosphorylation (Figure 2H, again an underestimate of total phosphate loss). 225

This implies that Scar is only phosphorylated after it has bound to Rac1, and 226

thus been activated. 227

Generalized inhibition of Rac1 activity is strongly likely to cause secondary 228

effects, as many proteins such as kinases (for example PAKs (31)) are 229

important for other aspects of motility and require Rac to be activated. To 230

verify that active Rac1’s effect on Scar phosphorylation is direct, we examined 231

cells in which the Pir121 protein was replaced by Pir121-EGFP, either wild 232

type or the Rac1 non-binding, A-site mutant (3, 5). Pak-CRIB-mRFP-mars2 (8) 233

localizes to the pseudopods and cell periphery in both strains (Figure 2I; 234

Panel I, Suppl. Movie 2), showing that Rac is similarly activated in WT and 235

mutant cells. However, the Scar complex is only activated and localized in 236

cells expressing the functional Pir121EGFP, not the A-site mutant (Figure 2I; 237

Panel II, Suppl. Movie 2). Scar phosphorylation is greatly diminished in the A 238

site mutant (Figure 2J), to a level comparable to that seen in Rac-inhibited 239

cells. This confirms that Scar phosphorylation occurs only after the complex is 240

activated by interaction with Rac1. 241

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242

To affirm the sequence of activation and phosphorylation, we tested the effect 243

of latrunculin, an inhibitor of actin polymerization (32, 33), that causes 244

exaggerated membrane recruitment of the Scar/WAVE complex in 245

mammalian cells (34) and Dictyostelium. In Dictyostelium, latrunculin causes 246

concentrated and persistent recruitment of Scar complex to the membrane 247

(Figure 2K, Suppl. Video 3). Upon latrunculin treatment Scar 248

phosphorylation rapidly increases; like the Scar recruitment, the 249

phosphorylation reaches a far higher level than is normally seen (Figure 2L). 250

Thus Scar phosphorylation again correlates with its recruitment and 251

activation, rather than upstream processes. 252

It is interesting to note that there is currently no biochemical assay for 253

Scar/WAVE activity. The only measure of activation is recruitment to the 254

extreme leading edge, and downstream consequences such as actin 255

polymerization. This phosphorylation assay could therefore be a potentially 256

useful indirect measure of recent Scar/WAVE activation in vivo. 257

Chemotactic signalling does not control Scar phosphorylation 258

Earlier reports described the MAP kinase ERK2, induced downstream of 259

chemotactic and growth factor signalling, as the principal driver of 260

Scar/WAVE phosphorylation (9, 11, 19). This is an important issue, because it 261

provides a clear connection between chemoattractant signalling and 262

pseudopod initiation and growth that is otherwise lacking. As described 263

earlier, we questioned this result, because MAP kinase sites are usually 264

restricted to a narrow consensus, whereas the sites we identified were 265

diverse. We therefore examined the effect of extracellular signals and MAP 266

kinases on Scar/WAVE phosphorylation. Growing Dictyostelium cells use 267

folate as a chemoattractant. During multicellular development they 268

12

downregulate folate receptors and express receptors to extracellular cAMP. 269

Treatment of growing cells with folate, or developed cells with cAMP, causes 270

increases in F-actin levels that are thought to be important for chemotaxis (35, 271

36). To provide a consistent narrative, we examined Scar phosphorylation 272

after both treatments. Despite massive activation of erkB protein, no change in 273

the Scar phosphorylation was seen in either folate- or cAMP-treated cells 274

(Figure 3 A & D), either visually from the gel or when expressed 275

quantitatively as the phosphorylated fraction (Figure 3B, C, E & F). 276

Knockouts of the Dictyostelium Gb protein show a complete loss of G-protein 277

signalling (37) and reduced ErkB activity (38); since there are also no tyrosine 278

kinase receptors in Dictyostelium these cells are complete chemotactic nulls. 279

However, Gb- mutant cells have normal levels of Scar phosphorylation 280

(Figure 3G &H), as well as apparently normal pseudopods (39). This confirms 281

that chemotactic signalling is not required for Scar phosphorylation. 282

We determined the importance of MAP kinase signalling for Scar/WAVE 283

phosphorylation in both Dictyostelium and mammalian cells. First we 284

examined the band shifts for Scar in western blots from erkA-, erkB- and erkAB- 285

nulls of Dictyostelium (40). The band shifts on the western blot and quantitated 286

peaks were not discernibly different from the WT parent (Figure 3I&J). Since 287

ErkA and ErkB are the only MAP kinases in Dictyostelium, MAP kinase 288

signalling is not important for Scar/WAVE phosphorylation. To confirm this 289

result is generally true, we examined the WAVE2 phosphorylation band shifts 290

in mammalian NIH3T3 and B16F1 cells after inhibition of Erk2 activity. 291

Treatment with U0126, an efficient inhibitor of MEK (41) immediately 292

abolished Erk2 phosphorylation (and thus activity) in both NIH3T3 and 293

B16F1 cells, but did not change the phosphorylation state of WAVE2 even 294

after prolonged incubation (Figure 3 K, L, M&N). These results confirm that 295

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the Scar/WAVE phosphorylations we observe are neither executed by Erk2 296

nor do they require Erk2 function. 297

Physical adhesion promotes Scar/WAVE phosphorylation 298

More generally, our data suggest Scar/WAVE phosphorylation is not 299

primarily regulated by extracellular signalling. Another strong candidate 300

regulator of pseudopod and lamellipod is cell:substratum adhesion. In the 301

absence of adhesion, cell protrusions collapse, and the Scar/WAVE complex is 302

strongly localised to the adherent edges of protrusions. We exploited the 303

ability of Dictyostelium to grow either in suspension or adhesion. First, we 304

compared the differences in Scar phosphorylation in cells grown in 305

suspension and adhesion in both vegetative and starved cells. The more 306

phosphorylated bands (* in Fig. 4A) are more intense in cells grown adherent 307

to Petri plates, when compared with suspension-grown cells. Similarly, 308

intensity plots of Scar from cells adherent to a surface clearly show more 309

peaks and greater intensities of more-phosphorylated peaks compared with 310

cells grown in suspension (Figure 4B). This observation suggested that 311

adhesion is a strong stimulant of Scar phosphorylation. To investigate this in 312

more detail, we allowed suspensions of cells to adhere to a plastic surface, 313

and observed a clear and highly significant (p= 0.0089; Kruskal Wallis test) 314

upregulation of Scar phosphorylation (Figure 4 C&D). The proportion of 315

intensity in phosphorylated bands increases from 25±3.98% to 42.5±1.99% 316

(Mean±SEM; Figure 4D). Conversely, when we examined adherent cells that 317

had been de-adhered by a stream of buffer from a pipette and kept in 318

suspension, we saw an obvious reduction in phosphorylated bands (Figure 319

4E). The proportion of intensity in phosphorylated bands decreased from 320

40±3.59 to 20±3.08 (mean, SEM; Figure 4F). Thus cell:substrate adhesion 321

causes activation of Scar phosphorylation. 322

14

We examined whether cell:substrate adhesion coupled to Scar activation via 323

adhesion-linked signalling, or through a physical process such as 324

deformation. This was enabled by talin A/B double knockout Dictyostelium 325

cells. Talin is a key couple between adhesion molecules like integrins and the 326

actin cytoskeleton (42, 43) and talA/B- mutant cells completely fail to adhere 327

when allowed to fall onto a substratum (44). Consequently they remain 328

extremely spherical and pseudopod-free when cultured, even in dishes where 329

normal cells adhere. However, we were able to force talA/B- cells into 330

adhesion to plastic, glass or 1 % agar surfaces by carefully aspirating the 331

liquid from the dishes so cells were compressed by capillary forces. Scar 332

phosphorylation increases sharply in cells that are making talin-independent 333

contacts with the substratum and the comparison of Scar phosphorylation 334

between suspension and attached talA/B- cells are identical to WT cells (Figure 335

