1

PDK1 regulates focal adhesion disassembly through modulation of 1

v3 integrin endocytosis 2

3

Laura di Blasio1,2,*

, Paolo Armando Gagliardi 1,2

, Alberto Puliafito 1,2

, Roberto Sessa 4

1,2,3, Giorgio Seano

1,2,4, Federico Bussolino

1,2,5 and Luca Primo

1,2,5,* 5

6

1Candiolo Cancer Institute FPO-IRCCS, Candiolo, Turin, Italy 7

2Department of Oncology, University of Torino, Turin, Italy 8

9

3 Current address: UC Berkeley, 689 Minor Hall, Berkeley, USA 10

4 Current address: Edwin Steele Laboratory for Tumor Biology, Department of 11

Radiation Oncology, Harvard Medical School, Massachusetts General Hospital, 12

Boston, USA 13

14

5 These authors contributed equally to this work. 15

16

* Correspondence should be addressed to: Laura di Blasio (laura.diblasio@ircc.it) and 17

Luca Primo (luca.primo@ircc.it); Str. Provinciale 142, km 3.95, 10060 Candiolo 18

(Torino), Italy; Tel +390119933505, Fax +390119933524 19

20

Running title: PDK1 regulates integrin endocytosis 21

© 2015. Published by The Company of Biologists Ltd.Jo

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JCS Advance Online Article. Posted on 14 January 2015

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SUMMARY 22

23

Non-amoeboid cell migration is characterised by dynamic competition among 24

multiple protrusions to establish new adhesion sites at the cell's leading edge. 25

However, the mechanisms that regulate the decision to disassemble or to grow nascent 26

adhesions are not fully understood. 27

Here we show that in endothelial cells (EC) 3-phosphoinositide-dependent protein 28

(PDK1) promotes focal adhesions (FA) turnover by controlling endocytosis of 29

integrin αvβ3 in a PI3K-dependent manner. We demonstrate that PDK1 binds and 30

phosphorylates integrin αvβ3. Down-regulation of PDK1 increases FA size and slows 31

down their disassembly. This process requires both PDK1 kinase activity and PI3K 32

activation but does not involve Akt. Moreover, PDK1 silencing stabilizes FA in 33

membrane protrusions decreasing EC migration on vitronectin. 34

These results indicate that modulation of integrin endocytosis by PDK1 hampers EC 35

adhesion and migration on extracellular matrix, thus unveiling a novel role for this 36

kinase. 37

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INTRODUCTION 38

39

In the currently accepted model of non-amoeboid cell migration, initiation of cell 40

locomotion involves a directional protrusion to form a lamellipodium, which is 41

stabilised through integrin-mediated adhesions (Lee et al., 1993). 42

In this process, integrins mediate dynamic interactions between the actin cytoskeleton 43

and the extracellular matrix (ECM) (Hynes, 1992), driving cell migration in many 44

physiological and pathological contexts, including tissue regeneration and repair, 45

embryonic development, inflammation, tumour angiogenesis and cancer metastasis 46

(Lauffenburger and Horwitz, 1996); (Ridley et al., 2003); (Friedl and Wolf, 2010). 47

Integrins are organized into large protein clusters named focal adhesions (FA), which 48

are complex structures that form at sites of integrin attachment to the ECM (Burridge 49

and Chrzanowska-Wodnicka, 1996). Nascent adhesions (also called focal contacts) at 50

the leading edge of migrating cells either mature into stabilised FA or turnover. While 51

FA formation mechanisms are largely known (Zamir and Geiger, 2001), the driving 52

mechanisms for FA disassembly are not yet well understood. In the tail FA may be 53

disassembled or left on the substratum as “footprints” (Regen and Horwitz, 1992); 54

(Smilenov et al., 1999). Microtubules contribute to focal adhesions disassembly either 55

through the modulation of Rho GTPase signalling (Broussard et al., 2008) or through 56

a FAK/dynamin pathway, independently of Rho and Rac activity (Ezratty et al., 2005). 57

Clathrin and some of its adaptors (e.g. AP-2 and Dab2) are also involved in this 58

process, by mediating integrin endocytosis from disassembling adhesion sites (Chao 59

and Kunz, 2009); (Ezratty et al., 2009). 60

During cell migration, integrins are continuously endocytosed and recycled back to 61

the plasma membrane (Pellinen and Ivaska, 2006); (Caswell and Norman, 2008); 62

(Caswell et al., 2009). In recent years it has been demonstrated that trafficking is used 63

to restrict integrins to cell protrusions (Caswell et al., 2007) and to guide the recycling 64

of other receptors, such as epidermal growth factor receptor (EGFR) (Caswell et al., 65

2008) and vascular endothelial growth factor receptor (VEGFR) (Reynolds et al., 66

2009). 67

Among integrins, v3 is particularly important in the vascular system (Avraamides 68

et al., 2008); (Hynes, 2007). This integrin is expressed at low level in quiescent 69

endothelial cells (EC) and is up-regulated during normal and tumour angiogenesis, 70

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which made it an attractive therapeutic target (Brooks et al., 1994). Indeed, v3 and 71

v5 antagonists are being used in clinical trials as anti-angiogenic drugs, including 72

the humanized monoclonal antibody Vitaxin and the RGD-mimetic Cilengitide 73

(Gutheil et al., 2000); (Nabors et al., 2007). However, neither v nor of 3/5 doubly 74

deficient mice displayed vascular network with obvious developmental defects (Bader 75

et al., 1998); (Hodivala-Dilke et al., 1999); (Huang et al., 2000). Thus, these and 76

others studies suggest that regulationofv3 integrin may play a critical role in 77

angiogenesis. 78

We recently showed that in EC the serine/threonine kinase PDK1 was required for 79

directional motility (Primo et al., 2007). PDK1 phosphorylates kinases of the AGC 80

family, including Akt (Alessi et al., 1997), and contains a C-terminal pleckstrin 81

homology (PH) domain (Mora et al., 2004) which binds with high affinity to the 82

phosphoinositide 3-kinase (PI3K) products, phosphatidylinositol 3,4,5-trisphosphate 83

(PIP3) and phosphatidylinositol 3,4-bisphosphate (Anderson et al., 1998). Although 84

several effectors have been proposed to be activated by PDK1 during cell migration, 85

the exact molecular mechanism is not known (Primo et al., 2007); (Pinner and Sahai, 86

2008); (Liu et al., 2009); (Gagliardi et al., 2014). 87

Here we present evidence for a completely new function of PDK1. We found that 88

PDK1 promotes FA turnover on vitronectin by controlling the endocytosis of integrin 89

αvβ3 in a PI3K-dependent manner. Notably, PDK1 dynamically localizes to FA just 90

before their disassembly and interacts with v3, modulating EC adhesion and 91

migration. 92

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RESULTS 93

94

v3-mediated cell adhesion is modulated by PDK1 95

To efficiently and persistently migrate, cells need to modulate integrin-mediated 96

contacts with the surrounding ECM molecules. Therefore, we investigated whether 97

PDK1 regulated EC adhesion on the ECM by silencing PDK1 expression with short 98

hairpin RNAs (shRNA). We identified two shRNAs, indicated as shPDK1_79 and 81, 99

which reduced PDK1 protein level of about 90% and 80%, respectively, compared to 100

EC expressing not-targeting shRNA (shScrl) (fig. S1A). EC adhesion was analysed in 101

real time by measuring cellular impedance with the xCELLigence system (see 102

Materials and Methods) on provisional ECM proteins vitronectin and fibronectin. 103

PDK1-silenced EC showed significantly increased adhesion ability, on both 104

vitronectin and fibronectin, compared to control cells (fig. 1A and B), while it did not 105

significantly change the spreading area of EC, excluding the possibility that cellular 106

impedance was affected by cell spreading (fig. S1B, C). In order to exclude off target 107

effects of shRNAs used to silence PDK1, we transduced shPDK1_79 cells with a 108

PDK1-wt cDNA resistant to silencing (fig. S1D). PDK1-wt re-expression rescued the 109

effect of PDK1 silencing, reducing the ability of shPDK1_79 to adhere on vitronectin 110

(fig. S1E). 111

Since fibronectin is the ligand of several integrins, while vitronectin binds specifically 112

v3 and v5 (Hynes, 2002), we sought to investigate whether PDK1 regulates 113

v3/v5-mediated cell adhesion. To this end, we analysed the distribution of these 114

integrins and the morphology of FA on vitronectin (fig. S1F). In EC v3 integrin 115

clearly localised to FA (fig. S1F); in contrast, v5 showed negligible accumulation 116

into FA, neither in basal culture condition nor in presence of VEGF stimulation (fig. 117

