The Formins FHOD1 and INF2 Regulate Inter

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RESEARCH ARTICLE The formins FHOD1 and INF2 regulate inter- and intra-structural contractility of podosomes Linda Panzer1,*, Leona Trübe1,*, Matthias Klose1, Ben Joosten2, Johan Slotman3, Alessandra Cambi2 and Stefan Linder1,‡

ABSTRACT Classically, podosome architecture is seen as bipartite (Linder Podosomes are actin-rich adhesion structures that depend on Arp2/3- and Aepfelbacher, 2003), consisting of a core of F-actin and complex-based actin nucleation. We now report the identification of the actin-associated proteins such as cortactin (Ochoa et al., 2000) or formins FHOD1 and INF2 as novel components and additional actin- gelsolin (Chellaiah et al., 2000), and a surrounding ring structure based regulators of podosomes in primary human macrophages. of plaque proteins including vinculin (Zambonin-Zallone et al., FHOD1 surrounds the podosome core and is also present at 1989) and paxillin (Pfaff and Jurdic, 2001). The structure is podosome-connecting cables, whereas INF2 localizes at the anchored to the ventral plasma membrane by transmembrane podosome cap structure. Using a variety of microscopy-based matrix receptors such as integrins (Pfaff and Jurdic, 2001; Teti methods; including a semiautomated podosome reformation assay, et al., 1989) and CD44 (Chabadel et al., 2007), with cytoskeletal measurement of podosome oscillations, FRAP analysis of single linkage provided by proteins such as talin (Zambonin-Zallone podosomes, and structured illumination microscopy, both formins et al., 1989) or kindlin-3 (also known as FERMT3) (Ussar et al., were found to regulate different aspects of podosome-associated 2006). However, recent structural analyses have revealed a more contractility, with FHOD1 mediating actomyosin contractility between complex picture of podosome architecture. In particular, plaque podosomes, and INF2 regulating contractile events at individual proteins were shown to be assembled not in a continuous ring, podosomes. Moreover, INF2 was found to be a crucial regulator of but in several clusters that surround the core structure (van den podosome de novo formation and size. Collectively, we identify Dries et al., 2013b). In addition, the core structure, being FHOD1 and INF2 as novel regulators of inter- and intra-structural dependent on Arp2/3 complex activity (Kaverina et al., 2003; contractility of podosomes. Podosomes thus present as one of the few Linder et al., 2000a), has been shown to be surrounded by a layer currently identified structures which depend on the concerted activity of unbranched filaments (Akisaka et al., 2008; Luxenburg et al., of both Arp2/3 complex and specific formins and might serve as a 2007). model system for the analysis of complex actin architectures in cells. Moreover, unbranched actin filaments were shown to connect individual podosomes into a higher-ordered network (Burgstaller KEY WORDS: FHOD1, Formins, INF2, Macrophages, Podosomes and Gimona, 2005; Luxenburg et al., 2007) and to exert forces based on actomyosin contractility (Bhuwania et al., 2012), thus INTRODUCTION probably coordinating turnover of individual podosomes with the Macrophages are highly invasive cells of the monocytic lineage. net movement of podosome groups. Actomyosin contractility at In order to fulfill their functions during immune surveillance, podosomes is regulated by the membrane-associated protein they have to cross tissue barriers and navigate through the dense supervillin (Bhuwania et al., 2012). Interestingly, supervillin was meshwork of the extracellular matrix (ECM) (Hynes, 2009), which shown to localize to yet another substructure of podosomes, a cap- often involves proteolytic cleavage of ECM material (Sabeh et al., like structure on top of the podosome core (Bhuwania et al., 2012), 2009). Accordingly, macrophages are able to form actin-rich which only partially overlaps with the core structure. A similar podosomes at the cell–substrate interface that function as both sites localization on top of the actin core of macrophage podosomes has of adhesion and hotspots of matrix degradation (Linder and been described for the formin FMNL1 (Mersich et al., 2010), Aepfelbacher, 2003; Linder and Wiesner, 2015; Murphy and indicating the potential existence of further cap proteins and Courtneidge, 2011). Podosomes are able to locally degrade the pointing to a likely function of this structure in the regulation of matrix through recruitment of matrix-lytic enzymes, in particular of unbranched actin filaments and actomyosin contractility at the matrix metalloproteinase family (Linder, 2007). For efficient podosomes. invasion, both podosome-localized ECM degradation and turnover Podosomes are highly dynamic structures, displaying several of the podosome structure itself have to be spatiotemporally levels of external and internal dynamics. The lifetime of podosomes coordinated. has been determined as 2–12 min, and podosomal actin can be exchanged three times during this period (Destaing et al., 2003). de novo 1Institute for Medical Microbiology, Virology and Hygiene, University Medical Moreover, besides formation, podosomes can also be Center Eppendorf, Martinistraße 52, Hamburg 20246, Germany. 2Department of formed by fission from pre-existing mother podosomes, which is Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud particularly evident in primary macrophages (Evans et al., 2003; University Medical Center, Geert Grooteplein Zuid 26-28, Nijmegen 6525 GA, The Netherlands. 3Erasmus University, Wytemaweg 80, Rotterdam 3015 CN, Kopp et al., 2006). In addition, podosomes have been shown to The Netherlands. undergo cycles of actomyosin-based internal stiffness (Labernadie *These authors contributed equally to this work et al., 2010), leading to protrusion of the podosome structure, as ‡ Author for correspondence ([email protected]) shown by deformation of pliable matrix (Labernadie et al., 2014), accompanied by oscillations of actin-based fluorescence (van den