4G). Intensity plots show no difference in phosphorylation between adherent 336

WT cells and talA/B- cells that have been forced to adhere (Figure 4 H, I & J). 337

This result implies that Scar/WAVE phosphorylation is mediated by the 338

physical process of attachment – through mechanochemical processes, for 339

example - rather than through canonical integrin adhesion signalling. 340

To determine if Scar/WAVE2 phosphorylation in mammalian cells is also 341

adhesion dependent, we compared the band shifts in cells that grow in 342

suspension (Jurkat and JVM3) and adhered to a surface (NIH3T3, the 343

pancreatic cancer lines PDACA & B, Cos7, MDA-MB-231 and B16F1). The 344

Jurkat and JVM3 cells have less intense upper bands, meaning highly 345

phosphorylated Scar/WAVE2 is less abundant. Both also have an additional 346

lower band (representing less-phosphorylated Scar/WAVE2) not seen in any 347

of the cells that grow in adhesion (Figure 4K). The intensity ratios of the 348

upper intense and lowest of the bands present in all cells showed a marked 349

increase in adherent cells (Black bars in Figure 4L) compared with suspension 350

15

cells (White bars in Figure 4L). We also modulated adhesion by varying cell 351

density – cells grown at high confluency have a lower adhesion area due to 352

space constraints imposed by neighbouring cells. The amount of Scar/WAVE2 353

phosphorylation decreases with confluency in both NIH3T3 and B16F1 cells 354

(Figure 4M, N & O). 355

Unphosphorylated and Phosphomimetic Scar Mutants are Fully 356

Functional 357

In essentially every lamellipod or pseudopod of every normal cell, actin and 358

Arp2/3 complex recruitment are mediated by the activation of the Scar/WAVE 359

complex (2). Phosphorylation of the polyproline domain has been described 360

as important for Scar/WAVE complex activation and formation of such 361

protrusions (9–11, 14, 19, 20). To determine the importance of polyproline 362

domain phosphorylation to cell migration in vivo, we constructed 363

unphosphorylatable (ScarS8A) and phospho-mimetic (ScarS8D) mutants, with 364

serines mutated to alanines and aspartates, respectively (Figure 1F & J) and 365

expressed them in scar- cells. Both mutants were expressed at wild type levels 366

(Figure 1J). 367

Cells lacking Scar are inefficient in migration, as they have small pseudopods 368

and migrate mainly by blebbing, and are less directional ((8)Figure 5A; Panel 369

I, Suppl. movie 4). In contrast, scar- cells rescued with ScarWT, ScarS8A and 370

ScarS8D formed longer pseudopods that moved faster and split frequently 371

(Figure 5A; Suppl. Video 4). Pseudopod formation was similar in cells rescued 372

with WT and either Scar mutant. The migration speed of knockout cells was 373

completely rescued by expression of wild type or either phosphomutant Scar 374

(Figure 5B), with phosphorylation changes causing a slight increase in cell 375

speed. The tortuosity of the cell perimeters, which describes the amount of 376

recent protrusion and shape change, was low in scar- cells but rescued by 377

16

ScarS8A and ScarS8D just as well as by ScarWT (Figure 5C). Thus, 378

phosphorylation is clearly neither a precondition for activity, nor a direct 379

cause of SCAR inactivation. 380

Unphosphorylatable Scar is Recruited in Larger and Longer-lived 381

Patches 382

Activated Scar complex localizes to the fronts of cells and causes actin 383

protrusions there (8). We have found that GFP-tagging Scar itself greatly 384

alters both Scar and pseudopod dynamics. Therefore to examine the effects of 385

Scar phosphorylation on the dynamics of Scar recruitment, we expressed Scar 386

mutants in Scar- cells in which Nap1 has been replaced by a single copy of 387

GFP-Nap1 (i.e. scar-/Nap1GFP cells; Ura et al., 2012). ScarWT, ScarS8A and ScarS8D 388

all localized efficiently at the pseudopod periphery, but there were marked 389

differences in the dynamics of the association (Figure 5D; Suppl. Video 5). As 390

usual (14) ScarWT was recruited in bursts of fairly short duration (8.2 s ± 5.9s, 391

mean±SD). The unphosphorylated Scar (ScarS8A) remained recruited for a 392

substantially longer duration (13.7s ± 11.63s, mean±SD). As well as being 393

longer-lived (Figure 5E), ScarS8A patches were also larger (Figure 5F). This led 394

to changes in patch frequency – the larger, longer-lived ScarS8A patches were 395

made at a lower frequency than ScarWT (Figure 5G). Conversely, localization 396

of ScarS8D was even briefer (6.37 ±1.94s, mean±SD) and patches were made at 397

an even higher frequency than ScarWT, though they were about the same size. 398

Based on these observations, we hypothesized that Scar/WAVE 399

phosphorylation is not required for its activation, but rather modulates cell 400

migration by controlling the dynamics of Scar/WAVE patches. To confirm 401

this, we constructed nearly completely un-phosphorylated and 402

phosphomimetic Scar by additionally mutating the 5 serine residues of the 403

acidic domain (Ura et al., 2012). Scar complex and F- actin were both 404

17

beautifully accumulated in the pseudopods of cells expressing ScarS13D and 405

ScarS13D (Suppl. Video 6). 406

To further confirm this observation in B16 F1 mouse melanoma cells, we 407

employed WAVE1/2 knock out cells and examined the effect of unphospho- 408

mutations in the proline-rich domain (WAVE2S8A/T1A; Figure 1N). WAVE1/2 409

KO cells transfected with empty vector did not form lamellipodia (Figure 5H). 410

However, expressing both WAVE2WT and WAVE2S8A/T1A in knockout cells 411

rescued lamellipodia formation. These results firmly indicate that Scar 412

phosphorylation is neither required for its activation, nor does it cause direct 413

inactivation. 414

Scar Phosphomutants Rescue scar-/wasp- Cells 415

In Dictyostelium cells lacking Scar, WASP can be repurposed to drive 416

pseudopod formation and Arp2/3 complex activation (8, 45), which 417

complicates the phenotypes of scar mutants. Scar-/wasp- cells cannot move 418

and cannot grow; we have therefore developed a tetracycline-inducible 419

double knockout (scartet/wasp-) cell line to test Scar function without WASP 420

complementation (45). We exploited this line to test unphosphorylatable Scar 421

mutants’ ability to support growth and pseudopod formation. Scartet/wasp- 422

cells were transfected with ScarS8A and ScarS8D then deprived of doxycycline 48 423

h prior to experiments to remove native Scar. As usual (46) both mutants 424

express at wild type levels (Figure 6A). Importantly, expression of both 425

ScarS8A and ScarS8D is dominant; expression of the native Scar is suppressed 426

even in the presence of doxycycline (Figure 6A). 427

Similar to ScarWT, both ScarS8A and ScarS8D rescued the growth of Scartet/wasp- 428

