1

FHOD1 regulates stress fiber organization by controlling transversal arc 1

and dorsal fiber dynamics 2

3 4

Nina [Schulze]1, Melanie [Graessl]1, Alexandra [Blancke Soares]2, Matthias [Geyer]3, Leif 5

[Dehmelt]4, Perihan [Nalbant]1,* 6

7

8 1Department of Molecular Cell Biology, Center for Medical Biotechnology, University of 9

Duisburg-Essen, 45141 Essen, Germany 10 2Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany 11 3Research Center Caesar, 53175 Bonn, Germany 12 4Department of Systemic Cell Biology, Max-Planck-Institute of Molecular Physiology, and 13

Dortmund University of Technology, Fakultät Chemie, Chemische Biologie, 44227 14

Dortmund, Germany 15

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*Corresponding author: 22

Perihan Nalbant, Ph. D. 23

Molecular Cell Biology 24

University of Duisburg-Essen 25

Universitätsstrasse 2 26

45141 Essen, Germany 27

Phone: +49 201 183 3206 28

Fax: +49 201 183 7339 29

Email: perihan.nalbant@uni-due.de 30

31

Running title: FHOD1 in stress fiber organization 32

This is an Open Access article distributed under the terms of the Creative Commons Attribution License(http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium providedthat the original work is properly attributed.

© 2014. Published by The Company of Biologists Ltd.Jo

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JCS Advance Online Article. Posted on 30 January 2014

2

Summary 33

34

The formin FHOD1 can act as a capping and bundling protein in vitro. In cells, active FHOD1 35

stimulates the formation of ventral stress fibers. However, the cellular mechanism by which 36

this phenotype is produced and the physiological relevance of FHOD1 function were not 37

understood so far. Here, we first show that FHOD1 differentially controls the formation of 38

two distinct stress fiber precursors. On the one hand, it inhibits dorsal fiber growth, which 39

requires polymerization of parallel, long actin filament bundles. On the other hand, it 40

stimulates transversal arcs that are formed by fusion of short antiparallel actin filaments. This 41

combined action is critical for stress fiber maturation and their spatio-temporal organization 42

and lack of FHOD1 function perturbs dynamic cell behavior during cell migration. 43

Furthermore, we show that the GBD-FH3 domains are responsible for stress fiber association 44

and co-localization with Myosin. Surprisingly, FHOD1 that lacks those domains nevertheless 45

retains its full capacity to stimulate arc and ventral stress fiber formation. Based on our 46

findings we propose a mechanism, in which FHOD1 promotes the formation of short actin 47

filaments and transiently associates with transversal arcs to tightly control their formation and 48

turn-over into mature ventral stress fibers in space and time during dynamic cell behavior. 49

50

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Introduction 51

52

Diaphanous related formins (DRFs) form an evolutionarily conserved family of actin 53

regulators, which are involved in numerous cellular processes such as morphogenesis, cell 54

division and cell polarity. In general, formins are well known for their ability to act as 55

processive actin nucleators, or “leaky cappers” for actin filaments: they catalyze the 56

nucleation of new actin filaments and stay attached to the growing filament barbed end 57

(Kovar and Pollard, 2004; Higashida et al., 2004). However, the efficiency of actin nucleation 58

and subsequent, processive monomer addition varies greatly among individual family 59

members (Paul and Pollard, 2009). In addition to their nucleation activity, several formins 60

have been described to have other modulatory roles such as actin filament bundling or actin 61

severing/de-polymerization (Harris et al., 2006; Harris et al., 2010), suggesting that they 62

might also play additional roles in cells (Harris et al., 2004; Chhabra and Higgs, 2006; 63

Chesarone et al., 2010). 64

65

For multiple family members, the highly conserved FH2 domain was shown to be sufficient to 66

catalyze actin nucleation in vitro. The FH1 domain is thought to promote processive filament 67

elongation by recruiting profilin bound monomeric actin (Sagot et al., 2002; Paul and Pollard, 68

2008; Romero et al., 2004). Two additional modules shared among DRFs, the C-terminal 69

DAD (diaphanous autoregulatory domain) and the N-terminal DID (diaphanous inhibitory 70

domain) region, interact with each other, keeping the protein in the inactive conformation 71

(Alberts, 2001; Faix and Grosse, 2006; Schönichen and Geyer, 2010). In mDia1, this auto-72

inhibitory, intramolecular interaction was shown to be released by binding of the Rho GTPase 73

RhoA to the N-terminal GTPase binding domain (GBD) stimulating the actin nucleating 74

activity of this formin and promoting actin polymerization into stress fibers from focal 75

adhesions (Watanabe et al., 1997; Hotulainen and Lappalainen, 2006). ROCK, another 76

prominent effector of RhoA, phosphorylates Myosin II and increases its motor activity to 77

promote bundling and contractility of existing actin filaments (Ishizaki et al., 1997). Thus, 78

RhoA controls stress fiber formation via the concerted activation of at least two downstream 79

effectors: Rho kinase (ROCK) and mDia1 (Watanabe et al., 1999). 80

81

The formin FHOD1 is also thought to play a role in stress fiber formation (Koka et al., 2003). 82

FHOD1 shares a similar domain organization with other DRFs, and is also activated by a 83

mechanism involving release of auto-inhibition (Schönichen et al., 2006). Truncation of the 84

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C-terminal amino acids 1012-1164 including the DAD region and a short sequence with 85

unknown function (called X) leads to constitutive activation of this formin and formation of 86

prominent stress fibers throughout the cell (Koka et al., 2003; Gasteier et al., 2003). The 87

RhoA effector ROCK phosphorylates FHOD1 at three residues within this C-terminal region 88

(S1131, S1137, T1141) ablating the inhibitory interaction between the N-terminal FH3 89

domain (DID in mDia1) and the DAD-region (Takeya et al., 2008). Similar to the C-terminal 90

truncation mutant, overexpression of the corresponding triple phosphomimetic mutant induces 91

the formation of thick linear stress fibers, suggesting that ROCK mediated phosphorylation is 92

involved in the activation of FHOD1 (Takeya et al., 2008). In addition, the Rho GTPase Rac1 93

was suggested to release the auto-inhibition of FHOD1 by binding to the GBD region similar 94

to the interaction between RhoA and mDia1 (Westendorf, 2001). However, there is little 95

experimental evidence to support this claim and it is currently unclear, how Rac1 activity 96

affects FHOD1 in cells. Active FHOD1 has been shown to localize to stress fibers and its 97

overexpression in cells induces the formation of thick rigid actin fibers (Koka et al., 2003; 98

Gasteier et al., 2005). Truncation of a region encompassing the GBD-, FH3 and a part of the 99

coiled-coil domain (1-573) was shown to ablate FHOD1 stress fiber binding (Takeya et al., 100

2003; Schönichen et al., 2013), however, the functional role of stress fiber association was not 101

explored in cells. 102

103

Based on a recent study by Krainer et al. FHOD1 expression was among the highest of all 15 104

human formins tested in a large variety of cell lines and tissues suggesting its global 105

physiological relevance in cytoskeletal organization (Krainer et al., 2013). Yet, it is currently 106

not known how FHOD1 is involved in the regulation of the complex actin meshwork and its 107

coordinated reorganization during cellular morphogenesis or directional cell migration. In 108

adherent cells, several distinct types of stress fibers exist, each of which could be regulated 109

differentially by endogenous FHOD1. Contractile transversal arcs are curved structures 110

formed by the anti-parallel assembly of short actin filaments (Zhang et al., 2003). After their 111

initial formation in the lamellipodium, they undergo retrograde flow towards the cell center 112

(Heath, 1983; Small et al., 1998; Hotulainen and Lappalainen, 2006). Myosin II motors and 113

the filament bundling protein α-actinin are incorporated into arcs in a periodically alternating 114

pattern (Hotulainen and Lappalainen, 2006). During their retrograde flow arcs are not linked 115

to focal adhesions but associate with dorsal stress fibers which are also generated in the 116

leading edge of motile cells. These fibers are associated to focal adhesions with their distal 117

ends whereas their proximal end is linked to the retrograde flow of the transversal arc 118