Received 16 July 2015; Accepted 24 November 2015 Dries et al., 2013a). Journal of Cell Science

298 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 298-313 doi:10.1242/jcs.177691

Several lines of evidence thus suggest the presence of unbranched and INF2 regulating contractile events at individual podosomes. actin filaments at podosomes: (i) the identification of connecting FHOD1 and INF2 thus present as novel regulators of inter- and cables that mediate contact between individual podosomes intra-structural contractility of macrophage podosomes. (Burgstaller and Gimona, 2005; Luxenburg et al., 2007), (ii) the observed contractility of individual podosomes (Labernadie et al., RESULTS 2014, 2010; van den Dries et al., 2013a), which is probably based on FHOD1 and INF2 localize to distinct substructures of the presence of unbranched actin filaments (Akisaka et al., 2008; macrophage podosomes Luxenburg et al., 2007), (iii) the presence of proteins such as myosin Previous mass spectrometry analyses revealed the presence of IIA (also known as MYH9) and supervillin (Bhuwania et al., 2012) several formins in podosome-enriched cell fractions of primary that regulate actomyosin contractility, thus necessitating the macrophages (Cervero et al., 2012), with the formins DAAM1, DIA1 presence of unbranched F-actin, (iv) the presence of the formin (DIAPH1), DIA2 (DIAPH2), FHOD1, FMNL1, FMNL2 and INF2 FMNL1, a regulator of unbranched F-actin, at podosomes (Mersich being detected in at least one of three experiments performed in et al., 2010), and (v) as revealed by proteomic data, the presence of parallel. In order to screen for potential localization of these formins further formin isoforms in podosome-enriched cell fractions from at macrophage podosomes, we first overexpressed respective EGFP macrophages (Cervero et al., 2012). Collectively, these findings fusions in primary human macrophages. As formins are mostly indicate, in addition to Arp2/3 and associated proteins, the likely autoinhibited by intramolecular binding between their Dia inhibitory presence of regulators of unbranched filaments at podosomes. domain (DID) and Dia autoregulatory domain (DAD) domains Formins are a family of actin regulators defined by the presence of (Fig. 1A) (Higgs, 2005; Kuhn and Geyer, 2014), both wild-type and a formin homology (FH)-2 domain involved in actin binding constitutively active constructs were used, lacking either the (Schonichen and Geyer, 2010). The 15 mammalian formin isoforms C-terminal DAD domain or bearing respective mutations in their form several subgroups, including among others the diaphanous DID domain (Fig. 1A). In addition to the reported localization of formin (DIA, also known as DIAPH), formin-like protein (FMNL), FMNL1 (Mersich et al., 2010), we detected prominent enrichment of dishevelled-associated activator of morphogenesis (DAAM), both FHOD1 and INF2 at podosomes. Western blots of macrophage inverted formin (INF2) or FH1/FH2-domain-containing protein lysates also confirmed that both proteins are expressed endogenously (FHOD) groups. Formins fulfill a variety of actin-associated (Fig. 1B,C). Moreover, INF2 can be expressed in at least two functions such as nucleation, elongation, capping, severing or isoforms, distinguished by the presence or absence of a C-terminal bundling of actin filaments, with the actual activities depending on CaaX box. Prenylation of the cysteine residue within this motif the individual isoform (Schonichen and Geyer, 2010). However, in mediates binding to the endoplasmic reticulum (Ramabhadran et al., contrast to Arp2/3-complex-generated branched actin networks, 2011). Therefore, isoform-specific antibodies were used, revealing formins are primarily associated with unbranched actin filaments that only the non-CaaX-containing isoform