cells (Figure 6B). To study the migratory phenotype, cells were examined 429

migrating under agarose up a folate gradient. As previously observed (45), 430

repressed Scartet/wasp- cells made long, stable filopods, but could not form 431

18

pseudopods or migrate. However, cells expressing either ScarS8A or ScarS8D 432

showed clear localisation of Scar complex to protrusions and F-actin 433

polymerisation (Figure 6 C&D; Suppl. Video 7), and robust pseudopod 434

formation (Figure 6E; Suppl. Video 8). Both mutants rescued cell migration 435

effectively (scar-/wasp- cells do not migrate under the agar, so their speed 436

cannot be measured in this assay, but it is essentially zero). ScarS8A supported 437

a substantially higher speed (8.35 ± 3.2µm/min, mean±SD) than either WT 438

(3.71 ± 2µm/min, mean±SD) or ScarS8D (5.2 ± 2.1 µm/min, mean±SD; Figure 6F). 439

These results firmly indicate that unphosphorylatable and phosphomimetic 440

mutants are fully functional for growth and cell migration in cells without 441

requiring cooperation from WASP. 442

Phosphorylation as a Degradation Signal 443

Scar/WAVE complex dynamics at the pseudopod periphery are poorly 444

understood. In particular, the regulation of Scar/WAVE removal – which 445

must be rapid, to allow the dynamic behaviour observed in moving cells - is 446

mysterious (24). Our results suggest that phosphorylation is among the 447

mechanisms that control the rapid changes in Scar complex localization (Figs 448

2K, 5 and 6). One way this could be achieved is by promoting breakdown of 449

the complex. This seems particularly appropriate as we previously found that 450

mutations in the acidic domain rendered Scar unstable (14) as well as 451

excessively active. Furthermore, re-examining our earlier data (Figures 4D 452

and 4F, for example) we noticed a surprising change – under conditions 453

where Scar phosphorylation decreases, such as deadhesion, there is an acute 454

increase in the total amount of protein (Figure 7A). Similarly a drop in Scar 455

phosphorylation causes protein levels to increase acutely (Figure 7B). We 456

therefore compared the stability of ScarWT, ScarS8A and ScarS8D during 457

starvation, with and without cycloheximide to inhibit protein synthesis. 458

19

Starvation makes cells move more rapidly (47) and increases Scar levels 459

(Figure 7 C&D). In the absence of cycloheximide, ScarWT & ScarS8A levels 460

increased as usual during starvation (Figure 7 C&D). ScarS8D levels, however, 461

decreased (Figure 7C&D). When cells were starved with cycloheximide, 462

amounts of ScarWT, ScarS8A and ScarS8D all decreased, but the loss was 463

substantially stronger in ScarS8D (Figure 7C & E). This result shows that 464

phosphomimetic Scar is less stable, implying that the reason ScarS8D makes 465

smaller and shorter-lived pseudopods is that it is degraded more rapidly. 466

20

Discussion 467

We have used an improved assay for phosphorylation of Scar/WAVE’s 468

polyproline domain to open a window on pseudopod dynamics. This has 469

revealed a number of surprises. Phosphorylation occurs on multiple sites 470

after Scar/WAVE is activated by proteins such as activated Rac, and reveals 471

an adhesion-dependent, signalling-independent pathway. It is clear that 472

these phosphorylations are not important for Scar/WAVE to be activated, and 473

it seems likely that they do not inhibit its activation, either. Rather, 474

phosphorylation appears to be a relatively subtle tool by which cells can 475

manipulate the dynamics (both recruitment and loss) of Scar/WAVE patches 476

and thus pseudopods. Pseudopod lifetime intersects with a wide range of cell 477

biology – it underlies the mechanism of chemotactic steering (48, 49), and 478

regulates cell polarity, persistence (50) and shape change (51) – and mutants 479

with uncontrolled pseudopod lifetime have serious motility defects (4). Thus 480

although the function of Scar/WAVE phosphorylation is different from what 481

has been believed, it is fundamentally physiologically important. 482

483

Our finding that signalling does not greatly alter the rate of Scar/WAVE 484

phosphorylation, and thus activation, does not imply that signalling is not 485

important to pseudopod formation and evolution. Chemotaxis – cells 486

migrating up gradients of soluble signalling molecules – clearly happens, and 487

is clearly mediated by pseudopods. However, we (48) and others (49) have 488

found that normal chemotaxis rarely involves initiation of new pseudopods – 489

rather, pseudopod generation is mostly random, but localised receptor 490

activation modulate the positions where pseudopods evolve, their lifetime 491

and stability after they are formed, and sometimes the pattern in which new 492

split pseudopods are directed. None of these measured processes require 493

21

extracellular signals causing direct activation of pseudopod catalysts; rather, 494

more subtle changes like the precise subcellular localisation and timing of 495

Scar/WAVE activation could be biased by chemoattractant signalling. 496

Equally, chemoattractants could work through pathways other than 497

Scar/WAVE activation, biasing the growth and lifetimes of pseudopods rather 498

than their initiation. This is in full agreement with the pseudopod-centred 499

view of chemotaxis (52), which emphasizes that pseudopods are self-500

organized, rather than the outcome of external signals. 501

502

The kinase sites around the polyproline domain have little in common with 503

one another, and do not match the consensus sequences for most kinases that 504

are active in the actin domain. We hypothesize that the phosphorylation sites 505

are sterically inaccessible until the Scar/WAVE complex is activated. Once the 506

complex is active it adopts an open conformation, whereupon any and all of a 507

large range of constitutive kinases such as CK2 and GSK3 could act on it. In 508

this case the phosphorylation would be a passive reflection of the fact that the 509

complex had been activated, rather than a driver or even modulator of the 510

initial activation process. This explanation is comfortably consistent with the 511

wide range of data we have obtained, though it paints a different picture of 512

Scar/WAVE activation from what we had expected. 513

514

This work showcases our ability to use scar-/wasp- cells to test the functioning 515

of mutated Scar proteins, and the phenotypic changes caused by alterations in 516

the sequence. Previously, results have been complicated because WASP 517

changes its organization and cell biological role when the Scar complex is not 518

present (8). We have therefore been working against an ill-defined 519

background of partial rescue. In this work we show that either 520

phosphomimetic or unphosphorylatable Scar is fully able to rescue the 521

22

mutant phenotypes, because scar-/wasp- Dictyostelium cells cannot grow, do 522

not make pseudopods, and barely move. Similarly, the use of 523

WAVE1/WAVE2 knockout B16-F1 cells allows detailed study of 524

physiologically normal levels of WAVEs without partial complementation. 525

We therefore look forward to a new focus on physiological function and a 526

more direct connection between the dynamics of the Scar/WAVE complex, the 527

behaviour of pseudopods and lamellipods, and cell migration. 528

529

Our results imply that cell:substrate adhesion is a crucial driver of 530

Scar/WAVE activation. Adhesion has been an undeservedly neglected area of 531

Scar/WAVE biology. It seems intuitively obvious that pseudopod formation 532

should be regulated by adhesion – if pseudopods or lamellipods are 533

generated in a location where adhesion is difficult or impossible, they will be 534

inefficient or ineffective at producing cell migration. Cells should in 535

retrospect therefore be expected to monitor adhesion, and to favour 536

pseudopods that can attach. The results in Figure 4 showing normal 537

Scar/WAVE activation in talin knockouts offer one reason why connections 538

have so far been missed – activation does not require the integrin-linked 539

signalling pathways that dominate adhesion of (for example) mammalian 540

fibroblasts (53). Instead, we hypothesize that the connection is physical, by 541