S1F) (Friedlander et al., 1995). Then, we concentrated our attention on the effect of 118

PDK1 silencing on v3 and v5 distribution in EC plated on vitronectin (fig. 1C). 119

As expected, shScrl EC displayed v3 localization to small FA mainly along the cell 120

periphery; instead, both shPDK1 EC showed longer and thicker v3-positive FA, 121

sometimes localised along the whole basal side of the cells (fig. 1C). Moreover, 122

PDK1 silencing increased average size, total area and number of v3-positive FA 123

(fig. 1D). Conversely, the distribution of v5 integrin between control and silenced 124

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cells did not change (fig. S1G). The re-expression of PDK1-wt in shPDK1_79 EC 125

plated on vitronectin restored the physiological aspect of v3-positive FA (fig. 1E, 126

F). 127

When plated on fibronectin, shPDK1 EC displayed FA that were comparable to those 128

observed in cells plated on vitronectin (fig. S2A, right panels, and B). In contrast, 129

when EC were plated on collagen type-I, PDK1 silencing had no effects on FA 130

organisation (fig. S2A, left panels, and B). Interestingly, the v3 inhibitor cRGDfv 131

completely neutralised the effect of PDK1 silencing on vitronectin (fig. S1H and J), 132

while we could not measure any effect on collagen type-I (fig. S1I and K). These 133

results indicate that PDK1 down-regulation increases EC adhesion on vitronectin by 134

regulating organisation and size ofv3-containing FA. 135

136

PDK1 regulates FA disassembly 137

Since an enlargement of FA may indicate a change in the turnover kinetics of their 138

components (Webb et al., 2002), we further investigated the role of PDK1 in this 139

process, by taking advantage of a method to monitor the synchronised disassembly of 140

adhesions (Bershadsky et al., 1996); (Ezratty et al., 2005). This method is based on 141

the finding that microtubule depolymerisation promoted by nocodazole stimulates FA 142

formation. Upon nocodazole removal, microtubule regrow and FA rapidly and 143

synchronously disassemble. By adapting this approach, we found that the nocodazole 144

treatment of EC for 1 hour stabilised paxillin and vβ3-containing FA and, 145

concurrently, completely disassembled microtubules (fig. S2C and 2A, C). Then, 146

approximately 30 minutes after nocodazole washout, while microtubules regrew, FA 147

disassembled (fig. S2C and 2B, C). Upon microtubules disassembly, control and 148

PDK1-knockdown EC showed similar FA size (fig. 2A, C). Moreover, consistently 149

with the idea that depletion of PDK1 affects FA turnover, we found that FA 150

disassembly was slower in PDK1-knockdown EC than in control EC, even though 151

microtubules regrowth was normal (fig. 2B, C). Conversely, v5 integrin was 152

neither accumulated in paxillin-positive FA after nocodazole treatment nor clearly 153

changed its localisation during nocodazole washout (fig. S2D). Thus, these data 154

suggest that PDK1 promotes FA disassembly. 155

156

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PDK1 associates withv3 integrin and dynamically localises to FA 157

PDK1 usually localises throughout the cytoplasm and accumulates at the plasma 158

membrane through the binding with PIP3 produced by PI3K (Mora et al., 2004). Thus, 159

we wondered whether PDK1 moved to FA during their disassembly. Paxillin-GFP-160

expressing EC were treated with nocodazole and PDK1 and v integrin localisation 161

was analysed by immunofluorescence staining and confocal microscopy (fig. 3A, B). 162

Cells treated with nocodazole showed an enrichment of PDK1 in FA identified by 163

paxillin-GFP and anti-v integrin staining (fig. 3A, white arrows). When nocodazole 164

was removed, PDK1 quickly disappeared from FA (fig. 3B). In untreated cells, 165

cultured in basal medium and plated on vitronectin, the co-localisation of PDK1 with 166

paxillin and αvβ3 was still visible but less pronounced (fig. 3C). Such qualitative 167

observations were corroborated by a quantification of the amount of PDK1 or v 168

found in single FA with respect to the cytoplasmic region. The results of this 169

quantitative analysis confirmed the qualitative observations (further details are given 170

in fig. S3A, B and Materials and Methods). 171

PDK1 localisation to FA led us to investigate whether PDK1 and v3 integrin 172

physically associated in nocodazole-treated cells. We co-immunoprecipitated 173

endogenous PDK1 with v3 integrin in nocodazole treated EC and we found the 174

amount of co-immunoprecipitated PDK1 to be increased in EC overexpressing PDK1 175

(fig. 3D and S2E). After nocodazole removal, v3 integrin was no longer able to co-176

immunoprecipitate with endogenous PDK1 while it still maintained, although reduced, 177

the ability to co-immunoprecipitate with overexpressed PDK1 (fig. 3D and S2E). 178

The association of PDK1 with v3 was confirmed also in untreated cells (fig.3E). 179

Concordantly to co-localisation experiments, the amount of PDK1 co-180

immunoprecipitated with v3 in basal culture conditions was very limited (fig.3E). 181

The specificity of PDK1/v3 co-immunoprecipitation was further demonstrated by 182

experiments showing the absence of PDK1 in v3-immunoprecipitated from PDK1-183

silenced cells (fig. 3F). In addition, endogenous PDK1 was not immunoprecipitated 184

by anti-21 antibody suggesting that PDK1 was able to bind specifically v3 185

integrin (fig. S2G). Moreover, in 293T cells, that express v but not 3 integrin 186

(Taherian et al., 2011), PDK1 was found in v3-immunoprecipitated only after the 187

ectopical expression of 3 integrin (fig. S2F). To further confirm the interaction, we 188

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performed pull-down experiments with the v and 3 cytoplasmic tails (fig. 3G, H). 189

GST-fused cytoplasmic tail of 3, which maintain the ability to interact with Talin, 190

efficiently associated with PDK1 (fig. 3G). Moreover, a biotinylated peptide of the 191

cytoplasmic tail of v integrin, but not of 2, was able to co-precipitate both 192

overexpressed and endogenous PDK1 (fig.3H). For reasons that remain to be 193

investigated, we could hardly detect v3 integrin by PDK1 immunoprecipitation, 194

even in nocodazole treated cells (fig. S2H). 195

The in vivo association of PDK1 with v3 was also analysed by performing 196

proximity ligation (PLA) experiments. PLA positive spots were detected in presence 197

of anti-PDK1 and either anti-v or anti-3 antibodies, both at the periphery and in the 198

cytoplasm of the cells plated on vitronectin (fig. 3I). Absence or small amount of 199

spots was detected with anti-PDK1 antibody coupled with not-immune IgG or anti-3 200

antibody, respectively (fig. 3I). Taken together, these results indicate that PDK1 201

associates with the cytoplasmic domain of v3 integrin and dynamically localizes to 202

FA. 203

204

PDK1 controls v3 endocytosis during FA disassembly 205

To elucidate the mechanism by which PDK1 regulates FA disassembly, we first tested 206

whether PDK1 knockdown affected the activity of FAK, a key regulator of FA 207

turnover. However, the phosphorylation of FAK on Tyr397, which is critical for FAK 208

activation and FA disassembly (Webb et al., 2002) was not affected by PDK1 209

silencing (fig. S3C). Recently, it has been demonstrated that FA disassembly requires 210

endocytosis of integrins (Chao and Kunz, 2009); (Ezratty et al., 2009). Since we 211

observed that the plasma membrane expression of 3 integrin was slightly increased 212

in PDK1-silenced EC (fig. S3D), we sought to determine whether PDK1 regulated 213

endocytosis of v3 integrin thereby affecting FA turnover. We then used both the 214

antibody-chase (fig. 4A, B) and biochemical internalisation assay (fig. 4C) to 215

determine whether PDK1 regulated endocytosis of v3 integrin. 216

In antibody-chase assay, we observed by indirect immunofluorescence that the 217

amount of anti-3 antibody internalised after acid wash was reduced in PDK1 218

silenced cells (fig. 4A, 37°C plus acid wash, B). As expected, cells treated in the same 219

manner but kept at 4°C did not internalise v3 integrin (fig. 4A, 4°C). To better 220

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characterise this effect, we performed a quantitative biochemical internalisation assay 221

based on surface protein biotinylation, followed by cell surface reduction and 222

detection of internalised biotinylated integrin by capture enzyme-linked 223

immunoadsorbent assay (ELISA) (Roberts et al., 2001). This experiment, performed 224

in presence of receptor recycling inhibitor primaquine (PQ), showed that PDK1 225

silencing decreased the amount of v3 integrin internalised by EC (fig. 4C). In 226

contrast, the internalisation of 5 integrin was not affected by PDK1 knockdown 227

either in presence or not of PQ (fig. S3E). 228

Different v3 integrin internalisation routes have been described. Actually, v3 229

integrin can be internalised in a cholesterol- and caveolin1-dependent manner, by 230

clathrin-coated structures or by macropinocytosis (Caswell et al., 2009). To define 231

which v3 internalisation routes are predominant in EC, and potentially regulated by 232