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meshwork (Small et al., 1998; Hotulainen and Lappalainen, 2006). In contrast to arcs, dorsal 119

stress fibers are formed by polymerization of long parallel actin filaments at focal adhesions. 120

Lastly, ventral stress fibers are coupled with both ends to focal adhesions and thought to be 121

formed by fusion of transversal arcs with two flanking dorsal stress fibers or by end-to-end-122

fusion of two dorsal stress fibers with each other (Small et al., 1998; Hotulainen and 123

Lappalainen, 2006). Stress fibers induced by active FHOD1 mutants might be related to such 124

ventral stress fibers. However, the precise formation mechanism and spatial organization of 125

these thick actin fibers are still poorly understood. Based on its homology to other DRFs, 126

FHOD1 would be expected to play a role in those processes by nucleating and stimulating 127

actin polymerization in stress fibers. However, a recent study by Schönichen et al. showed 128

that FHOD1 does not act as an actin filament nucleator, but instead can cap and bundle actin 129

filaments in vitro (Schönichen et al., 2013). It is currently unclear, what role those molecular 130

functions of FHOD1 play in cells, and if they are involved in the enhanced formation of 131

ventral stress fibers by the activated formin. 132

133

Here we used a combination of live cell microscopy, expression of truncation mutants and 134

acute pharmacological perturbation to investigate FHOD1 function in cells. Our studies 135

revealed that this formin distinctly regulates the formation and growth of different stress fiber 136

types. RNAi-mediated silencing of FHOD1 expression resulted in the collapse of the cellular 137

stress fiber meshwork and defects in cell migration and cell spreading. Together, our work 138

supports a model, in which FHOD1 plays a central role in the spatial and temporal 139

coordination of cellular stress fiber dynamics. 140

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Results 141

142

FHOD1 depletion leads to the collapse of the stress fiber meshwork in U2OS cells 143

144

To study the cellular mechanism how FHOD1 induces stress fibers in cells, we first used 145

RNAi to deplete the endogenous formin. Efficient knock-down of the protein was confirmed 146

by western blot analysis (Fig. S1A). First, transversal arcs and dorsal stress fibers were 147

quantified in RNAi treated cells. We distinguished between those distinct stress fiber types 148

based on their association pattern to focal adhesions (Hotulainen and Lappalainen, 2006): 149

actin arcs are not associated to focal adhesions but instead linked to dorsal stress fibers, and 150

dorsal stress fibers are associated with their distal end to focal adhesions and with their 151

proximal end to actin arcs. After FHOD1 depletion, both well-organized actin arcs and dorsal 152

stress fibers were observed less frequently (Fig. 1A,B). Instead, cells preferentially formed 153

thick and less dynamic peripheral actin bundles (Fig. 1A, yellow arrow, B). These findings 154

were confirmed with four individual siRNA oligonucleotides to exclude off-target effects 155

(Fig. S1B). As reported earlier (Small et al., 1998; Hotulainen and Lappalainen, 2006), 156

transversal arcs were localized on the dorsal cell surface (Fig. 1C). We next analyzed, if 157

FHOD1 plays a role in the spatial organization of arcs within individual cells. We restricted 158

our morphometric measurements to those cells that were still able to form arcs, and found that 159

FHOD1 depletion decreased the arc-covered cell area (Fig. 1D, see Fig. S1C for definition of 160

area quantification). Furthermore, the average actin fluorescence intensity was reduced in this 161

area, suggesting that the density of arcs is reduced as well (Fig. 1E). Interestingly, we 162

observed a substantial increase of cells that contained prominent stellate accumulations of 163

actin fibers (Fig. 1F-H). The exclusive presence of the focal adhesion marker paxillin at the 164

distal ends of the individual fibers and the strong Myosin II staining in the center suggests that 165

those unusual structures might be generated by collapse of the transversal arc and dorsal stress 166

fiber meshwork (Fig. 1G,H). Live-cell imaging revealed that these structures are only formed 167

transiently (Fig. 1I) to leave behind the less dynamic peripheral actin bundles described above 168

(Fig. 1A, yellow arrow). In addition to the clear long-term effects of FHOD1 depletion on 169

actin network organization, Cytochalasin D washout experiments revealed impaired stress 170

fiber recovery in cells lacking FHOD1, suggesting that this formin is not only critical for 171

long-term network organization, but also for the dynamic de novo formation of stress fibers 172

(Fig. S2). These striking defects in actin stress fiber organization were accompanied by a 173

significant decrease in migration efficiency (Fig. S3A-D). 174

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175

The observed disruption in actin network organization was also paralleled by alterations in the 176

size of focal adhesions. We quantified this effect by measuring the average number and focal 177

adhesion size in confocal micrographs of mKate-Paxillin expressing cells (Fig. 2A). We 178

found that the average size of focal adhesions in FHOD1 depleted cells was significantly 179

decreased (Fig. 2B) while the number of focal adhesions per cell was significantly increased 180

(Fig. 2C). In contrast, average cell area was not altered after formin depletion (ntsiRNA: 1740 181

µm2 ± 123.8; FHOD1 siRNA: 1679 µm2 ± 97.05 (mean ± s.e.m.)). This apparent change in 182

focal adhesion organization was accompanied by only moderately perturbed cell spreading 183

(Fig. 2D,E; Fig. S3E). We found that the early phase of U2OS cell spreading on collagen type 184

I was significantly impaired as reflected by a smaller average adhesion area compared to 185

control cells (Fig. 2D,E, 30 min). Interestingly, this difference was ablated at a later time-186

point (Fig. 2D,E, 60 min) suggesting compensation by other, possibly Rac1 mediated 187

mechanisms (Price et al., 1998; del Pozo et al., 2000). 188

189

190

FHOD1 promotes transversal arc formation and their turnover into ventral stress fibers 191

192

In order to study the cellular mechanism, by which FHOD1 affects the cellular stress fiber 193

organization, we combined acute Cytochalasin D washout experiments with the expression of 194

FHOD1 mutants. Compared to control EGFP-transfected cells, overexpression of wild-type 195

FHOD1 did not significantly alter stress fiber recovery dynamics (Fig. 3A,B). To study the 196

effect of activated FHOD1 we used a point-mutant (FHOD1 V228E) which induces an open 197

conformation and strongly stimulates stress fiber formation in cells (Schulte et al., 2008). In 198

contrast to C-terminally truncated constitutively active FHOD1 (FHOD1 1-1011), this point-199

mutant retains an intact C-terminus. After Cytochalasin D washout, FHOD1 V228E induced 200

enhanced generation of dorsal transversal arcs as compared to EGFP control and wild-type 201

FHOD1 (Fig. 3A,B). This observation is further supported by an analysis of confocal F-actin 202

image stacks, which revealed enhanced F-actin staining on the dorsal cell surface induced by 203

FHOD1 V228E (Fig. 3C). 204

205

In addition, live-cell imaging during acute Cytochalasin D washout revealed that the enhanced 206

arc formation was paralleled by an accelerated transformation of arcs into straight stress fibers 207

(Fig. 3C-E and Movie S1). This can explain the known long-term effect of enhanced stress 208

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fiber formation after overexpression of constitutively active FHOD1 mutants (Koka et al., 209

2003; Gasteier et al., 2003; Takeya and Sumimoto, 2003; Schulte et al., 2008). Our data now 210

show that FHOD1 stimulates the generation of transversal arcs, which then more rapidly 211

mature into linear stress fiber bundles (Fig. 3D-F). 212

213

We next examined the effect of FHOD1 activation or inactivation on dorsal stress fibers. In 214

cells lacking this formin, we found that the average growth rate of dorsal stress fibers and the 215

average maximal fiber-length were significantly increased (Fig. 4A,B). Furthermore, in 216