direct mechanical coupling. This is a well-documented and common 542

mechanism for coupling proteins and cell behaviour (54). Overall, this work 543

exposes a new and conserved mechanism for regulating migration, which will 544

influence cells moving in many physiological contexts. We look forward to 545

seeing new biological examples emerge. 546

547

23

548

549

24

Acknowledgements 550

We thank Prof. Robert R. Kay for erkA and erkB knockout cells, Dr. Rachana 551

Patel for providing NIH3T3 cells, Drs Margaret O’Prey, Heather Spence and 552

Jamie Whitelaw for help in microscopy and mammalian cell culture, and Dr. 553

Simona Buracco for discussions about protein stability. 554

Funding 555

This work was supported by Cancer Research UK core grant number A17196 556

and Multidisciplinary Award A20017 to RHI, by the Deutsche 557

Forschungsgemeinschaft, grant GRK2223/1 to KR, and a grant from the NIH 558

(GM063691) to BG. 559

560

Authors’ contribution 561

RI and SPS conceived and designed experiments. SPS performed experiments. 562

PT assisted SPS during in experiments and provided essential reagents. SL 563

performed MS. SPS wrote the manuscript. RI and LM corrected and improved 564

the manuscript. MS, QT, BG and KR generated B16-F1 mutant cell lines and 565

plasmids 566

Declaration of Interests 567

The authors declare no competing interests 568

569

25

Figure Legends 570

Figure 1: Multiple phosphorylations in Scar/WAVE. 571

A) Western blot of Dictyostelium Scar using normal (0.3%) bis-acrylamide gels. 572

Whole cells were boiled in sample buffer and immediately separated on 10% 573

bis-tris gels, blotted and probed with an anti-Scar antibody, yielding a single 574

diffuse band. Scan of signal intensity shows a single, slightly diffuse peak. 575

Mccc1 is a loading control (55). 576

B) Western blot of Dictyostelium Scar using low (0.06%) bis-acrylamide gels. 577

Whole cells were boiled in sample buffer and immediately separated on 10% 578

bis-tris gels, yielding a multiple discrete bands. Scan of signal intensity shows 579

separate peaks. 580

C) Phosphatase treatment. Whole Dictyostelium cells were lysed in TN/T 581

buffer, then incubated with & without lambda phosphatase, boiled in sample 582

buffer, and analysed using low bis-acrylamide gels. The scan shows multiple 583

peaks resolved into one and shifted downwards after phosphatase treatment. 584

D) Western blot of mammalian WAVE2 in mammalian cells using normal 585

(0.3%) bis-acrylamide gels. Whole MDA-MB-231 and NIH 3T3 cells were 586

boiled in sample buffer, separated on 10% bis-tris gels, blotted and probed 587

with an anti-WAVE2 antibody, yielding a single band. Scan of signal intensity 588

shows a single peak. 589

E) Western blot of mammalian WAVE2 in mammalian cells using low (0.06%) 590

bis-acrylamide gels. Whole MDA-MB-231 and NIH 3T3 cells were boiled in 591

sample buffer and separated on 10% bis-tris gels, yielding at least 5 bands. 592

Incubation with lambda phosphatase resolves the bands into lower bands. 593

Scans of signal intensity show clear multiple peaks resolving incompletely 594

towards one lower peak. All experiments were repeated thrice with 595

comparable results. 596

26

F) Schematic of Dictyostelium Scar architecture and identified phosphorylated 597

residues. 598

G-K) Deletions of different amino acids. Lysates from Scar- cells expressing 599

ScarWT and Scar lacking the VCA domain (G), with Tyr 88, 129 & 201 600

substituted with F (H), Ser 287/290/301 substituted with Ala (H), Ser 601

287/290/301/335/339 substituted with Ala or Asp (I), Ser 602

287/290/301/335/339/384/388/389 substituted with Ala or Asp (J), and Ser 603

287/290/301/335/339/384/388/389/430/431/432/437/440 substituted with Ala or 604

Asp (K), were boiled in sample buffer and analyzed on low (0.06%) bis-605

acrylamide 10% bis-tris gels. Extra bands in Scar are completely lost after 606

substitution of 13 serines. 607

L) Phosphatase treatment of ScarWT, ScarS8A and ScarS8D. Lambda phosphatase 608

treated cell lysates were analysed using low (0.06%) bis-acrylamide 10% bis-609

tris gels. Unlike ScarWT, ScarS8D shows little removal of additional bands after 610

phosphatase treatment. 611

M) Schematic of human WAVE2 architecture and phosphorylated Ser/Thr 612

residues identified in published and high-throughput screens. 613

N) Loss of extra WAVE2 bands in B16F1 mouse cells after substitution of 614

polyproline serines. WAVE1/2KO cells were transfected with intact WAVE2 615

or WAVE2 with Ser 293/296/298/308/343/351/429/442 and Thr 346 substituted 616

with alanine, then lysed, boiled in sample buffer, separated using low (0.06%) 617

bis-acrylamide 10% bis-tris gels, blotted and probed with an anti-WAVE2 618

antibody. 619

620

621

622

Figure 2: Rac1 binding and Scar/ WAVE phosphorylation. 623

27

A) Effect of Rac1 inhibition by EHT 1864 on Pak-CRIB-mRFPmars2 624

localization in Dictyostelium. Dictyostelium cells expressing Rac1 marker, Pak-625

CRIB-mRFPmars2 were imaged by AiryScan confocal microscopy. Pak-CRIB-626

mRFPmars2 (Red) rapidly delocalises after addition of EHT1864 (EHT1864, 50 627

µM) treatment (Scale bar= 10 µm). 628

B) Effect of Rac1 inhibition by EHT 1864 on lamellipod formation in B16 629

melanoma cells. B16F1 cells were seeded on Laminin A coated glass, and 630

treated with EHT1864 (50 µM) for 6h. Cells were fixed and stained with Alexa 631

fluor 568-phalloidin. Untreated cells retained broad lamellipods with intense 632

F- actin at the leading edge, while lamellpods and F-actin are compromised in 633

the EHT1864 treated cells. 634

C-E) Effect of Rac1 inhibition by EHT 1864 on Dictyostelium Scar 635

phosphorylation. Cells were treated with 50 µM EHT1864 for the indicated 636

times, and lysates were analysed for Scar band shifts by western blotting 637

using low (0.06%) bis-acrylamide gels. Disappearance of extra bands after 638

EHT 1864 treatment indicates loss of phosphorylated Scar. Graph in D shows 639

density quantitation of western blots at different times. E shows ratio of 640

upper (more phosphorylated) band aggregate intensity to total Scar (bars 641

show mean±SEM, n=3). 642

F-H) Effect of Rac1 inhibition by EHT 1864 on B16 melanoma WAVE2 643

phosphorylation. Cells were treated with 50 µM EHT1864 for the indicated 644

times, and lysates were analysed for WAVE2 band shifts by Western blotting 645

using low (0.06%) bis-acrylamide gels. Disappearance of extra bands after 646

EHT 1864 treatment indicates loss of phosphorylated WAVE2. Graph in G 647

shows density quantitation of western blots at different times. H shows ratio 648