PDK1, we performed antibody-chase internalisation assay of 3 integrin in presence 233

of different endocytosis inhibitors (fig. S4A-C). Cyclodextrin, which block 234

cholesterol-dependent endocytosis, strongly inhibited 3 internalisation. In contrast, 235

the macropinocytosis inhibitor, amiloride, had no influence on 3 endocytosis. 236

Treatment of EC with dynasore, which inhibit dynamin-mediated endocytosis, had 237

only a minor effect (fig. S4A, B). 238

Hence, we tested whether PDK1 modulates v3 integrin endocytosis during 239

nocodazole washout. Simultaneously with nocodazole removal, anti-β3 antibody was 240

added to the cells. (fig. 4D-G). In shScrl EC, after 30 minutes of nocodazole washout, 241

3 integrin was endocytosed and simultaneously FA disassembled (fig. 4D, G and 242

S3F). Conversely, 3 integrin internalisation during nocodazole washout was 243

impaired in PDK1-silenced EC resulting in more stable FA (fig. 4E-G, and S3F). It is 244

worth noting that 3 integrin internalised during FA disassembly followed previously 245

described endocytosis route (Caswell et al., 2009). Indeed, after nocodazole washout, 246

3 integrin localised to Rab4- and Rab5-positive early endosomes and partially to 247

Rab11-positive recycling endosomes, irrespectively of primaquine addition (fig. S3G-248

I). 249

In biochemical internalisation assay, PDK1-silencing significantly reduced the 3 250

integrin internalisation after nocodazole removal. As shown in fig. 4H, the addition of 251

PQ increased the percentage of internalised 3 integrin during nocodazole washout; 252

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notably, PDK1-knockdown EC still displayed reduced ability to internalise 3 253

integrin (fig. 4H). Therefore, PDK1 controls v3 integrin endocytosis taking place 254

during FA disassembly. 255

256

Both PH domain and kinase activity of PDK1 are required for controlling 257

integrin v3 endocytosis 258

PDK1 is characterised by a centrally located kinase domain, a C-terminal PH domain, 259

and a docking site, named PIF-binding pocket, which is located on the small lobe of 260

the kinase domain (Tian et al., 2002); (Biondi et al., 2001). To determine which 261

region was required for v3 endocytosis regulation by PDK1, we infected 262

shPDK1_79 EC with lentivirus coding for different PDK1 mutants resistant to 263

silencing (Gagliardi et al., 2012) (fig. S4D). Then, shScrl EC and shPDK1_79 EC 264

expressing PDK1 wild-type (wt), PDK1 constitutively anchored to the plasma 265

membrane (caax), PDK1 lacking the PH domain (ΔPH), PDK1 kinase-dead (KD) or 266

PDK1 with a mutation on the PIF pocket (L155E) were assayed in the antibody-chase 267

and biochemical internalisation assays (fig. 5A-B and S4E, respectively). The re-268

expression of PDK1-wt, PDK1-caax and PDK1-L155E was able to recover the 269

normal level of v3 endocytosis; conversely, PDK1-ΔPH and PDK1-KD did not 270

rescue the defective v3 integrin internalisation caused by endogenous PDK1 271

silencing (fig. 5A-B, S4E and not shown). The same results were obtained when we 272

tested the v3 internalisation during FA disassembly (fig. S4F). Moreover, PDK1-273

ΔPH mutant lost the ability to localise on the plasma membrane in proximity of FA 274

(fig. 5C) and co-immunoprecipitated with v3 less efficiently than PDK1-wt and 275

PDK1-KD (fig. S4G and not shown). 276

Thus, membrane localisation through PIP3 binding and kinase activity are necessary 277

to PDK1 for regulating v3 endocytosis. 278

279

PI3K but not Akt inhibition reduces integrin v3 endocytosis 280

Following PI3K activation and production of PIP3, both PDK1 and Akt localise to the 281

plasma membrane by means of their PH domains. The proximity of the two proteins 282

at plasma membrane allows the phosphorylation of the activation loop of Akt by 283

PDK1(Collins et al., 2003); (McManus et al., 2004). Thus, we verified whether the 284

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inhibition of either PI3K or Akt with chemical compounds was able to phenocopy the 285

effect of PDK1 silencing on v3 endocytosis (fig. 5D and S4H). The block of PI3K 286

activity with LY294002 slightly, but significantly, inhibited endocytosis of antibody-287

bound 3 integrin (fig. 5D and S4H). Surprisingly, Akt inhibition increased the rate of 288

v3 internalisation (fig. 5D and S4H). Neither of the two inhibitors had effects on 289

endocytosis of transferrin (fig. 5D). Taken together, these data demonstrates that PI3K 290

activation is necessary for v3 endocytosis and that PDK1 regulates this process in a 291

PI3K-dependent way, but without the requirement of downstream activation of Akt. 292

293

3 integrin is phosphorylated by PDK1 294

Given that kinase activity of PDK1 was determinant for the regulation of v3 295

integrin endocytosis, we wondered whether PDK1 was able to phosphorylate 3 296

integrin cytoplasmic domain in cultured cells. It has been previously shown that 297

threonine 753 of 3 integrin was phosphorylated in vitro by PDK1 and Akt (Kirk et 298

al., 2000). Therefore, we verified whether PDK1 was able to phosphorylate β3 299

integrin also in vivo. In absence of specific antibodies anti-phospho threonine 753, we 300

used an anti-phospho Akt-substrate antibody that recognizes peptides and proteins 301

containing phospho-Ser/Thr preceded by Lys/Arg at positions -5, such as threonine 302

753 of β3. We validated this antibody by expressing the cytoplasmic tail of integrin 303

3 in 293T cells and treating the cells with protein phosphatase 2A (PP2A) inhibitor 304

Calyculin-A or PDK1 inhibitor BX-795 (fig. 6A). The cytoplasmic tail of 3 integrin 305

showed basal phosphorylation that was enhanced by treatment with Calyculin-A (fig. 306

6A, Cal). Conversely, the treatment with the PDK1 inhibitor reduced the 307

phosphorylation of 3 integrin cytoplasmic tail, suggesting this antibody as specific 308

for the phospho-threonine 753 (fig. 6A, PDKi). Thus, we analysed the 309

phosphorylation of endogenous 3 integrin immunoprecipitated from EC. EC treated 310

with PDK1 inhibitor showed a reduced phosphorylation of β3 (fig. 6B). These data 311

were further confirmed by PLA experiments performed with anti-β3 and the same 312

anti-phospho Akt-substrate used in immunoblot (fig. 6C). Untreated cells showed 313

more spots of threonine phosphorylated 3 integrin than BX-795-treated cells (61 ± 8 314

spots/cell vs 36 ± 5, N=10); conversely, the inhibition of Akt had only a partial effect 315

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(42 ± 7 spots/cell) (fig. 6C). In addition, we observed a reduced phosphorylation of 3 316

immunoprecipitated from PDK1-silenced EC compared to control EC (fig. 6D). 317

To determine whether threonine 753 residue and its phosphorylation by PDK1 was 318

important for 3 endocytosis, we infected EC with either wild-type or T753A mutant 319

of 3 integrin. Both wild-type and T753A 3 integrin were correctly targeted to the 320

plasma membrane and localised to FA (fig. S4I-J and 6E). As expected, the Ser/Thr 321

phosphorylation of T753A 3 integrin was reduced compared to wild-type protein (fig. 322

6F). Thus, we analysed internalisation of ectopic wild-type and T753A mutant of 3 323

integrin (fig. 7A-D and S3J). While wild-type 3 integrin internalization was 324

comparable to that of the endogenous protein, T753A 3 integrin showed a reduced 325

rate of endocytosis (fig. 6A-B) and less accumulation in endocytic vesicles (fig. 6C-D 326

and S3J). 327

Thus, PDK1 phosphorylates 3 cytoplasmic tail and this phosphorylation is important 328

for its endocytosis. 329

330

EC migration and FA dynamics in membrane protrusions are controlled by 331

PDK1 332

Next, we sought to determine if PDK1 was necessary to regulate FA dynamics in EC 333

and in particular for those forming in protruding regions. Therefore, we analysed 334

paxillin-positive FA by TIRF microscopy in live control and PDK1-silenced EC 335

seeded on vitronectin (fig. 8A-B and movies 1-2 in supplemental materials). Control 336

cells had adhesive structures with heterogeneous size and migrate by assembling and 337

disassembling focal structures within protrusions (fig. 8A). The majority of FA in 338

control EC was small and dot-like; conversely, shPDK1_79 EC displayed a 339

significant fraction of FA longer than 0.75 m (fig. 8B). To quantify the effect of 340