FHOD1-depleted cells, the majority of mature dorsal stress fibers bent or buckled instead of 217

growing straight and perpendicular to the cell edge as in control cells (Fig. 4C,D and Movie 218

S2). This suggests that the formation of dorsal stress fibers is stimulated in the absence of 219

FHOD1 and that their regulation is perturbed leading to aberrant turnover dynamics. 220

Conversely, dorsal stress fibers were observed less frequently and their average length was 221

decreased after Cytochalasin D washout if activated FHOD1 was expressed (Fig. 4E-G). In 222

contrast to other formins, such as mDia1 which was proposed to stimulate actin 223

polymerization by processive actin filament elongation (Higashida et al., 2004), this 224

inhibitory effect of FHOD1 might be due to an alternative mechanism involving filament end 225

capping. 226

227

228

FHOD1 localizes to contractile anti-parallel actin arrays at sites of Myosin association 229

230

To gain more insight into the mechanism of FHOD1 mediated actin arc formation we next 231

studied its intracellular localization via confocal scanning and TIRF microscopy at low 232

expression levels. Wild-type FHOD1 is largely cytosolic, however, due to the increased 233

signal-to-background ratio in TIRF microscopy, we found that wild-type FHOD1 was 234

distributed in an irregular, punctate pattern, with some preference for ventral stress fibers. 235

Those structures are known to form anti-parallel actin filament arrays (i.e. arrays of mixed 236

actin filament polarity (Naumanen et al., 2008)) (Fig. 5A, yellow arrow). Actin arcs, which 237

also contain anti-parallel actin arrays undergo rapid retrograde flow along the dorsal side of 238

the cell and are therefore hardly detected via TIRF microscopy. The constitutively active 239

mutant FHOD1 1-1011 was strongly associated with both ventral stress fibers and arcs and 240

therefore easily detected in the complete cell volume via confocal scanning microscopy (Fig. 241

5B, yellow and green arrows, respectively). 242

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243

Interestingly, active FHOD1 did not bind to the majority of dorsal stress fiber structures, 244

which are thought to be composed of parallel actin arrays (i.e. unidirectional actin filament 245

arrays) (Fig. 5B, orange arrows). Thus, the preferential binding of FHOD1 to ventral stress 246

fibers and actin arcs compared to dorsal fibers suggests that FHOD1 selectively binds to anti-247

parallel actin arrays. However, FHOD1 did bind to the distal ends of dorsal fibers that 248

overlapped with focal adhesions (Fig. 5B, red arrows). Interestingly, similar to FHOD1, 249

Myosin was also strongly localized to the distal ends of dorsal stress fibers that substantially 250

overlap with focal adhesions (Fig. 5C, red arrows, S4A), suggesting coordinated 251

functionalities of the two proteins in these areas. 252

253

Arcs are formed in the lamellipodium by bundling and fusion of short α-actinin bound actin 254

filaments that undergo rapid retrograde flow (Hotulainen and Lappalainen, 2006; Burnette et 255

al., 2011; Tojkander et al., 2011). Myosin II is incorporated into these actin filament bundles 256

generating an alternating pattern with α-actinin. As FHOD1 associates with stress fibers, it 257

might play a role in coordinating those processes. Previously, it was unclear, if FHOD1 co-258

operates with α-actinin mediated filament assembly or Myosin mediated contractility. Here, 259

we found that activated FHOD1 (EGFP-FHOD1 V228E) substantially co-localized with 260

Myosin and was excluded from α-actinin enriched regions (Figs 5D, S4B). Thus, our results 261

suggest that FHOD1 might co-operate with Myosin to regulate the formation of contractile 262

stress fibers. 263

264

265

Recruitment of FHOD1 to stress fibers and Myosin co-localization requires an N-terminal 266

targeting region 267

268

Stress fiber localization of FHOD1 is mediated by sequences within its N-terminal region 1-269

573 that consists of multiple parts including the GBD, FH3-, linker and helical domain 270

(Takeya and Sumimoto, 2003; Schönichen et al., 2013). The FH3 domain adjacent to the 271

GBD bears the capacity of FHOD1 to interact with the C-terminal DAD to mediate auto-272

inhibition (Takeya and Sumimoto, 2003; Schönichen et al., 2006; Schulte et al., 2008). The 273

helical domain was suggested to be responsible for FHOD1 side-binding to actin filaments in 274

vitro and might therefore mediate filament bundling (Schönichen et al., 2013). However, the 275

precise N-terminal region that is responsible for stress fiber localization was not mapped so 276

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far. To address this question, we generated various FHOD1 truncation mutants and analyzed 277

their subcellular localization (Figs 6A,B, S5B). 278

279

As expected, full-length, activated FHOD1 V228E robustly associated with stress fibers 280

similar to the previously described constitutive active mutant FHOD1 1-1011 (Schulte et al., 281

2008; Koka et al., 2003; Gasteier et al., 2003; Takeya and Sumimoto, 2003) (Figs 6A,B). In 282

agreement with previous studies, the entire N-terminus (1-573) was found along stress fibers 283

(Fig. 6B). Here, we show that GBD alone (1-115) was predominantly cytosolic, whereas 284

GBD-FH3 (1-339) yielded distinct association with stress fibers (Fig. 6B) and co-localization 285

with Myosin II (Fig. 6C). In contrast, a construct lacking these domains (340-1164) was again 286

mostly cytosolic as judged by confocal microscopy (data not shown) and did not localize 287

along stress fibers. However, using TIRF microscopy, we found a weak but reproducible 288

accumulation of FHOD1 340-1164 along the entire length of focal adhesions (Figs 6B, S5C). 289

Interestingly, when stress fiber formation was stimulated acutely by Cytochalasin D washout, 290

this N-terminal truncation mutant strongly accumulated in the lamellipodium (Fig. 6D). 291

Lamellipodia were rarely observed after long-term expression of FHOD1 340-1164, however, 292

if they were present we also observed enrichment of this mutant in this cell region in steady 293

state conditions (data not shown). Together, these observations show that the GBD-FH3 294

domains can mediate FHOD1 stress fiber association and co-localization with Myosin II 295

independent of the two domains that were previously proposed to mediate F-actin interaction: 296

the FH2 domain (617-1011) and the helical domain (396-573) (Pruyne et al., 2002; 297

Schönichen and Geyer, 2010; Schönichen et al., 2013). In agreement with this, a construct 298

containing only the linker region and the helical domain (340-573) localized mostly diffusely 299

in the cells (Fig. S5B). 300

301

As shown recently, truncation of the entire N-terminus (574-1164) effectively ablated stress 302

fiber association (Fig. 6B) (Schönichen et al., 2013). Here, the majority of this truncated 303

FHOD1 was mostly cytosolic, but also found in dot-like accumulations throughout the cell 304

body with a preference for focal adhesions suggesting that this construct has a tendency to 305

form aggregates but nevertheless retains some subcellular targeting functionality (Fig. 6B). 306

307

We next studied the functional role of the FHOD1 N-terminal stress fiber localization domain 308

and the helical domain. Surprisingly, in long-term expression as well as acute Cytochalasin D 309

washout experiments FHOD1 340-1164 which lacks the stress fiber targeting GBD-FH3 310

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domains (Figs 6B), nevertheless stimulated the formation of thick linear stress fibers to a 311

similar extent as the constitutively active FHOD1 V228E (Fig. 7A-C). FHOD1 340-1164 still 312

encompasses the helical domain (396-573) which was suggested to bind actin filaments in 313

vitro via side-binding and therefore might mediate filament bundling (Schönichen et al., 314

2013). Thus, the helical domain might play a functional role in stress fiber formation. Indeed, 315

the truncated mutant that lacks the helical domain (FHOD1 574-1164) failed to stimulate 316

stress fiber formation (Fig. 7A,B). 317

318

Thus, FHOD1 can induce stress fibers independently of the N-terminal GBD-FH3 domains 319