of upper (more phosphorylated) band aggregate intensity to total WAVE2 649

(bars show mean±SEM, n=3). 650

651

28

I) Requirement for Rac1-binding PIR121 A site for Scar complex localization 652

in Dictyostelium cells. Pir121- cells expressing Pir121-eGFP with and without 653

mutated A site (K193D/R194D) were coexpressed with Pak-CRIB- RFPmars2 654

and imaged by confocal microscopy while migrating under agarose up a 655

folate gradient. Upper panel: unaltered PIR121-eGFP; lower panel: Pir121-656

eGFPA site. Rac1 localizes (Red) to the membrane in both Pir121-EGFPWT and 657

Pir121-EGFPK193D/R194D. Scar complex (green) is recruited to sites of Rac1 658

activation only when Rac can bind. 659

J) Requirement for Rac1-binding PIR121 A site for Dictyostelium Scar 660

phosphorylation. WT, Pir121- and Pir121- cells expressing Pir121 with and 661

without mutated A site (K193D/R194D) were analysed for WAVE2 band shifts 662

by Western blotting using low (0.06%) bis-acrylamide gels. Phosphorylated 663

Scar bands are absent when PIR121’s A site is mutated. 664

K) Localization, distribution and hyperactivation of Scar complex after 665

latrunculin A treatment. Dictyostelium cells expressing eGFP-NAP1 were 666

imaged using an AiryScan confocal microscope. 5µM latA was added at t=0. 667

Scale bar = 10 µm. Scar complex activation at the cell membrane increases 668

after addition of Latrunculin. 669

L) Increased Scar phosphorylation after latrunculin A treatment. Dictyostelium 670

cells were treated with latA for the indicated times, then Scar band shifts were 671

analyzed by Western blotting using low (0.06%) bis-acrylamide gels. 672

Phosphorylated bands become markedly more abundant after latA treatment. 673

All experiments were repeated three times. 674

675

Figure 3: Signalling and Erk independent phosphorylation of Scar/WAVE. 676

A-C) Folate stimulation and Scar phosphorylation in Dictyostelium. Washed 677

growing cells were treated with 50µM folate for the indicated times, then Scar 678

band shifts were analyzed by Western blotting using low (0.06%) bis-679

29

acrylamide gels. Folate does not enhance Scar phosphorylation (B; bands 680

show mean ±SD, n=3) despite substantial signalling response shown by erkB 681

phosphorylation (C). 682

D-F) cAMP stimulation and Scar phosphorylation in Dictyostelium. 683

Aggregation-stage cells were treated with 50µM folate for the indicated times, 684

then Scar band shifts were analyzed by Western blotting using low (0.06%) 685

bis-acrylamide gels. cAMP does not enhance Scar phosphorylation (E) despite 686

very substantial signalling response shown by erkB phosphorylation (bars 687

show mean±SD; n=3). 688

G,H) Scar phosphorylation in WT and Gβ- cells. WT and Gβ- cell band shifts 689

were analyzed by Western blotting using low (0.06%) bis-acrylamide gels. 690

Both show similar band shifts of Scar, as reflected in the quantitation of the 691

upper band (H; bars show mean±SD; n=3) 692

I) Scar phosphorylation in MAP kinase (ERK) mutants. Scar-, WT, erkA-, erkB- 693

and erkAB- cells were analyzed by Western blotting using low (0.06%) bis-694

acrylamide gels. Single mutants show similar phosphorylation; double 695

mutant shows more, as shown by densitometry of the lanes (J). 696

K-N) WAVE2 phosphorylation in B16 (K,L) and NIH3T3 (M,N) cells after 697

inhibition of Erk activation. Cells were treated with U0126 for the indicated 698

times, then analyzed by Western blotting using low (0.06%) bis-acrylamide 699

gels. Erk and phospho-Erk levels were analyzed using normal gels and 700

antibodies against pan-Erk and phospho-Erk. Quantitation of the 701

phosphorylated bands shows no change despite essentially complete 702

inhibition of Erk (bars show mean±SD, n=3). 703

704

Figure 4: Physical adhesion enhances Scar/ WAVE phosphorylation. 705

A,B) Wild type Dictyostelium cells were grown in shaken suspension or 706

adhering to Petri dishes, then analyzed by Western blotting using low (0.06%) 707

30

bis-acrylamide gels. Adherent cells have more and intense bands of 708

Scar/WAVE. Densitometry of lane intensities (mean±SEM, n=3) shows a large 709

difference in the intensities of phosphorylated Scar bands. 710

C,D) Acute effect of adhesion on Scar/WAVE phosphorylation. Cells growing 711

in suspension were allowed to adhere in Petri dishes and lysed at indicated 712

time points. Cell lysates were analyzed by Western blotting using low (0.06%) 713

bis-acrylamide gels. The number and intensity of bands (*) increases with 714

time of adhesion. Quantitation of lane intensities (mean±SEM, n=3) confirms a 715

progressive increase in the intensities of phosphorylated Scar bands. 716

E,F) Acute effect of de-adhesion on Scar/WAVE phosphorylation. Cells grown 717

in Petri dishes were dislodged by a jet of medium from a P1000 pipette and 718

maintained in suspension (2x106 cells/ml) by shaking at 120 rpm. Cell lysates 719

were analyzed by Western blotting using low (0.06%) bis-acrylamide gels. 720

Quantitation of lane intensities (mean±SEM, n=3) confirms a progressive loss 721

in intensity of phosphorylated Scar bands. 722

G-J) Relative requirement of Talin and adhesion. WT and talA-/ talB- cells were 723

allowed to make physical contact with plastic Petri dishes, and where 724

appropriate forced to adhere by withdrawing the liquid with 3MM paper. 725

Cell lysates were analyzed by Western blotting using low (0.06%) bis-726

acrylamide gels. Densitometry of lane intensities shows a large difference 727

between WT and mutant cells on Petri dishes (I), but not when adhesion was 728

forced (J). 729

K,L) WAVE2 phosphorylation in adherent and nonadherent mammalian 730

cells. Lysates from cells that grow in suspension (Jurkat and JVM3) and 731

adhesion (NIH3T3, PDAC-A, PDAC-B, Cos7, MDA-MB231 and B16F1) lysates 732

were analysed for WAVE2 band shifts by Western blotting using low (0.06%) 733

bis-acrylamide gels. Suspension-growing cells have additional lower bands 734

(arrow) and less intense phosphorylated (*) band compared to adherent cells. 735

31

Graph (L) shows the ratio of upper intense band (*) and lowest band of 736

various mammalian cells. 737

M-O) Effect of culture density on WAVE2 phosphorylation. NIH3T3 and 738

B16F1 cells were grown at indicated cell densities and lysates were analysed 739

for WAVE2 band shifts by Western blotting using low (0.06%) bis-acrylamide 740

gels. WAVE2 phosphorylation decreases as the cell density increases. * 741

indicates phospho-WAVE2 bands. Graphs show ratio of upper intense band 742

(*) and lowest band. All graphs are representative of three independent 743

experiments. 744

745

Figure 5: Scar/WAVE phosphorylation mutants are functional. 746

A-C) Rescue of pseudopod formation by mutated Scar. Scar- cells were 747

transfected with ScarWT, ScarS8A and ScarS8D, and allowed to migrate under 748

agarose up a folate gradient while being observed by DIC microscopy. All 749

rescued cells formed actin pseudopods; those formed by ScarS8A expressing 750

cells were broad and elongated. Panel B shows quantitation of speeds and C 751

the tortuosity (total distance migrated/net distance migrated) of paths in B 752

(mean ± SD; n= 140Scar- 170 WT, 413S8A, 437S8D over 3 independent experiments; 753