was detectable in (Chhabra and Higgs, 2007). macrophage lysates (Fig. 1C). In the current study, we report the identification of the formins Co-staining of macrophages expressing EGFP–FHOD1 or FHOD1 and INF2 as new podosome components. FHOD1 has EGFP–INF2-nonCaaX with Alexa-Fluor-568–phalloidin or emerged as a regulator of stress fibers (Koka et al., 2003), through Alexa-Fluor-647–phalloidin to label podosome cores showed that regulating the dynamics of actin transverse arcs and dorsal fibers each formin localizes to different regions at podosomes. EGFP– (Schulze et al., 2014). Notably, FHOD1 has recently been shown to FHOD1 was present at a shell-like localization surrounding the regulate maturation of integrin-based adhesion sites (Iskratsch et al., F-actin-rich core (Fig. 1D–F; Fig. S1C–E) and partially colocalizing 2013). In vitro, FHOD1 acts as capping and bundling protein for with proteins of the podosome ring structure such as vinculin or actin filaments (Schonichen et al., 2013), although the exact talin (not shown). This was especially evident in three-dimensional biochemical activity of FHOD proteins is currently unclear reconstructions of single podosomes (Fig. 1D″–F″) and also (Bechtold et al., 2014). In contrast to these more accessory confirmed by fluorescence intensity measurements (Fig. 1G). In activities of FHOD1, INF2 has been shown to be involved in addition, FHOD1 was also present at podosome-connecting actin polymerization and severing of actin filaments (Gurel et al., 2014). cables (Fig. 1F′; for visualization of cables, see also Fig. S1J). The This unique set of abilities enables the formation of transient actin latter phenotype was particularly striking in cells expressing non- filaments that are, for example, involved in fission of mitochondria autoinhibited EGFP–FHOD1ΔC, which in most cases led to a (Korobova et al., 2013, 2014). Importantly, INF2 can be expressed prominent formation of these cables (Fig. 1D–F; Fig. S1F–H) in two splice variants, which mostly differ in the presence or (note that these phenotypes represent endpoints of a continuum absence of a C-terminal CaaX box motif (where C is the cysteine of possible phenotypes that depends on respective expression residue that is prenylated, a is any aliphatic amino acid, and the levels). By contrast, EGFP–INF2-nonCaaX localized to podosome residue at X determines which enzyme acts on the protein) (Chhabra cores (Fig. 1H–K), without prominent localization to connecting et al., 2009; Ramabhadran et al., 2011) which, upon prenylation, cables. Importantly, three-dimensional reconstructions revealed that mediates localization to the ER (Chhabra et al., 2009). Mutations in INF2 only partially colocalized with podosomal F-actin, and was INF2 have been linked to the disorders focal and segmental mostly present as a cap-like structure on top of the podosome core glomerulosclerosis (Brown et al., 2010) and Charcot–Marie–Tooth (Fig. 1H″–J″). Localization of FHOD1 and INF2 to these podosome disease (Boyer et al., 2011). substructures was also confirmed for endogenous proteins by using Using primary human macrophages, we now find that FHOD1 respective antibodies (Fig. S2A–H). and INF2 localize to different substructures of podosomes. FHOD1 Furthermore, FMNL2, a formin previously found to be enriched surrounds the podosome core and is also present at podosome- at ventral membranes of macrophages (Cervero et al., 2012), did not connecting cables, whereas INF2 localizes at the podosome cap localize to podosomes (Fig. S3A–F), supporting the specificity of structure. Accordingly, both formins were found to regulate the localization of FHOD1 and INF2. Also, addition of the formin different aspects of podosome-associated contractility, with inhibitor SMIFH2 (25 µM) to macrophage cultures led to strongly