PDK1 silencing, we compared silenced and control cells by isolating the images of 341

several protrusions per filmed cell. Each protrusion was identified as a subregion of 342

the cell expanding and retracting. Images were then segmented and the ratio of large 343

and long to small and round adhesive structures was calculated for each protrusion. 344

Typical protrusions are composed of small dotty-like structures normally identified as 345

focal complexes which can either disassemble or mature into elongated and thick 346

structures normally called focal adhesions. shPDK1_79 EC had substantially larger 347

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and thicker adhesive structures in the protrusions (fig. 8C). Notably, the re-expression 348

of PDK1 wild-type in shPDK1_79 EC completely recovered the normal aspect of FA 349

(fig. 8C and movie 3 in supplemental materials); conversely, the re-expression of 350

PDK1-ΔPH had no effect on FA structure (fig. 8C and movies 4 in supplemental 351

materials). 352

It is known that alterations of FA dynamics affect the cell ability to directionally 353

migrate. Thus, we evaluated the effect of PDK1 silencing in EC stimulated to migrate 354

on vitronectin. The PDK1 knockdown strongly reduced the ability to migrate 355

compared to normal EC (fig. 8D). 356

We therefore conclude that PDK1, through PIP3-binding, modulates FA dynamics in 357

cell protrusions and regulates EC motility. 358

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DISCUSSION 359

360

In this study, we showed that endocytosis of β3 integrin is controlled by PDK1, which 361

associates and phosphorylates this integrin, promoting its internalisation. The ability 362

of EC to adhere and to directionally move on the ECM strictly relies on the integrin 363

trafficking and FA dynamics. This prevailing idea is supported by several pieces of 364

evidence demonstrating that integrin recycling can contribute to cell migration 365

(Caswell and Norman, 2006); (Pellinen and Ivaska, 2006); (Nishimura and Kaibuchi, 366

2007) and that integrins are endocytosed into Rab-labeled endocytic compartments 367

during growth factor stimulation (Roberts et al., 2001); (Pellinen et al., 2008). Integrin 368

internalisation occurs through both clathrin-dependent and clathrin-independent 369

mechanism (Ezratty et al., 2009); (Nishimura and Kaibuchi, 2007); (Fabbri et al., 370

2005); (Shi and Sottile, 2008); (Gu et al., 2011). Nevertheless, how integrin 371

endocytosis and recycling are dynamically regulated in a spatially and temporally 372

restricted manner is not completely clear. In our experimental conditions αvβ3 373

internalisation mainly depends on membrane lipid-raft, but not on macropinocytosis. 374

Our experimental evidence suggests that PDK1 could regulate αvβ3 integrin 375

endocytosis by direct association. Moreover, kinase-inactive PDK1 is unable to 376

stimulate integrin endocytosis, suggesting that a phosphorylated substrate is involved 377

in this process. However, inactivation of Akt, one of the most relevant PDK1 378

substrates, does not reduce integrin endocytosis rate, indicating an Akt-independent 379

role for PDK1 in this process. Accordingly we were able to detect PDK1-dependent 380

phosphorylation on Thr 753 of β3 integrin, which has been previously identified as in 381

vitro PDK1 phosphorylation target (Kirk et al., 2000). While it has been reported that 382

phosphorylation of this threonine/serine-rich region decreases the binding of integrins 383

for filamin, the importance of this phosphorylation in vivo remains to be determined 384

(Kiema et al., 2006). 385

Although no clear evidence of a direct PDK1 involvement in the integrin function is 386

currently available, an interaction between PDK1 and the calcium-activated tyrosine 387

kinase Pyk2 regulating the integrity of FA has been observed (Taniyama et al., 2003). 388

Furthermore, the homologous of mammalian PDK1 in Saccharomyces cerevisiae (the 389

Pkh1/2 kinases) has been shown to be involved in the endocytic trafficking pathway 390

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(Friant et al., 2001); (deHart et al., 2002), but the molecular details are yet to be 391

elucidated. 392

Conversely, the crucial role of membrane localisation of PDK1 via PI3K activation in 393

regulating both substrate specificity and spatially restricted activation has been clearly 394

established (Currie et al., 1999). Notably, the PH domain of PDK1, which 395

preferentially binds PIP3 at the membrane (McManus et al., 2004), was required for 396

αvβ3 integrin endocytosis. Consistently, inhibition of PI3K activity reduces αvβ3 397

integrin endocytosis, although less than PDK1 silencing. Therefore these results 398

support a model in which both membrane translocation mediated by PH domain and 399

integrin interaction are involved in endocytosis. A similar mechanism has been 400

described for kindlin-2, which interacts through its FERM domain with integrin αII-401

3 but requires PIP3 binding to its PH domain for regulation of integrin activity (Liu 402

et al., 2011). 403

Although the process of FA disassembly remains to be fully elucidated, integrin 404

endocytosis and microtubule targeting are known to regulate FA turnover (Ezratty et 405

al., 2005); (Nishimura and Kaibuchi, 2007). In absence of PDK1, EC showed FA with 406

altered morphology, prevalently longer and more stable than in normal EC. This 407

phenotype can be attributed to altered FA dynamics (Chao and Kunz, 2009). The slow 408

FA disassembly during microtubule re-growth and the reduced integrin endocytosis in 409

EC knockdown for PDK1 suggest that FA disassembly, instead of their assembly, is 410

regulated by PDK1. In normal culture condition PDK1 is only relatively weakly 411

enriched in FA. Nevertheless, when the FA turnover was synchronised by nocodazole 412

treatment, it was possible to appreciate a clear localisation of PDK1 to FA, which 413

rapidly disappeared during nocodazole washout. This is consistent with the highly 414

dynamic association between PDK1 and integrin αv3, which occurs in proximity of 415

the plasma membrane. 416

Whether and how integrin endocytosis contributes to FA dynamics is still debated, but 417

recent evidence shows a critical role for endocytosis in FA disassembly induced by 418

nocodazole washout (Ezratty et al., 2009). Accordingly, PDK1 could regulate FA size 419

and turnover by affecting integrin endocytosis. In this context, the role of the PH 420

domain of PDK1 is particularly interesting. If PDK1 lacks the ability to bind PIP3, it 421

is no longer able to promote integrin endocytosis or FA disassembly. From this point 422

of view, the PIP3 binding ability of PDK1 could contribute to control FA dynamics to 423

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a specific intracellular compartment, for example defined by the localised PI3K 424

activity in response to chemotactic stimulation. Although this concept has not been 425

specifically tested here, the rostro-caudal PIP3 gradient in polarised cells, maintained 426

by PTEN activity in the rear of the cell, could be transformed in a PDK1-dependent 427

manner into differentially behaving cell–matrix adhesion sites. 428

The process of endothelial migration through provisional matrix, such as fibronectin 429

and vitronectin, is a critical event in the formation of new vessels in pathological 430

conditions (Silva et al., 2008). High expression levels of αv3 integrin characterise 431

angiogenic endothelium and blocking αv integrin function by disrupting ligand 432

binding can produce an anti-angiogenic effect (Brooks et al., 1994). Recently, 433

trafficking of αv3 integrin to and from the cell surface, has been demonstrated to 434

influence persistent cell migration, endothelial cell motility and angiogenesis (Caswell 435

et al., 2007); (di Blasio et al., 2010); (Reynolds et al., 2009). Accordingly, our results 436

show that an alteration of v3 endocytosis causes defects in EC migration on 437

provisional ECM molecules such as vitronectin. Taken together, these pieces of 438

evidence suggest that PDK1 regulation of αv3 endocytosis would directly affect 439

angiogenesis, integrating PI3K signalling activated by membrane receptors with ECM 440

dynamic interaction. 441

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MATERIALS AND METHODS 442

443

Cell culture and transfection 444

Human EC were isolated from umbilical cord veins and cultured as previously 445

described (Primo et al., 2007). 293T cells were grown in IMDM (Sigma) 446

supplemented with 10% FBS, L-Glutamine (2 mM, Sigma) and antibiotics. EC were 447

transfected by using Lipofectamine Plus reagent (Life Technologies) according to the 448

manufacturer’s instructions. The following constructs were used: paxillin-GFP, 449

vinculin-RFP, Rab4-, 5-, 11-GFP (kindly provided by Serini G.) and different mutants 450