(1-339), which are responsible for stress fiber targeting. Our results therefore suggest that the 320

helical domain 396-573 plays a key role in the enhanced stress fiber formation by the FHOD1 321

340-1164 mutant, potentially via its accumulation in the lamellipodium (Fig. 6D), which 322

might lead to the formation of short actin filament bundles via its proposed role in actin 323

bundling (Schönichen et al., 2013). 324

325

We next characterized the spatial organization of the thick actin stress fiber bundles that are 326

induced by activated FHOD1 V228E in more detail using confocal F-actin image stacks. We 327

found that the majority of those bundles were localized on the ventral side of cells, where they 328

associated with focal adhesions on both ends. Thus, those bundles were mainly composed of 329

ventral stress fibers (Fig. 7D, left). Interestingly, we also observed a minor fraction of thick 330

actin bundles that formed an arch-like structure, being localized dorsal only in their central 331

region (Fig. 7D, right, white arrow) and ventral in the flanking fiber ends (Fig. 7D, right, 332

white arrow tip), which were associated with focal adhesions. Thus, while sharing partial 333

dorsal localization with actin arcs, those arch-like structures also shared properties of ventral 334

stress fibers. 335

336

337

The C-terminal FHOD1 domain is required for efficient formation of ventral stress fibers 338

339

The C-terminal DAD region of FHOD1 mediates its auto-inhibitory conformation by 340

interacting with the N-terminal FH3 domain (Takeya and Sumimoto, 2003; Schönichen et al., 341

2006). The C-terminally truncated FHOD1 construct (FHOD1 1-1011) was therefore used in 342

previous studies as a model for constitutively active FHOD1 with open conformation. To test 343

if the FHOD1 C-terminus might play additional functional roles, we compared stress fiber 344

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formation of FHOD1 1-1011 with the FHOD1 point mutant (V228E), which contains the 345

entire C-terminus. Similar to FHOD1 1-1011, FHOD1 V228E is in the active conformation, 346

as this mutant blocks the intramolecular auto-inhibitory interaction (Schulte et al., 2008). We 347

analyzed the effect of these FHOD1 constructs on actin organization by quantifying the 348

number of cells that contain an increased number of mature ventral stress fibers (Fig. 8A). As 349

expected, wild-type FHOD1 only weakly enhanced formation of ventral stress fibers, whereas 350

the FHOD1 1-1011 mutant significantly stimulated their formation almost 3 fold as compared 351

to EGFP (Fig. 8A). Interestingly, the FHOD1 V228E mutant had an even more pronounced 352

effect on ventral stress fiber formation. This was surprising, as confocal imaging and cross 353

correlation analysis of FHOD1 fluorescence with the corresponding actin signals revealed that 354

the FHOD1 V228E mutant was only weakly associated with stress fibers as compared to 355

FHOD1 1-1011 (Fig. 8B,C). 356

357

While FHOD1 1-1011 lacks the three C-terminal residues phosphorylated by the upstream 358

regulator ROCK, these sites are still present in FHOD1 V228E. To study the effects of ROCK 359

inhibition on localization of those mutants, we combined acute pharmacological inhibition of 360

the kinase with live-cell TIRF microscopy. We found that wild-type FHOD1 rapidly 361

dissociated from actin fibers after addition of the ROCK inhibitor Y27632 (50 µM, 5 min). 362

This suggests that FHOD1 phosphorylation by ROCK antagonizes constitutive de-363

phosphorylation, thereby allowing dynamic control of FHOD1 stress fiber association (Fig. 364

8D,E). As expected, FHOD1 1-1011 remained mostly at stress fibers after ROCK inhibition. 365

However, FHOD1 V228E dissociated substantially from stress fibers after Y-27632 treatment 366

similar to wild-type FHOD1 (Fig. 8D, arrows, E). This finding suggests that even in the 367

FHOD1 V228E mutant, phosphorylation of the C-terminus still controls the actin binding 368

function mediated by the N-terminal GBD-FH3 domains. 369

370

Together, our observations suggest that the C-terminus plays not only an important role in 371

FHOD1 auto-inhibition, but also a more direct functional role in the stimulatory effect of 372

FHOD1 on stress fiber formation. 373

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Discussion 374

375

Here, we show that FHOD1 controls the coordinated maturation of stress fibers by its distinct 376

effects on two dynamic precursor stress fiber types: stimulation of transversal arcs and slower 377

growth of dorsal fibers. This combined action of FHOD1 leads to efficient formation of less 378

dynamic ventral stress fibers. Our detailed analysis of this process offers novel mechanistic 379

insight into the role of FHOD1 in the dynamic processes that build contractile actin structures 380

in cells. 381

382

Interestingly, FHOD1 was preferentially associated with actin structures that have anti-383

parallel orientation, such as ventral stress fibers and transversal arcs. Conversely, FHOD1 was 384

largely absent from dorsal fibers, which mainly consist of parallel actin-bundles (Cramer et 385

al., 1997; Svitkina et al., 1997; Pellegrin and Mellor, 2007). This preference of FHOD1 for 386

anti-parallel actin fibers is shared by Myosin II (Verkhovsky and Borisy, 1993), which has to 387

bind such anti-parallel fibers to generate contractile forces (Clark et al., 2007). The only 388

region close to dorsal stress fibers, which showed FHOD1 and Myosin II association, 389

overlapped substantially with focal adhesions suggesting that a subpopulation of anti-parallel 390

oriented actin filaments albeit with yet unknown function might be present in that region. It is 391

currently unclear, how FHOD1 might be able to selectively associate with anti-parallel actin 392

bundles. One possibility would be binding to Myosin II either directly or indirectly via 393

adapter proteins. Our observation, that FHOD1 is co-localized with Myosin II rich stripes and 394

not with α-actinin within stress fibers, supports this idea. The N-terminal GBD-FH3 region is 395

required for FHOD1 co-localization with Myosin II and might thus mediate such coordinated 396

recruitment. 397

398

Both Myosin II and FHOD1 are activated by phosphorylation via the serine/threonine kinase 399

ROCK (Amano et al., 1996a; Kimura et al., 1996; Hannemann et al., 2008; Takeya et al., 400

2008). The upstream activator of ROCK, the small GTPase RhoA, is known to form spatial 401

activity gradients that correlate with cell motility, cell shape and polarity (Pertz et al., 2006; 402

Nalbant et al., 2009). Thus, the RhoA/ROCK pathway is a likely candidate to coordinate the 403

spatio-temporal organization of stress fiber formation via FHOD1 and their contractility via 404

Myosin II in those cellular processes. 405

406

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Although regulation of FHOD1 by ROCK phosphorylation was proposed earlier (Hannemann 407

et al., 2008; Takeya et al., 2008), the functional role of this modification was not fully 408

explored. On the one hand, the effect of ROCK on subcellular FHOD1 localization and the 409

time-scale at which ROCK mediates FHOD1 regulation were unknown. On the other hand, 410

experiments that implemented C-terminal truncation mutants or phosphomimic mutations are 411

difficult to interpret as they might affect other functions of the C-terminus that are not directly 412

related to auto-inhibition. Indeed, the C-terminal DAD region of the formin mDia1 was 413

recently suggested to play an active role in filament nucleation (Gould et al., 2011). Here, we 414

show that in comparison to the C-terminal truncation (FHOD1 1-1011) the point mutation 415

V228E is an even stronger activator of stress fiber formation, although it is significantly less 416

co-localized with stress fibers. This suggests that the C-terminus of FHOD1 indeed plays 417

additional roles in FHOD1 function, potentially by augmenting actin monomer recruitment 418

similar as in the formins mDia1 and FMNL2 (Gould et al., 2011; Block et al., 2012). 419