****p≤0.0001, * p≤0.05, one-way ANOVA, Dunn’s multiple comparison test) 754

D) Subcellular localization of Scar complex. ScarWT, ScarS8A and ScarS8D were 755

expressed in Scar-/Nap1-/eGFP-NAP1 cells, allowed to migrate under agarose 756

up a folate gradient, and examined by AiryScan confocal microscopy. All 757

show efficient Scar/WAVE complex (green) localization to the pseudopods. 758

E,F,G) Lifetimes, size and generation rates of mutant Scar patches. Scar-759

/Nap1-/eGFP-NAP1 cells expressing ScarWT, ScarS8A and ScarS8D were allowed 760

to migrate up folate gradients under agarose, EGFP localization was observed 761

by AiryScan confocal microscopy (1f/3s). Patch lifetime was measured by 762

counting the number of frames a patch showed continuous presence of 763

32

labelled Scar complex, size was measured by hand from single frames, and 764

generation rate calculated from the number of patches lasting at least 2 765

frames. ScarS8A shows increased and ScarS8D diminished lifetime compared 766

with ScarWT . ScarS8A patches are longer-lived than ScarS8D and ScarWT , and 767

ScarS8A generates more and ScarS8D less frequently than ScarWT (mean ± SD; 768

n>25 cells; * p≤0.05, ** p≤0.01, **** p≤0.0001, one-way ANOVA, Dunn’s 769

multiple comparison test). 770

H) Rescue of lamellipodia formation by WAVE2 phosphomutants. B16F1-771

WAVE1-/2- cells expressing WAVE2WT and WAVE2S8A/T1A were allowed to 772

adhere on laminin A coated coverslips, fixed and stained with Alexa 568-773

phalloidin. WAVE2WT and WAVE2S8A/T1A both rescue lamellipodia formation. 774

775

Figure 6: Scar phosphomutants rescue growth and pseudopods in scar-776

/wasp- cells. 777

A) Expression of ScarWT, ScarS8A and ScarS8D in cells lacking endogenous Scar 778