of PDK1 fused to YFP (Gagliardi et al., 2014). 451

Lentivirus-driven PDK1 shRNA and PDK1 cDNA infection 452

Short hairpin RNAs (shRNA) against human PDK1 (2 constructs, TRCN0000039779 453

and TRCN0000039781 named shPDK1_79 and shPDK1_81, respectively) were 454

purchased from TRC-Hs1.0 library (Sigma). Self-inactivating lentiviral particles were 455

produced, as previously described (Gagliardi et al., 2012). 456

Silencing-resistant myc-tagged PDK1 wild-type, PDK1-caax, PDK1-ΔPH, PDK1-KD 457

(K110N) and PDK1-L155E, previously cloned into PINCO retroviral vector (Primo et 458

al., 2007), were subcloned into a third-generation lentiviral vector 459

pCCL.sin.cPPT.polyA.CTE.eGFP.minhCMV.hPGK (Amendola et al., 2005); 460

(Follenzi et al., 2000) as described in (Gagliardi et al., 2012). 461

3 integrin point mutation and cloning 462

3 integrin, previously cloned into pcDNA3 vector (Woods et al., 2004), was 463

subcloned into the third-generation lentiviral vector PWPXL (http://tronolab.epfl.ch/) 464

with In-Fusion® HD EcoDry™ Cloning-Kit (Clontech Laboratories). To add the Flag-465

tag and to mutate the threonine 753 residue to alanine, the following primers were 466

designed: “3 FW” GCCTAAGCTTACGCGTATGCGAGCGCGACCGAGGCCC, 467

“3 wt RE” AGGTTGATTATCATATGTTACTTGTCATCGTCATCCTTGTAA 468

TCAGTGCCCCGGTACGTGATATTGGTGAAGGTAGA and “3 T753A RE” 469

AGGTTGATTATCATATGTTACTTGTCATCGTCATCCTTGTAATCAGTGCCC470

CGGTACGTGATATTGGTGAAGGCAGA. The acceptor plasmid PWPXL was 471

digested in MluI and NdeI sites. 472

Adhesion assay 473

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Cell adhesion was evaluated with xCELLigence system (Roche). 96-well microtiter 474

plates that are specifically designed to measure cellular impedance (E-Plate, Roche) 475

were coated with vitronectin, fibronectin or collagen type-I (0.25 g/ml) for 1 hour at 476

37°C and then saturated with 3% bovine serum albumin. 1x104 EC were resuspended 477

in 0.1 ml of serum free medium and transferred on E-Plates after background 478

measurement. When used, the v3 inhibitor cRGDfv (200nM, Merck-Millipore) was 479

added before transferring cells on E-Plates. The extent of cell adhesion and spreading, 480

measured as changes in impedance, was monitored every 15 seconds for a total period 481

of 2 hours. The measured impedance was expressed as relative impedance (Cell 482

Index). The Cell Index at each time point is defined as (Rn-Rb)/(15Ω), where Rn is 483

the cell-electrode impedance of the well when it contains cells and Rb is the 484

background impedance of the well with the media alone. As a control, the same 485

number of cells were plated on 96-wells plates coated with vitronectin, fibronectin or 486

collagen type-I. After 2 hours of adhesion, cells were fixed, stained and photographed 487

to measure to spreading area. 488

Focal adhesion disassembly assay 489

This assay was performed as described in (Ezratty et al., 2005) with modifications. 490

Briefly, EC were seeded on vitronectin-coated glass coverslips (1 g/ml) and treated 491

with 4 M nocodazole (Sigma) for 1 hour to completely depolymerise microtubules. 492

The drug was washed out with serum free medium, and microtubules were allowed to 493

re-polymerise for 30 minutes. Cells were then fixed with 4% paraformaldehyde in 494

PBS for 10 minutes followed by immunofluorescence staining. 495

Immunofluorescence microscopy 496

To analyse focal adhesions organisation, EC were plated on vitronectin, fibronectin or 497

collagen type-I (1 g/ml)-coated glass coverslips. After 3 hours of adhesion, cells 498

were fixed and stained as previously described (di Blasio et al., 2010). The following 499

antibodies were used: anti-v3 (MAB1976, Millipore), anti-paxillin (BD 500

Biosciences), anti-tubulin (Santa Cruz Biotechnology), anti-PDK1 (Sigma and BD 501

Biosciences), anti-5 (Cell Signaling Technology and Millipore), anti-v (AB1923, 502

Millipore), anti-phospho FAK (Millipore) and anti-Flag (Cell Signaling Technology). 503

Analysis was performed using a confocal laser-scanning microscope (TCS SP2 with 504

DM IRE2; Leica) equipped with 63x/1.40 HCX Plan-Apochromat oil-immersion 505

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objective. Confocal images are the maximum projections of a z section of 1.50 µm. 506

The images were arranged and labelled using Photoshop software (Adobe). Focal 507

adhesions average size, area/cell and number/cell were quantified in 50 cells per 508

sample, captured with the same zoom, from three independent experiments, using 509

Image J. 510

To measure the degree of colocalization of PDK1 and v3 integrin, we used paxillin 511

staining to define ROIs corresponding to focal adhesions by filtering with a high pass 512

filter to enhance high paxillin-GFP regions followed by manual thresholding. Average 513

PDK1 and v3 intensities within each FA were divided by the average intensity 514

within cells. Cells that underwent nocodazole washout were not added in the 515

quantification as focal adhesions were absent in these cells and high paxillin staining 516

would not be a well-defined, biologically known, ROI. 517

To generate simulated random images first a random image was generated and 518

multiplicative noise was added to it. Finally median filtering was applied to recreate 519

patches of the same size of those found in PDK1 images. A representative image is 520

shown in fig. S3A. 521

The internalisation of β3 integrin was performed as previously described (di Blasio et 522

al., 2010). When indicated, 24 hours before the assay, EC were transfected with GFP-523

tagged Rabs protein. To visualise 3 tagged with Flag, anti-Flag antibody was used 524

(Cell Signaling Technology). The same protocol was followed for the analysis of Tfn-525

555 (20 m/ml, Life Technologies) endocytosis. The inhibitors used are: amiloride 526

(50 M, Sigma), dynasore (80 M, Sigma), cyclodextrin (5 mM, Sigma), LY-294002 527

(10 M, Sigma), Akti (InSolution™ Akt inhibitor VIII, 5 M, Calbiochem). 528

Vesicles area/cell was quantified in 50 cells per sample, captured with the same zoom, 529

from three independent experiments, using Image J. 530

Immunoprecipitation and western blots 531

After 1 hour treatment with nocodazole in 10% FBS medium at 37°C (4 m, Sigma), 532

cells were washed with serum free medium and either transferred to ice (time 0 533

minutes washout) or to 37°C with fresh 10% FBS medium for 30 minutes (time 30 534

minutes washout). Then cells were washed twice with cold PBS, and lysed in EB 535

buffer as previously described (di Blasio et al., 2010). 1 mg of proteins was incubated 536

with anti-αvβ3 (MAB1976, Millipore), anti-PDK1 (BD Biosciences), anti-21 537

of a z section of 1.50 µm.

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(Millipore), anti-3 (N20, Santa Cruz Biotechnologies) or negative control mouse IgG 538

for 2 hours; immune complexes were recovered on anti-mouse IgG-Agarose (Sigma). 539

To analyse the membrane localisation of wild-type and T753A mutant of 3 integrin, 540

we surface labelled EC at 4°C with 0.2 mg/ml N-Hydroxysulfosuccinimide (NHS)-541

SS-biotin (Pierce) in PBS for 30 minutes and then immunoprecipitated all biotinylated 542

proteins by Streptavidin-agarose conjugate (Millipore). Proteins were separated by 543

SDS-PAGE electrophoresis, blotted and

incubated with primary antibody (anti-PDK1 544

[Cell Signaling Technology], anti-v [AB1930, Millipore], anti-3 [N20, Santa Cruz 545

Biotechnologies], anti-1 [Millipore], anti-pSer/Thr Akt-substrate [Cell Signaling 546

Technology], anti-Flag [Cell Signaling Technology]). 547

Peptide and GST pulldown 548

Peptide pulldowns were carried out using synthetic, N-terminally biotin coupled 549

peptide corresponding to the cytoplasmic portion of v and 2 integrin (biotin-550

YRMGFFKRVRPPQEEQEREQLQPHENGEGNSET and biotin-551

WKLGFFKRKYEKMTKNPDEIDETTELSS, Genscript) as previously described 552

(Schiller et al., 2013). 553

For GST pulldown assay, cytoplasmic tail of 3 integrin was cloned in pDONR/zeo 554

using Gateway BP Clonase (Life technologies). To perform the BP recombination we 555

previously performed a PCR reaction using the following primers: “attB1-3 cyto” 556