Alternatively, the sensitivity of FHOD1 stress fiber association to ROCK inhibition suggests 420

that dynamic binding and unbinding of FHOD1 to stress fibers by 421

phosphorylation/dephosphorylation cycles might be necessary to fully activate FHOD1 422

function. 423

424

One of the most intriguing observations in this study was that FHOD1 has at least two distinct 425

cellular functions: a) stimulating stress fibers that contain anti-parallel filaments (arcs and 426

ventral stress fibers) and b) inhibiting the growth of stress fibers that contain long parallel 427

filaments (dorsal stress fibers). Our studies give critical novel insight into the molecular basis, 428

how FHOD1 could perform those functions: 1) We were able to narrow down the region of 429

FHOD1 that mediates its association with stress fibers and co-localization with Myosin II to 430

amino-acids 1-339. 2) We found that a truncation mutant lacking the GBD/FH3 domains 431

accumulated in the lamellipodium and along the entire length of focal adhesions. 3) We found 432

that persistent stress fiber association is not necessary for stimulation of stress fiber formation. 433

4) Further truncation of a helical domain (396-573), which was recently suggested to mediate 434

actin filament bundling in vitro (Schönichen et al., 2013), ablated FHOD1 mediated stress 435

fibers stimulation. Together, these observations suggest that the helical, FH1-, FH2- and C-436

terminal domains are critical for both the stimulatory function of FHOD1 for arcs and their 437

turn-over into ventral stress fibers. Previous studies proposed that the short bundled actin 438

filaments that form transversal arcs originate from the lamellipodium (Hotulainen and 439

Lappalainen, 2006; Burnette et al., 2011; Tojkander et al., 2011). Therefore, the 440

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lamellipodium localization of the highly active truncation mutant 340-1164 during acute arc 441

stimulation suggests that FHOD1 might mediate bundling of preformed short actin filaments 442

via the helical domain at the leading edge of the cell, which are then efficiently assembled 443

into transversal arcs at the interface to the lamellum. 444

445

Classically, formins are thought to act as processive actin capping proteins that can stimulate 446

actin polymerization (Zigmond et al., 2003; Shemesh et al., 2005b; Otomo et al., 2005). 447

However, recent biochemical studies show that purified active FHOD1 inhibits actin 448

polymerization in addition to filament bundling (Schönichen et al., 2013). In cells, dorsal 449

stress fibers require rapid filament elongation at the associated focal adhesion by the efficient 450

processive capping protein mDia1 (Hotulainen and Lappalainen, 2006; Oakes et al., 2012). At 451

those focal adhesions, FHOD1 might act as a capping protein, which would inhibit growth of 452

associated dorsal stress fibers by competing with mDia1. Our observation that a FHOD1 453

mutant lacking the stress fiber targeting GBD-FH3 domains associates more prominently with 454

focal adhesions in steady state conditions further supports this idea. In the lamellipodium, 455

linear filaments are generated by mDia2 or FMNL2 (Yang et al., 2007; Tojkander et al., 2011; 456

Block et al., 2012). Here, FHOD1 might enrich the pool of short actin filaments either by 457

permanent or slow processive capping. Concomitantly, FHOD1 might promote transversal arc 458

assembly by bundling those preformed, short filaments. Thus, it is intriguing that FHOD1 by 459

the same molecular activity might control opposing processes at dorsal stress fibers and arcs. 460

Finally, while the central and C-terminal domains of FHOD1 are responsible for robust 461

filament formation, the N-terminal stress fiber targeting region (1-339) might facilitate precise 462

spatial FHOD1 targeting to fine tune its cellular function. These processes in the generation of 463

mature contractile stress fibers are summarized in a working model which integrates the two 464

proposed molecular functions of FHOD1 – filament capping and bundling – into a dynamic 465

process tightly controlled in space and time (Fig. S6). 466

467

In summary, our study establishes a key role for the formin FHOD1 in the spatio-temporal 468

control of actin filament meshwork dynamics in adherent cells. FHOD1 promotes the efficient 469

formation of transversal actin arcs in the leading edge and, by restricting the length of dorsal 470

stress fibers, their coordinated turn-over into mature contractile stress fibers. 471

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Materials and Methods 472

473

Cell culture and reagents 474

Human U2OS osteosarcoma cells (ATCC HTB-96) were maintained in DMEM-GlutaMAX 475

(Life Technologies) supplemented with 10% fetal bovine serum (PAN Biotech), 50 U/ml 476

penicillin, and 50 µg/ml streptomycin (Life Technologies) at 37°C and 5% CO2. Prior to cell 477

seeding, coverslips (#1.5 glass, Thermo Scientific) and dishes (#1.5 glass bottom, MatTEK 478

Corporation) were coated with 10 µg/ml collagen type I (C8919, Sigma-Aldrich) for 1 h at 479

37°C and washed with PBS. 480

Cells were treated with the mycotoxin Cytochalasin D (2 µM, 90 minutes) (C8273, Sigma 481

Aldrich) to allow complete depolymerization of F-actin. Treatment started 72 h after 482

transfection of siRNAs or 16 h after transfection of EGFP or EGFP-FHOD1 constructs. Stress 483

fiber recovery was stimulated by five repeated washing steps with growth medium. The 484

selective ROCK inhibitor Y-27632 (Y0503) was purchased from Sigma-Aldrich. 485

486

Constructs and siRNAs 487

EGFP-Actin was provided by Melissa Rolls (Pennsylvania State University, USA) and 488

mKate-Paxillin was a gift from Eli Zamir (MPI Dortmund, Germany). EGFP-NMHCIIA 489

(Addgene Plasmid 11347, Wei und Adelstein, 2000) and mCherry-NMHCIIA (Addgene 490

Plasmid 35687, Dulyaninova et al., 2007) plasmids were obtained from Addgene. mCherry-α-491

actinin 1 and mCherry-Actin constructs were generated by replacing the EGFP from pEGFP-492

α-actinin 1 (Addgene plasmid 11908, Edlund et al., 2001) and pEGFP-Actin with mCherry 493

from pmCherry-C1 (Clontech) using AgeI/BsrGI restriction sites. For FHOD1 localization 494

studies low expression EGFP-FHOD1-contructs (delCMV-EGFP-FHOD1) were used as 495

described recently (Schönichen et al., 2013). Low cellular expression using a truncated, less 496

efficient CMV promoter (delCMV) has been introduced by a previous study by Watanabe and 497

Mitchison (Watanabe and Mitchison, 2002). FHOD1 V228E mutant was generated by site-498

directed mutagenesis (QuikChange Mutagenesis Kit, Agilent Technologies). Truncated 499

FHOD1 mutants were generated by PCR from wild-type delCMV-EGFP-FHOD1 or FHOD1 500

V228E, respectively. Plasmid DNA constructs were transfected using Lipofectamine™2000 501

(Life Technologies). 502

All siRNAs were purchased from Qiagen. The FlexiTube GeneSolution set (GS29109) 503

recommended by Qiagen to specifically target human FHOD1 contained four different 504

siRNAs (termed #2, #5, #6 and #7 by the supplier), which were used either as a mix (referred 505

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to as siFHOD1) or as individual oligonucleotides: siFHOD1#2 (5’-506

CAGCGAGAGGAGCATCTACAA-3’), siFHOD1#5 (5’- 507

AGGGTCAACGCTATCTTGGAA-3’), siFHOD1#6 (5’-CCCGCCGTGTTGCCATGCTAA-508

3’) and siFHOD#7 (5’-CCCGTGCACCCAGGCTCTCTA-3’). AllStars negative control 509

siRNA (1027280) was used as control (ntsiRNA). With the exception of wound healing 510

assays, cells were transfected with 5 nM of the indicated siRNAs directly after plating (fast 511

forward transfection) using HiPerFect (Qiagen) and replated 24 h after transfection. 512

Experiments were carried out 72 h after siRNA transfection. Knockdown efficiency was 513

determined by Western blotting. 514

515

Immunofluorescence 516

Cells were fixed with 4% formaldehyde in PBS (20 minutes, 37°C), washed with PBS and 517

permeabilized using 0.2% Triton X-100 in PBS (10 minutes, room temperature (RT)). 518