and WASP. scartet/wasp- cells expressing ScarWT, ScarS8A and ScarS8D were 779

grown with or without doxycycline and analysed by Western blotting using 780

low (0.06%) bis-acrylamide gels. MCCC1 was used as a loading control. 781

B) Rescue of Scar/WASP mutant cell growth. scartet/wasp- cells were 782

transfected with ScarWT, ScarS8A and ScarS8D, expression of endogenous Scar 783

was blocked by removing doxycycline at t=0, and cell growth in suspension 784

was measured using a Casy cell counter (Innovatis). Points show mean±SEM, 785

n=3. 786

C) Localisation of Scar complex in scartet/wasp- cells expressing Scar 787

phosphomutants. ScarWT, ScarS8A and ScarS8D were co-expressed with 788

HSPC300-EGFP in Scartet/WASP- cells, and cells migrating under agarose up a 789

folate gradient were imaged by AiryScan confocal microsopy. 790

33

D) Localisation of F-actin in scartet/wasp- cells expressing Scar 791

phosphomutants. ScarWT, ScarS8A and ScarS8D were co-expressed with LifeAct-792

mRFPmars2 in scartet/wasp-, grown with doxycycline to maintain normal Scar, 793

then kept without doxycyline for 48hrs so only the mutant was expressed. In 794

cells without Scar or WASP, LifeAct was observed in thin spiky filament, but 795

the pseudopods of ScarWT, ScarS8A and ScarS8D expressing cells contained 796

normal F-actin levels. 797

E) Rescue of pseudopod formation by mutated Scar. scartet/wasp- cells were 798

transfected with ScarWT, ScarS8A and ScarS8D, and allowed to migrate under 799

agarose up a folate gradient while being observed by DIC microscopy. All 800

rescued cells formed actin pseudopods; those formed by ScarS8A expressing 801

cells were broad and elongated. ScarWT, ScarS8A and ScarS8D expressing cells 802

formed pseudopods. Panel F shows quantitation of speeds observed by 10 X 803

phase contrast microscopy). Almost no unrescued scartet/wasp- cells migrated 804

under the agar so no speed could be measured. (mean ± SD; n>278 cells from 3 805

independent experiments; **** = p≤0.0001, one-way ANOVA, Dunn’s multiple 806

comparison test). 807

808

Figure 7: Phosphorylation destabilizes Scar/WAVE. 809

A) Total Scar levels from adherent cells dislodged from the surface and 810

maintained in suspension. Data measured by densitometry from cells in Fig. 811

4F. Deadhesion, which diminishes phosphorylation, acutely increases SCAR 812

levels. 813

B) Total Scar levels from nonadherent cells acutely allowed to adhere to a 814

surface. Data measured by densitometry from cells in Fig. 4D. Adhesion, 815

which causes a strong increase in phosphorylation, acutely decreases SCAR 816

levels. 817

34

C-E) Reduced stability of phosphomimetic Scar. Scar- cells expressing ScarWT 818

ScarS8A and ScarS8D were starved with and without cycloheximide. Protein 819

samples were analysed for Scar expression at indicated time points using 820

normal 10% bis-tris gels. The graphs show densitometric quantitation of the 821

Scar bands from panel C, with and without cycloheximide (mean±SEM, n=3). 822

823

824

35

Supplementary Figure Legends 825

Supplementary Figure 1: GFP-TRAP pull down of EGFP-NAP1 cells. 826

A) Coomassie brilliant blue stained low-bis acrylamide PAGE gel of GFP-827

TRAP pull down samples. Lane 1,2,3 indicates samples from Scar-828

/EGFPNAP1 and lane 4, 5, 6 indicates Samples from EGFPNap1 cells. The Scar 829

band indicated on the gel was exiced for LC-MS/MS. 830

B) Representative western blot of above gel indicating phosphorylated Scar 831

bands. 832

833

Supplementary Figure 2: Incorporation of Scar mutants in the complex. 834

A) GFP-TRAP pull down was performed using MG132 treated and untreated 835

Dictyostelium Scar-/EGFP-NAP1 cells expressing ScarWT, ScarS8A and ScarS8D. 836

The eluates were analysed on low-bis PAGE western blotting for 837

Pir121/Nap1/Scar and ubiquitin. 838

B) Scar expression in the cell lysates was analysed by low-bis PAGE and 839

western blotting. 840

841

842

Video 1. Effect of EHT1864 on Rac1 and Scar complex localization. 843

Dictyostelium cells expressing PakCRIB-mRFPmars2 were allowed to migrate 844

under agarose up folate gradient and observed by AiryScan confocal 845

microscopy. Filmed at 1 frame/2s, Movie shows 10 frames/sec. EHT1864 was 846

added at frame 7 (after14 secs) in the video. 847

848

Video 2: Scar/WAVE and Rac1 activation in cells with mutant PIR121 A site. 849

Pir121 knock out cells expressing WT Pir121-EGFP and A site Pir121-EGFP 850

were further expressed with PakCRIB-mRFPmars2. Scar complex (green) and 851

36

PakCRIB-mRFPmars2 (Red) localization was visualized in migrating cells 852

under agarose up folate gradient. Filmed at 1 frame/2s, Movie shows 10 853

frames/sec. 854

855

Video 3: Effect of Latrunculin treatment on Scar complex localization. 856

eGFP-NAP1 cells were seeded on LabTekII rcover glass chambers and imaged 857

by AiryScan imaging. 5µm LatrunculinA was added to the cells undergoing 858

imaging. Filmed at 1 frame/15s, Movie shows 10 frames/sec. Latrunculin was 859

added at the frame 2 (after 15 sec). 860

861

Video 4: Pseudopod formation in Scar phosphomutants. 862

Scar- cells expressing ScarWT, ScarS8A and ScarS8D were allowed to migrate 863

under agarose up a folate gradient and observed by DIC. Filmed at 1 864

frame/2s, Movie shows 10 frames/sec. 865

866

Video 5: Scar complex localization in Scar phosphomutants. 867

Scar-/EGFP-Nap1 cells expressing ScarWT, ScarS8A and ScarS8D were allowed to 868

migrate under agarose up folate gradient and Scar complex activation in 869

pseudopods were observed by airyScan confocal microscopy. Filmed at 1 870

frame/2s, Movie shows 10 frames/sec. 871

872

Video 6: Scar complex activation in total Scar phosphomutants. 873

Scar-/EGFP-Nap1 cells expressing ScarS13A and ScarS13D were allowed to 874

migrate under agarose up folate gradient and Scar complex activation in 875

pseudopods were observed by airyScan confocal microscopy. Filmed at 1 876

frame/2s, Movie shows 10 frames/sec. 877

878

37

Video 7: Scar complex activation in Scar-/wasp- cells expressing 879

phosphomutant Scar. 880

Scartet/wasp- cells expressing ScarWT, ScarS8A and ScarS8D were allowed to 881

migrate under agarose up folate gradient and Scar complex activation in 882

pseudopods were observed by AiryScan confocal microscopy. Filmed at 1 883

frame/2s, Movie shows 10 frames/sec. 884

885

Video 8: Pseudopod formation in Scar-/wasp- cells expressing 886

phosphomutant Scar. 887

Scartet/wasp- cells expressing ScarWT, ScarS8A and ScarS8D were allowed to 888

migrate under agarose up folate gradient and were observed by differential 889

interference contrast microscopy. Filmed at 1 frame/2s, Movie shows 10 890

frames/sec. 891

892

38

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Tubulin

Phosphatase - + - +

NIH

3T3

NIH

3T3

MD

A-M

B-2

31

MD

A-M

B-2

31

WAVE2

ScarMccc1

Sca

r-

A B C

E D

Phosphatase - +

WT

WT

Sca

r-

*****

Figure 1 Singh et al.

***

0.0 0.1 0.2 0.3 0.4

50

100

150

200

250

Distance

Inte

nsity

(A.U

.)

0.0 0.2 0.4 0.6

100

200

300

Distance 0.0 0.2 0.4

100

200

300

Distance

-Phosphatase+Phosphatase

0.05 0.1 0.15 0.2 0.25

100

200

300

Distance

MDA-MB-231NIH3T3

Inte

nsity

(A.U

.)

Inte

nsity

(A.U

.)

Inte

nsity

(A.U

.) MDA-MB-231

0.05 0.1 0.15 0.2 0.25 0.350

100

150

200

250

Distance

NIH3T3-Phosphatase+Phosphatase

0.0 0.1 0.2 0.3

50

100

150

200

250

Distance

-Phosphatase+Phosphatase

Inte

nsity

(A.U

.)

WAVE2

Tubulin

B 1 255-256 376-377 443

SHD Poly-proline V C A

F

S S S S S SS S S S SS S

287 290 301 335 339 384 388 389 430 431 432 437 440

Y Y Y

88 129 210

S8S13

S5S3

Dd Scar

G HIn Scar-

IIn Scar-

Sca

rWT

Sca

rY88

F

Sca

rY88

/129

F

Sca

rY88

/129

/210

F

Sca

rS3A

Sca

rS5A

Sca

rS5D

Sca

r-

Sca

rWT

Sca

rdVC

A

****

****Scar

Mccc1

In Scar-

J

In Scar-

Sca

rWT

Sca

rS8A

Sca

rS8D

*

K

Sca

rS13

D

Sca

rS13

A

In Scar-

Scar

Mccc1

In Scar-

- + - + - + Phosphatase

Sca

rS8A

Sca

rS8D

Sca

rWT

B 1 170 218 411 498

SHD Poly-proline V C A

S S S S S S S T S S S S S S

293 296 298 308 343 346 351 429 442 482 484 488 489 497

S8/T1

WAV

E1/

2 K

O

WAV

E2W

T

WAV

E2S

8A/T

1A

WAVE2

Tubulin

M

N

Hs WAVE2

ScarMccc1

L

Pak

-CR

IB EHT1864 (50 μM) A B

C

I

Scar

Mccc1

PIR

121-

WT

A-

site

WT

A s

ite

PIR

121G

fp in

Pir1

21-

Figure 2 Singh et al.

F

K

Latrunculin A

0 10 20 30 min

5 min 10 min 15 min

19 min

Sca

r com

plex

J

L

EHT1864 (50 μM)

-15s 60s

Pak-CRIB Scar Complex Merged

Scar

Mccc1

Ax3

LatrunculinA

**** *

0 h 6 h 0 s 90 s

Scar

Mccc1

0 2 5 10 min EHT1864

EHT1864 0 2 4 6 h

WAVE2

***** *

**

pERK

ERK 0 2 4 6

0.0

0.5

1.0

1.5

2.0

Time (h)

Inte

nsity

(upp

er/lo

wes

t)

0.1 0.2 0.3

50

100

150

200

250

Distance

Inte

nsity

(A.U

.)

0246

0.0 0.1 0.2 0.3 0.4

100

200

300

Distance

Inte

nsity

(A.U

.)

02510

D E

G H

0 2 5 100

20

40

60

Time (min)

% P

hosp

hory

latio

n

19 min

A B

Mccc1

Scar

Folate 0 20 40 60 120 240 Sec

Figure 3 Singh et al.

C

Scar

Mccc1

Sca

r-

Erk

A-

Erk

B-

Erk

AB

-

WT

WAVE2

pErk

Erk

Uo126 0 5 10 20 30 60

Mccc1

Scar

WAVE2

pErk

Erk

Uo126 0 5 10 20 30 60

In Dictyostelium

B16F1

NIH3T3

D EMccc1

pERK

pERK

Mccc1

cAMP 0 20 40 60 120 240 sec

0 20 40 60 120 2400

10

20

30

40

50

Time (Sec.)

% P

hosp

hory

latio

n

Scar (Folate,50 μM)

0 20 40 60 120 2400

2

4

6

Time (Sec.)

Rel

ativ

e fo

ld c

hang

e

pErkB (Folate, 50 μM)

0 20 40 60 120 2400

10

20

30

Time (Sec.)

% P

hosp

hory

latio

n

Scar (cAMP, 1μM)

0

5

10

15

20 pERKB (cAMP, 1μM)

Time (Sec.)