GGGGACAAGTTTGTACAAAAAAGCAGGCTTAACCATGAAACTCCTCATCA557

CCATCCACGAC and “attB2-3 cyto” GGGGACCACTTTGTACAAGAAAG 558

CTGGGTCAGTGCCCCGGTACGTGATATTGGT. The construct was then 559

transferred through Gateway LR Clonase in the pDEST27 (GST-N-Term Tag, Life 560

Technologies). pDEST27 and pDEST27-3 cyto constructs were transfected in 293T 561

cells with Calcium Phosphate (Promega) and GST and GST-3 cyto were isolated and 562

used to pulldown proteins from EC extracts as described in (Gagliardi et al., 2014). To 563

analyse the phosphorylation of 3 cytoplasmic tail, 293T cells expressing pDEST27-564

3 cyto construct were treated with either the PDK1 inhibitor BX-795 (1 m, Axon 565

Medchem) or Calyculin-A (10 nM, Merck-Millipore) for 1 hour; GST-3 cyto was 566

isolated through Glutathione Sepharose 4B beads and phosphorylation was analysed 567

by western blot. 568

Proximity ligation assay (PLA) 569

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PLA was performed according to the Olink Bioscience’s protocol using Duolink 570

II reagents with minor changes. After blocking and primary antibody staining (anti-571

PDK1 + anti-v, anti-PDK1 + anti-3 [C-20, Santa Cruz Biotechnologies], anti-572

PDK1 + anti-3 [AB1920, Millipore] and PDK1 + aspecific rabbit IgG as negative 573

controls), PLA anti-mouse MINUS and anti-rabbit PLUS or PLA anti-goat MINUS 574

and anti-rabbit PLUS probes were diluted 1:5 in PBS 1% donkey serum and incubated 575

for 1 hour at 37°C in a pre-heated humidity chamber. Subsequent ligation and 576

amplification steps were performed using Duolink II Detection Kit according to the 577

manufacturer’s protocol. 578

To study the phosphorylation of 3 integrin, EC, seeded on vitronectin, were treated 579

with either the PDK1 inhibitor BX-795 or Akt inhibitor for 1 hour and then analysed 580

as described above with anti-3 + anti-pSer/Thr Akt substrate antibodies and PLA 581

anti-goat MINUS and anti-rabbit PLUS probes. 582

Endocytosis assay 583

Endocytosis assay were performed as previously described in (Roberts et al., 2001) 584

and modified in (di Blasio et al., 2010). When endocytosis during nocodazole washout 585

was assayed, EC were treated with nocodazole as described above and then labelled 586

with biotin. Levels of biotinylated integrin were determined by capture-ELISA with 587

anti-v3 (MAB1976, Millipore), anti-3 (BD Biosciences) and anti-5 (BD 588

Biosciences). To analyse the endocytosis of wild-type and T753A mutant of 3 589

integrin, total biotinylated proteins were immunoprecipitated by Streptavidin-agarose 590

conjugate and then ectopic 3 was visualised by SDS-PAGE electrophoresis and 591

immunoblotting with anti-Flag antibody. Bands intensities from three independent 592

experiments were quantified using Image J software. 593

Flow cytometry 594

To detect levels of 3 integrin cell surface expression, cells were detached with 595

EDTA 10mM in PBS and incubated with primary antibody (anti-β3, BD Biosciences) 596

for 30 minutes at 4°C, and Alexa Fluor 488-conjugated secondary antibody at 4° C for 597

30 minutes. Cells were analysed on a Beckman Coulter Cyan ADP. 598

Total internal reflection fluorescence (TIRF) microscopy 599

EC transfected with paxillin-GFP were seeded at low density on glass bottom plates 600

(MatTek Corporation) coated with vitronectin (0.75 μg/ml). After 1 hour of adhesion, 601

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the cells were placed on an inverted microscope equipped with a humidified chamber 602

at 37°C and 5% CO2, and visualised using True MultiColor Laser TIRF Leica AM 603

TIRF MC (Leica Microsystems). 604

To quantify FA size and shape we employed image analysis algorithm developed in 605

(Zamir et al., 1999); (Berginski et al., 2011). Briefly, images of paxillin-GFP EC, 606

acquired by means of TIRF microscopy, were filtered with a high pass filter to 607

enhance high paxillin-GFP regions. The width of the filter used was 1.3 m. Filtered 608

images were thresholded by an empirically determined value chosen to identify 609

adhesive structures. Identified structures were then filtered to eliminate segmentation 610

errors. In particular, objects with a Major to Minor axis ratio larger than 5 and larger 611

than 5 m2 were disregarded. Threshold values used to discriminate between 612

large/long and small/round adhesions were 0.75 m of length and a Major to Minor 613

axis ratio of 2.5. In practice these two fractions correspond to the adhesion that are 614

large and elongated, and the adhesions that are small and round in shape. Image 615

analysis routines were written in MATLAB. 616

Cell migration assay 617

EC were starved overnight and then seeded on the upper side of 8-µm pore filters of 618

Transwell chamber (Falcon, 5104 cells/well) coated on both sides with vitronectin (5 619

µg/ml). After 5 hours of incubation at 37°C, 5% CO2, cells on the upper side of the 620

filters were mechanically removed and those that had migrated to the lower side of the 621

filters were fixed in 2.5% glutaraldehyde for 30 minutes and stained with 0.1% crystal 622

violet. Four random fields of each sample in the lower surface of the filters were 623

counted at 10x magnification. 624

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ACKNOWLEDGEMENTS 626

627

This work was supported by: Associazione Italiana per la Ricerca sul Cancro (AIRC) 628

(IG 10133, 14635, 9158); Fondazione Piemontese per la Ricerca sul Cancro 629

(Intramural FPRC/LUPR/2010); MIUR- Fondo Investimenti per la Ricerca di Base 630

RBAP11BYNP (Newton) (to FB and LP); University of Torino- Compagnia di San 631

Paolo, grant RETHE (to FB), grant GeneRNet, (to LP); FP7-ICT-2011-8 Biloba 632

(contract 318035) and CNR (grant “Personalized Medicine”). PAG is supported by a 633

triennial FIRC fellowship (15026). 634

635

AUTHOR CONTRIBUTIONS 636

LDB and LP conceived the project and designed experimental details. LDB 637

performed experiments. PAG set up PDK1 silencing and GST pulldown experiment. 638

AP performed analysis of FA on live cells. GS set up confocal microscopy analysis. 639

RS set up FACS analysis. LDB and LP wrote the manuscript with contributions from 640

all authors. FB and LP obtained funding and supervised the study. 641

642

CONFLICT OF INTEREST 643

The authors declare that they have no conflict of interest. 644

645

ABBREVIATIONS LIST 646

EC, endothelial cells; FA, focal adhesions; PDK1, 3-phosphoinositide-dependent 647

protein; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PLA, proximity 648

ligation assay; TIRF, total internal reflection fluorescence; cRGDfv, cyclo (Arg-Gly-649

Asp-D-Phe-Val). 650

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Gagliardi, P. A., di Blasio, L., Orso, F., Seano, G., Sessa, R., Taverna, D., 731 Bussolino, F. and Primo, L. (2012). 3-phosphoinositide-dependent kinase 1 controls 732

breast tumor growth in a kinase-dependent but Akt-independent manner. Neoplasia 733

14, 719-731. 734

Gagliardi, P. A., di Blasio, L., Puliafito, A., Seano, G., Sessa, R., Chianale, F., 735 Leung, T., Bussolino, F. and Primo, L. (2014). PDK1-mediated activation of 736

MRCKalpha regulates directional cell migration and lamellipodia retraction. J Cell 737

Biol 206, 415-434. 738

Gu, Z., Noss, E. H., Hsu, V. W. and Brenner, M. B. (2011). Integrins traffic rapidly 739

via circular dorsal ruffles and macropinocytosis during stimulated cell migration. J 740

Cell Biol 193, 61-70. 741

Gutheil, J. C., Campbell, T. N., Pierce, P. R., Watkins, J. D., Huse, W. D., 742 Bodkin, D. J. and Cheresh, D. A. (2000). Targeted antiangiogenic therapy for cancer 743

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Huang, X., Griffiths, M., Wu, J., Farese, R. V., Jr. and Sheppard, D. (2000). 751

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Haemost 5 Suppl 1, 32-40. 759

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filamin binding to integrins and competition with talin. Mol Cell 21, 337-347. 762