Samples were washed and incubated in blocking solution (2% BSA in PBS, 1 h, RT) followed 519

by incubation with anti-Paxillin antibody (1:500 in blocking solution, 1h; clone 349, BD 520

Transduction Laboratory) or anti-NMHCIIa antibody (1:200 in blocking solution, 1h; 521

ab24762, Abcam), respectively. Coverslips were washed and incubated with AlexaFluor488- 522

or AlexaFluor633-conjugated goat-anti-mouse (1:500; Life Technologies) or AlexaFluor488-523

conjugated goat-anti-rabbit (1:1000; Life Technologies) secondary antibody together with 524

Rhodamine-Phalloidin to visualize filamentous actin (1:1,000; Life Technologies) in blocking 525

solution (1 h, RT). Samples were washed and, if required, stained with 4',6-diamidino-2-526

phenylindole (DAPI) (1:2,000, 10 minutes, RT; Sigma-Aldrich). Coverslips were mounted 527

using ProLong Gold (Life Technologies). 528

529

Microscopy 530

Epifluorescence and phase contrast imaging was carried out with an Eclipse Ti inverted 531

microscope (Nikon) equipped with a motorized stage, a build-in Perfect Focus System and a 532

CoolSNAP HQ2 camera (Photometrics). Images were acquired using a 20x/0.45 NA air or a 533

60x/1.40 NA oil immersion objective. Acquisition was controlled by NIS-Elements Imaging 534

Software (Nikon). Random positions were chosen using the “multi-position tool”. 535

Confocal laser scanning microscopy was performed on a TCS SP5 AOBS system (Leica 536

Microsystems) supported by LASAF software (Leica Microsystems) and equipped with an 537

HCX PL APO 63x/1.4 NA oil immersion objective. Laser lines used for excitation were 538

488 nm (EGFP, Alexa Fluor 488), 561 nm (RFP, Rhodamine, mCherry) and 633 nm (Alexa 539

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Fluor 633). Confocal Z-Stacks were acquired with 210 nm spacing and maximum projections 540

were generated with ImageJ software (http://rsbweb.nih.gov/ij/). Color-coded maximum 541

projections of confocal Z-Stacks were generated using the “Time Series Color Coder” Macro 542

(http://cmci.embl.de/downloads/timeseriescolorcoder) with a modified LUT. 543

544

Time-lapse TIRF microscopy was performed on an Eclipse Ti-E (Nikon) inverted microscope 545

with a motorized TIRF Illuminator Unit (Nikon), an Andor AOTF Laser Combiner (Andor 546

Technology), and a Clara Interline CCD camera (Andor Technology). Laser lines used for 547

excitation of EGFP and mCherry were 488 nm and 561 nm, respectively. Images were 548

acquired using an Apo TIRF 100×1.49 NA oil immersion objective (Nikon). Acquisition was 549

controlled by Andor IQ Software (Andor Technology). 550

All microscopes were equipped with temperature-controlled incubation chambers. Time-lapse 551

microscopy experiments were carried out at 37°C in CO2-independent medium (HBSS buffer, 552

10% FBS, 2 mM L-Glutamine, 10 mM HEPES, 1 mM MgCl2, 1 mM CaCl2, supplemented 553

with 10 µl/ml Oxyrase (Oxyrase, Inc.)) with indicated frame rates. 554

555

Wound healing assay 556

IBIDI culture inserts (IBIDI) were used to study wound healing efficiency. Cells (8x103) were 557

seeded in each compartment of the culture inserts and directly transfected with 10 nM of 558

siRNAs using HiPerFect (Qiagen). Inserts were removed 72 h after siRNA transfection and 559

growth medium was replaced by growth medium supplemented with 10 mM HEPES. Cell 560

migration was monitored by time-lapse phase contrast microscopy (frame rate: 30 seconds). 561

For each condition, ten different positions along the wound were recorded in each experiment. 562

Migration efficiency was quantified by measuring the migrated distance (ImageJ). Lines were 563

drawn along the wound edge at start and end points of migration assay and the average 564

movement was calculated from all positions. 565

566

Cell adhesion and spreading 567

For adhesion and spreading assays, cells were detached from culture dishes using 568

Trypsin/EDTA and plated onto collagen type I coated glass coverslips in growth medium 72 h 569

after siRNA transfection. After 15, 30 or 60 minutes, coverslips were rinsed once with PBS, 570

fixed with 4% formaldehyde and stained with DAPI (nuclei) and Rhodamine-Phalloidin (F-571

actin). Epifluorescence images were acquired using a 20x/0.45 NA air objective. Adhered 572

cells were quantified from 25 random regions for each condition and time point. To quantify 573

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cell spreading, 20 random positions were acquired using a 60x/1.40 NA oil immersion 574

objective for each condition and cell area was measured using ImageJ software. Focal 575

adhesion size and number per cell were quantified from confocal images of cells expressing 576

mKate-Paxillin using ImageJ. Thresholds were set manually to isolate focal adhesions 577

properly. Average area and number of focal adhesion per cell were quantified from binary 578

images using the analyze particle tool in ImageJ. Particles smaller than 0.25 µm2 and larger 579

than 10 µm2 were excluded to avoid unspecific background signal and not properly separated 580

neighboring adhesions. 581

582

RNA isolation, reverse transcription and quantitative real-time PCR analysis 583

Total RNA was extracted using the RNeasy Mini kit (Qiagen). Reverse transcription was 584

performed with Superscript®II Reverse Transcriptase (Life Technologies) according to the 585

manufacturer’s protocols using 500 ng total RNA. Quantitative real-time PCR was performed 586

with a StepOnePlus™ Real-Time PCR system (Life Technologies) using the Fast SYBR® 587

Green Master Mix (Life Technologies) and human FHOD1 specific primer pairs (forward: 5´-588

TACACGGTCACCCTCATCAA-3´, reverse: 5´-AGTGCATCCGTCACATCGTA-3´). 589

GAPDH, ACTB and RRN18S were used as housekeeping genes (QuantiTect Primer Assays, 590

Qiagen). Relative quantification was performed using the efficiency corrected relative 591

quantification method (Pfaffl, 2001). Primer efficiencies were calculated for each primer pair 592

by performing dilution series experiments. 593

594

Western blot analysis 595

Cells were washed once with ice-cold PBS and lysed in ice-cold radioimmunoprecipitation 596

assay buffer (50 mM Tris, pH7.5, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 597

1 mM EDTA, 1x protease inhibitor cocktail and 1x PhosSTOP phosphatase inhibitor cocktail 598

(Roche)). Insoluble cell debris were removed by centrifugation at 13,000 g for 10 minutes at 599

4°C. Protein concentration in the supernatants was determined by the Bradford method 600

(BioRad). Equal amounts of total protein were mixed with 5x Laemmli sample buffer, boiled 601

at 95°C for 10 minutes and separated by SDS-PAGE. After electrophoresis, proteins were 602

transferred on a PVDF membrane (Thermo Scientific) using a Biometra fastblot B34 blotting 603

device (Biometra). Blots were blocked for 1 h at RT with 5% nonfat dry milk in TBS-T 604

(20 mM Tris, pH 7.6, 137 mM NaCl and 0.1% Tween-20) and incubated overnight at 4°C 605

with the primary antibodies anti-FHOD1 (1:200; FM3521; ECM Bioscience) or anti-α-606

Tubulin (1:20,000; clone B-5-1-2, Sigma-Aldrich). Membranes were washed three times with 607

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TBS-T and incubated with HRP-conjugated anti-mouse secondary antibody (1:20,000; Santa 608

Cruz Biotechnology, Inc.) for 1 h at RT. After additional washing steps with TBS-T and TBS 609