Rel

ativ

e fo

ld c

hang

e

0 20 40 60 120 240

Scar

Mccc1

WT

Gβ−

WT

15 min

Gβ-0

10

20

30

40

% P

hosp

hory

latio

n

0.05 0.15 0.25 0.35 0.4550

100

150

200

250

300

Distance

Inte

nsity

(A.U

.) WTErkA-ErkB-ErkAB-

0 5 10 20 30 600.0

0.5

1.0

1.5

2.0

Time (min)

Inte

nsity

(Top

/bot

tom

)

0 5 10 20 30 600.0

0.5

1.0

1.5

2.0

2.5

Time (min)

Inte

nsity

(Top

/bot

tom

)

F

B16F1

NIH3T3

G H I J

K L

M N

a

b

A

FE

G

L

N

WAVE2

Tubulin

JV

M3

Jur

kat

NIH

3T3

PD

AC

A

PD

AC

B

Cos

7

MD

A-M

B-2

31

B16

F1

Figure 4 Singh et al.

0 2 5 10 20 30

Time after adhesion (min)

Mccc1

Scar *******

a

b

0 1 2 5 10 15

Scar

Mccc1

Time after deadhesion

***** *

0 2 5 10 20 300

10

20

30

40

50

Adhesion (Time, min)

% P

hosp

hory

latio

n0 1 2 5 10 15

0

10

20

30

40

50

Suspension (Time, min)%

Pho

spho

ryla

tion

0.0 0.1 0.2 0.3

100

200

300

Distance

Vegetative

Suspension Adhesion

Inte

nsity

(A.U

.)

25 50 75 100 Confluency (%)

WAVE2

Tubulin

WAVE2

Tubulin

B16F1

NIH3T3

25 50 75 1000.0

0.5

1.0

1.5

2.0

Confluency (%)

Inte

nsity

(Upp

er/lo

wes

t) B16F1

25 50 75 1000.0

0.5

1.0

1.5

2.0

2.5

Confluency (%)

Inte

nsity

(Upp

er/lo

wes

t) NIH3T3

JVM3

Jurka

t

NIH3T

3

PDAC-A

PDAC-B

COS-7

MDA-MB-23

1

B16F1

0

1

2

3

Inte

nsity

(Upp

er/lo

wes

t)

*

Suspension Adhesion

Scar

Mccc1

WT

Talin

A/B

-

Suspension

WT

Talin

A/B

-

WT

adhe

sion

Talin

A/B

- fo

rced

adh

esio

n

Petri Plate

H

Distance

Suspension

0.0 0.1 0.2 0.30

20

40

60

Inte

nsity

(A.U

.) WTTalinA-/B-

Mccc1

Scar

Sus

pens

ion

Adh

esio

n

******

D

B

C

Distance

Plate

0.0 0.1 0.2 0.30

10

20

30

40

WTTalinA-/B-

I

Distance

Forced adhesion

0.2 0.4 0.6

20406080

100WTTalinA-/B-

J

K

M O

B

D

************

*

******

**** ****ns

0 15 30 45 60 s S

carW

T S

carS8

ASc

arS8

D

In S

car-

Sca

r-

AIn

Sca

r-

C

Figure 5 Singh et al.

0 6 18 24s 12

E

F G

Scar-0

5

10

15

20202530

Spee

d (μ

m/m

in)

Scar-0.0

0.2

0.4

0.6

0.8

1.0

1.2

Tortu

osity

ns

********

********

****

********

*****ns

ns

0

5

10

15

20

Acc

umul

atio

n si

ze (μ

m)

0

5

10

15

20

Patc

h /m

in

F-A

ctin

WAVE1/2 KO WAVE2WT WAVE2S8A/T1AH

0

25

Patc

h lif

e tim

e (S

ec)

Sca

rWT

Sca

rS8A

Scar

S8D

ScarWT ScarS8A ScarS8D

ScarWT ScarS8A ScarS8D

ScarWT ScarS8A ScarS8D

ScarWT ScarS8A ScarS8D ScarWT ScarS8A ScarS8D

Scar

Mccc1

Doxycycline + - + - + - + -

In Scar-/WASP-

A B

0 15 30 45 60 s

In S

car-

/WA

SP-

Sca

r-/W

ASP

-

In Scar-/WASP-

Scar

com

plex

E

F- A

ctin

In Scar-/WASP-

Figure 6 Singh et al

C D

F

Scar-/WASP-

******** ****

0

4

8

12

16

Spee

d

0 24 48 72 96 120 1440

10

20

30

40

50

Time (h)

Cel

l no.

(x10

5 )

Scartet-/WASP-

WT

S8A

S8D

Sca

rWT

Sca

rS8A

Scar

S8D

ScarWT ScarS8A ScarS8D ScarWT ScarS8A ScarS8D

ScarWT ScarS8A ScarS8D

ScarWT ScarS8A ScarS8D

A

- CycloheximideWT S8AS8D

0 2 4 6 0 2 4 6 0 2 4 60.0

0.5

1.0

Time (h)

Rel

ativ

e fo

ld c

hang

e

+ Cycloheximide

B

FIgure 7 Singh et al

C

WT S8AS8D

0 2 4 6 0 2 4 6 0 2 4 60.0

0.5

1.0

1.5

Time (h)

Rel

ativ

e fo

ld c

hang

e

ScarWT

Mccc1

ScarS8A

Mccc1

ScarS8D

Mccc1

0 2 4 6 2 4 6 h

- Cycloheximide + Cycloheximide

0 2 5 10 20 300.0

0.5

1.0

Adhesion time (min)

Fold

cha

nge

(Tot

al S

car)

0 1 2 5 10 150

1.0

2.0

Suspension time (min)

Fold

cha

nge

(Tot

al S

car)

D E

Table 1 Phosphorylated Residues in Scar

Peptide Sequence Identification Source

Y88 PSIEDYHRNTS By MS and Screening

Y129 SINTVYEKCKP By MS and Screening

Y210 VTKVRYDPVTG By MS and Screening

S287/S290 PPLNTSTPSPSSSF By MS and Screening

S301 QGRPPSTGFNT By MS and Screening

S335/S339 ANNRLSVHNSAPIVA Screening S384/S388/S389 ASGARSDLLSSIMQGM By MS and

screening Phosphorylated Residues in WAVE2 S293/S296/S298 GPKRSSVVSPSHPPPA Chen et al., 2010,

Lebensohn and Kirschner 2009, Phosphosite.org,

S308 APPLGSPPGPK Lebensohn and Kirschner 2009

S343/T346/S351 PVGFGSPGTPPPPSPPSFP Mendoza et al., 2011, Danson et al., 2007

S429 TKPKSSLPAVS Mertins et al., 2016, Phosphosite.org.

S442 RSDLLSAIRQG Mertins et al., 2016, Phosphosite.org.

Supp table 1; supp figs 1-3

A

B

Suppl. figure 1 Singh et al.

50

80

120

140

Scar

EGFP-NAP1PIR121

Scar-/EGFPNap1 EGFPNap1

50

80

140

Scar-/EGFPNap1 EGFPNap1

InputSup Pulldown PulldownSupInput

PulldownSupInputPulldownInputSup

1 2 3 4 5 6

C

Scar

Mccc1

In Scar-

Sca

rWT

Sca

rS8A

Sca

rS8D

Sca

r

Sca

rWT

Sca

rS8A

Sca

rS8D

Scar

Pir121Nap1

Ub

Scar

Mccc1

Ub

Gfp Trap pull down Whole cell lysate- MG132 + MG132 + MG132- MG132

A

Suppl. Figure 2 Singh et al

B

Sca

r

Sca

rWT

Sca

rS8A

Sca

rS8D

Sca

r

Sca

rWT

Sca

rS8A

Sca

rS8D

Sca

r

Sca

rWT

Sca

rS8A

Sca

rS8D