Kirk, R. I., Sanderson, M. R. and Lerea, K. M. (2000). Threonine phosphorylation 763

of the beta 3 integrin cytoplasmic tail, at a site recognized by PDK1 and Akt/PKB in 764

vitro, regulates Shc binding. J Biol Chem 275, 30901-30906. 765

Lauffenburger, D. A. and Horwitz, A. F. (1996). Cell migration: a physically 766

integrated molecular process. Cell 84, 359-369. 767

Lee, J., Ishihara, A. and Jacobson, K. (1993). How do cells move along surfaces? 768

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Liu, J., Fukuda, K., Xu, Z., Ma, Y. Q., Hirbawi, J., Mao, X., Wu, C., Plow, E. F. 770 and Qin, J. (2011). Structural basis of phosphoinositide binding to kindlin-2 protein 771

pleckstrin homology domain in regulating integrin activation. J Biol Chem 286, 772

43334-43342. 773

Liu, Y., Wang, J., Wu, M., Wan, W., Sun, R., Yang, D., Sun, X., Ma, D., Ying, G. 774 and Zhang, N. (2009). Down-regulation of 3-phosphoinositide-dependent protein 775

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PtdIns(3,4,5)P3 binding to PDK1 PH domain defined by knockin mutation. Embo J 780

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Nabors, L. B., Mikkelsen, T., Rosenfeld, S. S., Hochberg, F., Akella, N. S., Fisher, 784 J. D., Cloud, G. A., Zhang, Y., Carson, K., Wittemer, S. M. et al. (2007). Phase I 785

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endosomes is necessary for cell adhesion and spreading. Curr Biol 11, 1392-1402. 811

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fibronectin-based microenvironments. Nat Cell Biol 15, 625-636. 815

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mediates growth factor-induced Ral-GEF activation by a kinase- independent 831

mechanism. Embo J 21, 1327-1338. 832

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PKD1/PKCmu promotes alphavbeta3 integrin recycling and delivery to nascent focal 837

adhesions. Embo J 23, 2531-2543. 838

Zamir, E. and Geiger, B. (2001). Molecular complexity and dynamics of cell-matrix 839

adhesions. J Cell Sci 114, 3583-3590. 840

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Molecular diversity of cell-matrix adhesions. J Cell Sci 112 ( Pt 11), 1655-1669. 842

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843

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FIGURE LEGENDS 844

845

Figure 1. PDK1 regulates v3-dependent endothelial cells adhesion. A, B) 846

Control (shScrl) and PDK1-knockdown (shPDK1_79 and 81) EC adhering on 847

vitronectin and fibronectin was monitored for 2 hours using xCELLigence system. 848

Data were plotted as the mean Cell Index of three independent wells at each time 849

points; p-value was calculated at 40 minutes, * P<0.0005, § P<0.005, † P<0.05 for 850

knockdown EC versus control EC. C) Paxillin-GFP (green) transfected control (shScrl) 851

and PDK1 knockdown (shPDK1_79 and 81) EC were seeded on vitronectin and 852

stained with anti-v3 (red, Dapi in blue); scale bar 20 μm. D) Quantification of 853

paxillin- or v-positive FA area/cell, number/cell and average size. Data were plotted 854

as mean ± SD; ° P<0.001, † P<0.05 for knockdown EC versus control EC. E) Control 855

(shScrl) EC transfected with YFP alone (green) and shPDK1_79 EC transfected with 856

YFP alone or PDK1-wt-YFP were seeded on vitronectin and stained with anti-v3 857

(magenta, Dapi in blue); scale bar 50 μm. F) Quantification of FA average size. Data 858

were plotted as mean ± SD; § P<0.005 for shPDK1_79 EC transfected with PDK1-wt 859

versus shPDK1_79 EC. 860

861

Figure 2. PDK1 regulates FA disassembly on vitronectin. A, B) Paxillin-GFP 862

(green) transfected control (shScrl) and PDK1 knockdown (shPDK1_79 and 81) EC, 863

seeded on vitronectin, were treated with nocodazole (t=0 min NOCO washout, A). 864

Then the drug was washed out (t=30 min NOCO washout, B) and cells were stained 865

with anti- tubulin (red) and anti-v (magenta, Dapi in blue); scale bar 20 μm. C) 866

Quantification of FA area/cell and average FA size. Data were plotted as mean ± SD; ° 867

P<0.001, † P<0.05 for knockdown EC versus control EC. 868

869

Figure 3. PDK1 transiently localises to focal adhesions and associates with v3 870

integrin. A, B) Paxillin-GFP (green) transfected EC, seeded on vitronectin, were 871

treated with nocodazole (t=0 min NOCO washout, A). Then the drug was washed out 872

(t=30 min NOCO washout, B) and cells were stained with anti-v integrin (turquoise) 873

and anti-PDK1 (red, Dapi in blue). The merged images originate from the overlap of 874

paxillin and PDK1 staining; arrows indicate the zoomed regions; scale bar 10 μm. C) 875

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Paxillin-GFP (green) transfected EC, seeded on vitronectin, were fixed after 2 hours 876

of adhesion and stained with anti-v3 integrin (turquoise) and anti-PDK1 (red, Dapi 877

in blue). The merged images originate from the overlap of paxillin and PDK1 878

fluorescence; arrows indicate the zoomed regions; scale bar 20 μm. D) Empty vector-879

infected (-) or PDK1-overexpressing (+) EC were treated with nocodazole (t=0 min 880

NOCO wash). After nocodazole removal (t=30 min NOCO wash), EC were lysed and 881

v3 was immunoprecipitated; immunocomplexes and corresponding lysates were 882

immunoblotted with the indicated antibody. E) Wild-type EC were lysed and v3 883

was immunoprecipitated; immunocomplexes and corresponding lysate were 884

immunoblotted with the indicated antibody. F) Control (shScrl) and PDK1 885

knockdown (shPDK1_79 and 81) EC were lysed and v3 was immunoprecipitated; 886

immunocomplexes and corresponding lysates were immunoblotted with the indicated 887

antibody. G) Wild-type EC were lysed and pulldown was carried out using GST-fused 888

cytoplasmic tail of 3 integrin; immunocomplexes and corresponding lysate were 889

immunoblotted with the indicated antibody. H) Empty vector-infected (-) or PDK1-890

overexpressing EC (+) were lysed and peptide pulldown was carried out using 891

synthetic peptide corresponding to the cytoplasmic portion of v and 2 integrin. 892

Western blots shown are representative of four experiments performed with similar 893

results. I) EC were seeded on vitronectin; then PLA assay was performed with the 894

following antibodies: anti-PDK1/anti-v, anti-PDK1/anti-3, anti-PDK1/anti-3 and 895

anti-PDK1/rabbit IgG as negative controls. Dapi is in blue, red spots represent PLA 896

signals; scale bar 20 m for PDK1/rabbit IgG, 50 m for other pictures 897

898

Figure 4. PDK1 controls v3 endocytosis during focal adhesion disassembly. A) 899

Control (shScrl) and PDK1 knockdown (shPDK1_79 and 81) EC were plated on 900

vitronectin and incubated with anti-β3 antibody either at 4°C as control or at 37°C in 901

presence of primaquine to allow internalisation of antibody-bound integrin (red, Dapi 902

in blue); as a control of 3 surface staining, acid wash was done or not; scale bar 50 903

μm. B) Quantification of total vesicles area/cell after 10, 20 and 30 minutes of 904

endocytosis. Data were plotted as mean ± SD; § P<0.005 for knockdown EC versus 905

control EC. C) Endocytosis of v3 in presence of primaquine was evaluated in 906

control (shScrl) and PDK1 knockdown (shPDK1_79 and 81) EC after 10, 20 and 30 907

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minutes. Internalised biotinylated integrin was quantified by ELISA. Data were 908

plotted as mean ± SD of the percentage of total biotinylated integrin v3 which has 909

been internalised; § P<0.005 for knockdown EC versus control EC. D, E, F) Control 910

(shScrl) and PDK1 knockdown (shPDK1_79 and 81) EC, seeded on vitronectin, were 911

treated with nocodazole (t=0 min). Then, during the drug washout (t=30 min), cells 912

were incubated with anti-3 antibody in presence of primaquine and internalisation of 913

antibody-integrin complexes were followed by indirect immunoflourescence. FA 914

disassembly was confirmed with anti-paxillin antibody (magenta, internalised 3 915

integrin in green, Dapi in blue); scale bar 50 μm. G) Quantification of vesicles 916

area/cell. Data were plotted as mean ± SD; † P<0.05 for knockdown EC versus 917

control EC. H) Control (shScrl) and PDK1 knockdown (shPDK1_79 and 81) EC were 918

treated with nocodazole and v3 endocytosis was followed during drug washout in 919

presence or not of primaquine. Internalised biotinylated integrin was quantified by 920