(20 mM Tris, pH 7.6, 137 mM NaCl), protein bands were visualized using ECL Western 610

blotting substrate (Pierce). Images were captured with a Fusion Fx7 system and quantified 611

with Bio-1D software (Peqlab). 612

613

Statistical analysis 614

Independent two-tailed student´s t-tests were used if not stated otherwise. In case of unequal 615

variances Welch´s t-test was used. The resulting p-values are indicated in the figures and 616

legends. 617

618

619

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Acknowledgments 620

621

The authors thank Michael Ehrmann, Hemmo Meyer and Andrea Vortkamp for helpful 622

discussions. This work was supported by the Deutsche Forschungsgemeinschaft Priority 623

Programme funding (SPP 1464) to PN (NA 413/3-1) and by a grant from the Deutsche 624

Forschungsgemeinschaft to MG (GE 976/4). 625

626

627

Author contributions 628

629

NS and PN conceived or designed the experiments. NS performed the majority of 630

experiments with contributions from MGraessl and ABS. MGraessl generated TIRF imaging 631

data on FHOD1 localization and regulation. ABS contributed data on FHOD1 localization and 632

stress fiber stimulation. NS performed most of the data analysis with contributions from 633

MGraessl, ABS and PN. NS generated the figures. MG and LD contributed reagents. MG 634

commented on the manuscript. LD and PN wrote the paper. 635

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Figure Legends 796

Figure 1: Stress fiber organization is perturbed upon depletion of FHOD1. (A) Representative 797

confocal images of the actin cytoskeleton in fixed siRNA treated U2OS cells. Stress fiber types were 798

identified by F-actin (rhodamine-phalloidin) and Paxillin staining (focal adhesion marker; not shown). 799

Green and red arrows indicate dorsal stress fibers and transversal arcs, respectively. Yellow arrow 800

points to thick peripheral stress fiber bundles. Scale bar, 20 µm. (B) Percentage of cells with dorsal 801

stress fibers (dSF), transversal arcs (TA), both stress fiber types (dSF + TA) or thick peripheral actin 802

bundles only (peripheral bundles; yellow arrow in A). Data are mean ± s.d. from 3 experiments (n > 803

360 cells for each condition). (C) 3D organization of transversal arcs in U2OS cells. Depicted is the 804

maximum projection of a confocal Z-Stack (F-actin), in which the Z coordinates are indicated by the 805

color bar. Scale bar, 10 m. (D) Arc-covered lamellum regions were measured based on confocal Z-806

stacks (F-actin) and normalized to the whole cell adhesion area (ImageJ, free-hand line tool) (See Fig. 807

S1C for illustration of the analysis process). Only cells that contain arcs were included in the analysis. 808

Data are mean ± s.e.m. of n = 90 cells for each condition from 3 experiments. (E) Mean actin intensity 809

in arc-covered lamellum regions from (D). Red lines represent mean values. (F) Quantification of cells 810

with stellate stress fiber aggregates. Data are mean ± s.d. from 3 experiments (n > 360 cells for each 811

condition). (G) Representative image of a stellate stress fiber aggregate in a FHOD1 depleted cell. 812

Maximum projection of a confocal Z-Stack showing F-actin (rhodamine-phalloidin, red) and focal 813

adhesions (anti-paxillin antibody, green). Scale bar, 10 µm. (H) Actin aggregates contain Myosin IIA. 814

Representative maximum projection of a confocal Z-Stack of a FHOD1 depleted cell expressing 815

EGFP-NMHC IIA (green). F-Actin was visualized by rhodamine-phalloidin staining (red). Myosin 816

IIA is excluded from most of the straight actin fibers (white arrows) whereas strong localization is 817

detected within the aggregate. Scale bar, 10 µm. (I) Representative time-lapse confocal images 818

depicting the disassembly of an actin aggregate in an EGFP-Actin expressing FHOD1 depleted cell. 819

Scale bar, 10 µm. n = 20 movies. *** = p<0.001; ** =p<0.01; * = p<0.05. 820

821

Figure 2: FHOD1 depletion leads to decreased focal adhesion size and moderate spreading 822

defects. (A-C) FA size and number per cell were quantified 72 h after siRNA transfection. (A) 823

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Representative confocal images of mKate-Paxillin and EGFP-Actin expressing control (ntsiRNA) and 824

FHOD1 depleted (siFHOD1) cells. Quantification by threshold based image analysis. Lower panel: 825

Binary images and outlines of focal adhesions. Scale bar, 10 µm. (B) Focal adhesion size and (C) 826

number were quantified using the analysis as shown in (A). Data are mean ± s.e.m. of n = 20 cells for 827

each condition from 5 experiments. (D-E) Cell spreading of ntsiRNA and FHOD1 depleted 828

(siFHOD1) cells. F-actin (rhodamine phalloidin; grey) and nuclear (DAPI; blue) staining at the 829

indicated time-points after replating. (D) Representative images during spreading. Scale bar, 20 µm. 830

(E) Quantification of cell area after replating. Data are mean ± s.e.m. of n > 100 cells (15 min) or 831

> 200 cells (30 and 60 min) from 3 experiments. ** = p<0.001** = p<0.01; * = p<0.05. 832

833

Figure 3: Active FHOD1 promotes transversal arc formation and enhanced maturation into 834

linear stress fibers. Acute effects of FHOD1 on stress fiber formation. Cells expressing EGFP, 835

EGFP-FHOD1 wild-type (FHOD1 WT), and active EGFP-FHOD1 V228E (FHOD1 V228E) were 836

treated with Cytochalasin D (2 µM, 90 min) to disrupt actin filaments. Stress fiber recovery was 837

stimulated by drug washout. Cells were either fixed and stained with rhodamine-phalloidin (F-actin) 838

(A -C) or subjected to confocal time-lapse imaging of the actin cytoskeleton (D-F). (A) Upper panel: 839

3D organization of stress fibers. Depicted are representative maximum projections of confocal Z-840

Stacks (F-actin) 30 min after washout, in which the Z-coordinates are indicated by the color bar. 841

Lower panel: Enlarged areas from boxed regions of upper panels. Scale bar, 10 µm. (B) Arc-covered 842

lamellum regions (in percent of total cell adhesion area) in fixed cells 30 minutes after drug washout 843

(see Fig. S1C for illustration of the analysis process). Data are mean ± s.e.m. of 100 cells for each 844

condition from 3 experiments. (C) FHOD1 V228E enhances stress fiber formation on the dorsal cell 845

side. Z-sections (middle) were generated from confocal Z-Stacks (F-actin) along lines indicated by red 846

arrows in the maximum projections (ImageJ, MultipleKymograph tool) (left). Scale bar, 10 µm. Mean 847

actin intensity was measured on the dorsal (red dotted line) or ventral (green dotted line) as indicated 848

in the schematic (middle) and plotted as “ratio dorsal/ventral” (right). n = 67 and 85 cells for the two 849

conditions, respectively (3 independent experiments; red lines indicate mean). (D) Representative 850

time-lapse confocal images of stress fiber recovery after Cytochalasin D washout in cells expressing 851

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mCherry-Actin and EGFP-FHOD1 constructs (not shown). Acquisitions started immediately after 852

washout. Scale bar, 10 µm. (E) Time-point of first linear stress fiber appearance in time-lapse images. 853

n = 40, 33, and 44 cells for the three conditions, respectively (3 experiments; red lines indicate mean). 854

(F) Data from D were plotted as Histogram. ns = not significant; *** = p<0.001; ** = p<0.01. 855

856

Figure 4: FHOD1 inhibits growth of dorsal stress fibers. (A-D) Time-lapse confocal imaging of 857

control (ntsiRNA) and FHOD1 depleted (siFHOD1) cells expressing EGFP-Actin and mKate-Paxillin. 858

(A) Representative confocal time-lapse images depicting dorsal stress fiber growth. Boxed regions 859

show enlarged dorsal stress fibers. Arrowheads indicate distal (red) and proximal (green) end of dorsal 860

fibers. (B) Length of nascent dorsal stress fibers at each frame after their initiation (duration 30 861

minutes; frame rate = 30 seconds). Data are mean ± s.e.m. for each time point of 25 control and 27 862