ELISA. Data were plotted as mean ± SD of the percentage of total biotinylated v3 921

integrin which has been internalised; § P<0.005 for knockdown EC versus control EC. 922

923

Figure 5. PDK1 needs both PH domain and kinase activity to control v3 924

integrin endocytosis. A) shPDK1_79 EC infected with lentivirus carrying different 925

PDK1 mutants resistant to silencing (PDK1-wt, PDK1-caax, PDK1-ΔPH, PDK1-KD) 926

and control EC (shScrl) were plated on vitronectin and incubated with anti-β3 927

antibody in presence of primaquine to allow internalisation of antibody-bound 928

integrin (red, Dapi in blue); scale bar 20 μm. B) Quantification of vesicles area/cell of 929

experiment shown in A. Data were plotted as mean ± SD; † P<0.05 for knockdown 930

EC versus control EC. C) EC were transfected with Vinculin-RFP (red) in 931

combination with different YFP-fused mutants of PDK1 (wt, PH and KD, in green), 932

seeded on vitronectin and fixed after 2 hours of adhesion; arrows indicate the zoomed 933

regions; scale bar 20 μm. D) Endocytosis of 3 and Tfn-555 was performed as 934

described in A with wild-type EC in presence of Akt inhibitor (Akti), LY294002 or 935

vehicle; then total vesicles area/cell was quantified. Data were plotted as mean ± SD; 936

† P<0.05 for treated cells versus control EC. 937

938

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Figure 6. 3 integrin cytoplasmic tail is phosphorylated by PDK1. A) 293T cells 939

expressing GST-3 cyto were treated for 1 hour with either the PDK1 inhibitor BX-940

795 (PDKi) or Calyculin-A (Cal); then lysed and purified with Glutathione Sepharose; 941

GST-proteins were immunoblotted with the indicated antibody. B) EC treated for 1 942

hour with the PDK1 inhibitor BX-795 (PDKi) were lysed and v3 was 943

immunoprecipitated; immunocomplexes and corresponding lysates were 944

immunoblotted with the indicated antibody. C) EC, seeded on vitronectin, were 945

treated with either the PDK1 inhibitor BX-795 (PDKi) or Akt inhibitor (AKTi) for 1 946

hour and then PLA assay was performed with the following antibodies: anti-3/anti-947

pSer/Thr Akt-substrate. Dapi is in blue, red spots represent PLA signals; scale bar 50 948

μm. D) Control (shScrl) and PDK1 knockdown (shPDK1_79 and 81) EC were lysed 949

and v3 was immunoprecipitated; immunocomplexes were immunoblotted with the 950

indicated antibody. E) 3 wild-type- and 3 T753A-expressing EC, seeded on 951

vitronectin, were fixed after 2 hours of adhesion and stained with anti-phospho FAK 952

(green) and anti-Flag (red, Dapi in blue); scale bar 20 μm. F) 3 wild-type- and 3 953

T753A-expressing EC were lysed and 3 was immunoprecipitated; 954

immunocomplexes were immunoblotted with the indicated antibody. 955

956

Figure 7. Threonine 753 of 3 integrin regulates its endocytosis. A, B) 957

Endocytosis of 3-Flag in presence of primaquine was evaluated in 3 wild-type- and 958

3 T753A-expressing EC after 10, 20 and 30 minutes. Total (tot) and internalised 959

biotinylated integrin was quantified by Streptavidin-agarose immunoprecipitation and 960

anti-Flag immunoblot (A). Data were plotted as mean ± SD of the percentage of total 961

biotinylated 3-Flag integrin which has been internalised (B); † P<0.05 for 3 962

T753A- EC versus 3 wild-type-EC. C) 3 wild-type- and 3 T753A-expressing EC 963

were plated on vitronectin and incubated with anti-β3 antibody at 37°C in presence of 964

primaquine to allow internalisation of antibody-bound integrin (green, Dapi in blue); 965

as a control ectopic 3 integrin was stained with anti-Flag antibody (red); scale bar 50 966

μm. D) Quantification of total vesicles area/cell after 30 minutes of endocytosis. Data 967

were plotted as mean ± SD; † P<0.05 for 3 T753A-expressing EC versus 3 wild-968

type-expressing EC. 969

970

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Figure 8. Adhesive structures in membrane protrusions and migration towards 971

vitronectin are regulated by PDK1. Representative shScrl (A) and shPDK1_79 (B) 972

EC transduced with paxillin-GFP and imaged by TIRF microscopy are shown. The 973

area marked by the dashed rectangle is enlarged in the right panels and representative 974

snapshots of the expanding protrusion are taken every 8 minutes for a total time of 25 975

minutes. Scale bar 10 m. (C) Images of several protrusions per filmed silenced and 976

control EC were analysed. Each protrusion was identified as a subregion of the cell 977

expanding and retracting. Images were then segmented and the ratio of large and long 978

to small and round adhesive structures was calculated for each protrusion. Each point 979

in the plot represents thus one protrusion. shScrl (N=22 protrusions with Na = 779 980

identified adhesive structures) versus shPDK1_79 (N =12, Na = 409), P= 0.0124; 981

shScrl versus shPDK1_79_PH (N = 10, Na = 344, P= 7.5623e-04). Statistical tests 982

were performed using a one-tailed t-test with a significance level of 0.05. (D) Control 983

(shScrl) and PDK1 knock-down EC (shPDK1_79 and 81) were used in a Transwell 984

migration assay, in presence of vitronectin coating. Data were plotted as mean ± SD;* 985

P<0.0005, § P<0.005 for knock-down EC versus control EC. 986

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FIG. 1

C D

shSc

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0.4

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18_1KDPhslrcShs shPDK1_79

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v

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αv

PDK1

Paxillin-GFPt=0 min NOCO washout

merge

t=30 min NOCO washout

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α2 αv beads

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PDK1

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shScrl 79 81 shScrlshPDK1

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PDK1+β3

PDK1+α3

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PDK1+αv

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input

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050

100150200250300350

shScrl shPDK1_79 shPDK1_81

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§§

β3 endocytosis during FA disassembly H

Paxillin Paxillin endo β3 Paxillinendo β3endo β3

tota

l ves

icle

s ar

ea/c

ell µ

m2

A B

C β3 endocytosis

0102030405060708090

% in

tern

alis

ed α

vβ3

shScrlshPDK1_79shPDK1_81

§§

4ºC 30 min

no a

cid

was

h

ac

id w

ash

18_1KDPhslrcShs shPDK1_79

37ºC 30 min

no a

cid

was

h

ac

id w

ash

18_1KDPhslrcShs shPDK1_79

β3 endocytosis during FA disassembly

FIG. 4

050

100150200250300350400450 shScrl

shPDK1_79shPDK1_81

§§

§

β3 endocytosis

αvβ

3

10 min 20 min 30 min

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

shScrl

shPDK1_79

PDK1-caaxPDK1-wt

PDK1-∆PH PDK1-KD

FIG. 5

A

C D

shPDK1_79

Vinc

ulin

-RFP

PDK

1-YF

Pm

erge

0

50

100

150

200

250

tota

l ves

icle

s ar

ea/c

ell µ

m2

- - wt caax ∆PH KD L155E

shScrl shPDK1_79

†

†

†

β3 endocytosis

B

050

100150200250300350400

tota

l ves

icle

s ar

ea/c

ell µ

m2 β3

Tfn-555†

†

Endocytosis

wt ∆PH KD

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

PLA β3 + pSer/Thr

vehicle AKTiPDKi

Flag

pSer/Thr

E F

- PDKi Cal

pSer/Thrβ3GST

GST-β3 cyto IP αvβ3 lysates

- PDKi - PDKi

pSer/Thr

β3

A CB

FIG. 6

IP αvβ3

shScrl 79 81shPDK1

Flag pFAK merge

β3 w

t β3

T75

3A - wt T753A

IP β3

β3

pSer/Thr

β3

β3

D

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

AStreptavidin IP lysates

tot 10 20 30 β3 wt β3 T753A

wt T753Atot

Flagtot 10 20 30

% in

tern

alis

ed β

3-Fl

ag

Bβ3-Flag endocytosis

Flag

endo

β3

β3 wt

mer

ge

β3 T753A C

0

50

100

150

tota

l ves

icle

s ar

ea/c

ell µ

2

β3 endocytosis

β3 wt β3 T753A

†

D

0

10

20

30

40

50

60

70

0 10min 20min 30min

β3 wt β3 T753A †

FIG. 7

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

0

0.1

0.2

0.3

0.4

0.5

Largeto

smallFAfra

ctions

0

20

40

60

80

100

mig

rate

d ce

lls

shScrlshPDK1_79shPDK1_81

*

§

A

B

DC Migration on vitronectin

FIG. 8

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t