FHOD1 depleted cells from 4 experiments. (C) Representative confocal time-lapse images showing 863

aberrant dorsal stress fiber bending in a FHOD1 depleted cell. (D) Quantification of cells with dorsal 864

stress fiber bending over time. Data are mean ± s.d. from 5 experiments (n > 40 cells for each 865

condition). (E-G) Constitutively active FHOD1 inhibits growth of dorsal stress fibers. Dorsal stress 866

fibers were quantified 30 min after Cytochalasin D washout by rhodamine-phalloidin (F-actin, green) 867

and Paxillin staining (focal adhesion marker, red). (E) Representative maximum projections of 868

confocal Z-Stacks showing stress fiber organization (F-actin, green) together with focal adhesions 869

(Paxillin, red) of FHOD1 WT or FHOD1 V228E expressing cells. Boxed regions are shown enlarged. 870

Red arrows point to typical dorsal stress fiber appearance for each condition. Scale bar, 10 µm. (F) 871

Quantification of dorsal stress fibers in fixed cells expressing EGFP-FHOD1 constructs 30 minutes 872

after Cytochalasin D washout. Data are mean ± s.d. from 3 experiments (n > 100 cells for each 873

condition). (G) Quantification of dorsal stress fiber length at 30 minutes after Cytochalasin D washout. 874

All detectable dorsal stress fibers were measured. Data are mean ± s.e.m. of 34-44 cells for each 875

condition from 3 experiments. Welch´s t-test was performed in (G). *** = p<0.001; ** = p<0.01; 876

ns = not significant. 877

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Figure 5: FHOD1 localizes to contractile stress fibers with anti-parallel actin arrays. (A) FHOD1 879

localizes to ventral stress fibers (yellow arrow). TIRF images of EGFP-FHOD1 (green) and RFP-880

Actin (red). Ventral stress fibers are attached to the cell cortex with both ends and therefore entirely 881

visible in the TIRF field. Scale bar, 20 µm. (B) Constitutively active FHOD1 1-1011 localizes to distal 882

ends of dorsal stress fibers. Maximum projections of confocal Z-stacks are shown. Overlay: F-actin 883

(red, phalloidin staining), EGFP-FHOD1 (1-1011) (green), Paxillin staining (blue). Yellow arrow: A 884

ventral stress fiber associated with both ends to the substrate. Orange arrow: main portion of a dorsal 885

stress fiber. Red arrow: distal end of a dorsal stress fiber. Scale bar, 10 µm. (C) Myosin II localization 886

at distal ends of dorsal stress fibers at the interface to focal adhesions (red arrows). Confocal single 887

plane images of F-actin (Rhodamine Phalloidin: red), anti-NMHC IIA (green) and anti-Paxillin (blue) 888

staining. (D) Active FHOD1 V228E co-localizes with Myosin on contractile stress fibers. Confocal 889

images of EGFP-FHOD1 V228E (green) and Myosin (mCherry-NMHC IIA; red) co-localization on 890

transversal arcs. Yellow arrow: Punctuate pattern with FHOD1 and Myosin co-localization. 891

Representative images of n = 60 cells from 4 experiments. Scale bars, 10 µm. 892

893

Figure 6: Localization of N-Terminally truncated FHOD1 constructs. (A) Domain architecture of 894

FHOD1 mutant constructs. Red star marks the V228E mutation to activate FHOD1. (B) TIRF images 895

of EGFP-FHOD1 mutants (green) and mCherry-Actin (red). The N-terminally truncated construct 896

340-1164 is not associated along stress fibers. Yellow arrows point to regions of FHOD1 897

accumulation at focal adhesions (n >60 cells from 4 experiments for each construct). (C) FHOD1 1-898

339 co-localizes with Myosin II. Single plane confocal images of EGFP-FHOD1 1-339 (green) and 899

Myosin (mCherry-NMHCIIA; red) (n = 36 cells from 3 experiments). (D) N-terminally truncated 900

FHOD1 localizes to the lamellipodium during acute stress fiber stimulation by Cytochalasin D 901

washout. Left: Representative confocal images of FHOD1 wild-type and FHOD1 340-1164 (EGFP: 902

green) with F-actin (Rhodamine Phalloidin: red). Right: Number of cells with accumulated EGFP-903

FHOD1 signal in the lamellipodium at the leading edge 30 minutes after washout of Cytochalasin D. 904

Data are mean ± s.d. of >50 cells for each condition from 3 experiments. ns = not significant; 905

* = p<0.05. Scale bars, 10 µm. 906

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907

Figure 7: Stress fiber binding is dispensable for robust stress fiber formation. (A) Representative 908

maximum projections of confocal Z-stacks (F-actin) of cells expressing EGFP-FHOD1 constructs. 909

Transfected cells are indicated by asterisks. (B) Cells entirely decorated with thick linear stress fibers 910

(red arrows in A) were quantified. Data are mean ± s.d. (n = 220-300 cells per condition from 3 911

experiments). (C) Acute stress fiber formation upon Cytochalasin D washout. Cells expressing EGFP 912

alone, EGFP-FHOD1 wild-type (FHOD1 WT) and active EGFP-FHOD1 mutants were treated with 913

Cytochalasin D (2 µM, 90 min) followed by extensive washout to stimulate stress fiber recovery. Cells 914

were fixed and stained with rhodamine-phalloidin (F-actin) 30 min after washout. Relative area of 915

cells covered with stress fibers was quantified. Data are mean ± s.e.m. of n = 56-74 cells for each 916

condition from 3 experiments. Welch´s t-test was performed in (C). ns = not significant; 917

*** = p<0.001. (D) 3D organization of stress fiber bundles stimulated by FHOD1 V228E. Depicted is 918

a single confocal plane on the ventral cell side (left) and maximum projections of confocal Z-Stacks 919

(F-actin, red; Paxillin, green). Z coordinates are indicated by the color bar (right). Scale bar, 10 m. 920

921

Figure 8: The C-terminal regulatory domain is required for FHOD1 effector function to 922

stimulate efficient stress fiber maturation. (A) FHOD1 V228E mutant is a more potent inducer of 923

ventral stress fibers as compared to the C-terminal truncated FHOD1 1-1011. Maximum projections of 924

confocal Z-stacks (F-actin) of cells transfected with EGFP-FHOD1 constructs and EGFP control were 925

generated.. Cells covered entirely with linear stress fibers (linear stress fiber phenotype) were 926

quantified. Data are means ± s.d. from 3 experiments (n = 46-62 cells). (B) Stress fiber association of 927

constitutively active FHOD1 mutants. Upper panel: Representative maximum projections of confocal 928

Z-stacks showing EGFP-FHOD1 and F-actin. Lower panel: Line scans were used to analyze the 929

correlation of FHOD1 (green) and F-actin (red) distribution (ImageJ, plot profile function). Lines were 930

drawn perpendicular to stress fibers from the leading edge to the center. Normalized intensity profiles 931

are shown. Scale bars, 10 µm. (C) Pearson´s correlation coefficient (GraphPad Prism) to quantify the 932

correlation of FHOD1 and F-actin intensities obtained from line scans (n = 26 for FHOD1 1-1011 and 933

24 FHOD1 V228E cells from 3 experiments). Red lines indicate mean. (D) TIRF images of a 934

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representative cell expressing EGFP-FHOD1 wild-type and mCherry-Actin prior and 5 min post 935

ROCK inhibition via Y-27632 (50 µM). Scale bar, 20 µm. (E) Quantification of stress fiber 936

dissociation of EGFP-FHOD1 wild-type and the two constitutively activated constructs EGFP-FHOD1 937

V228E, EGFP-FHOD1 1-1011. Average fluorescence intensity decrease along individual stress fibers 938

in each EGFP-FHOD1 TIRF image was measured using ImageJ software and normalized to the 939

change of the corresponding mCherry-Actin signal (n = 14–16 cells from 3-4 experiments for each 940

construct; red line marks mean). *** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns = not significant. 941

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