FHOD1 mediating actomyosin contractility between podosomes, pronounced reduction of podosomes (Fig. S3H–J), pointing to the Journal of Cell Science

299 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 298-313 doi:10.1242/jcs.177691

Fig. 1. See next page for legend. general importance of formin activity in podosome formation and/ INF2 regulates podosome size and ECM degradation or maintenance. We conclude that FHOD1 and INF2-nonCaaX are We next established siRNA-mediated knockdown for FHOD1 and expressed in primary macrophages, and localize to distinct INF2 to assess their impact on structural and functional parameters substructures of podosomes. (Note: for convenience, henceforth of podosomes, including number, size and lifetime, as well as ‘INF2’ refers to the INF2-nonCaaX splice variant, unless otherwise extracellular matrix degradation. For both formins, two sets of indicated.) individual siRNAs were established that led to efficient depletion Journal of Cell Science

300 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 298-313 doi:10.1242/jcs.177691

Fig. 1. EGFP-fused FHOD1 and INF2-nonCaaX localize to different not necessary for the upkeep of this structure. Collectively, these substructures of macrophage podosomes. (A) Domain structures of formin analyses revealed only a minor impact of FHOD1 on the tested constructs, fused to EGFP, and used in this study. FHOD1 features a GTPase podosome parameters. By contrast, INF2 emerged as a strong binding domain (GBD) involved in RhoGTPase binding, a formin homology 3 domain (FH3) containing the Diaphanous inhibitory domain (DID), followed by negative regulator of podosome size, and as a positive regulator of an additional actin binding site (ASBD) unique for FHOD1, formin homology podosomal matrix degradation. domains-1 (FH1) and -2 (FH2), involved in G- and F-actin binding, respectively, The previous experiments showed an influence of INF2 on and a C-terminal Diaphanous autoregulatory domain (DAD). Intramolecular podosome size, by measuring podosome core diameters (Fig. 2H). binding of DID and DAD domains leads to autoinhibition, whereas removal of However, this parameter does not distinguish between an overall Δ the DAD domain results in a non-autoinhibited conformation (FHOD1 C). increase in podosome size (i.e. a higher volume of podosome cores) Domains of INF2 are in part analogous to those of FHOD1, with the additional and an altered shape of podosomes (higher diameter, but lower height, presence of a dimerization domain (DD) and a WH2 domain involved in actin z binding and filament severing. Comparable to FHOD1, INF2 is regulated by leading to unaltered podosome volume). Therefore, optical -stacks of intramolecular autoinhibitory binding of DID and DAD domains. An A149D podosomes from INF2-depleted macrophages were obtained, and the mutation in the DID domain has been shown to result in activation of the protein height of podosome cores was measured. Indeed, the average height (Ramabhadran et al., 2012). Note that INF2 can be expressed as two splice of podosomes in INF2-knockdown cells was also significantly variants, which mostly differ in the presence or absence of a C-terminal CaaX increased (0.89±0.05 µm and 0.79±0.02 µm for INF2-specific box mediating binding to the ER. Numbers of first and last amino acid residues siRNAs versus 0.69±0.01 µm for controls), with a more detailed are indicated. (B,C) Western blots of macrophage lysates developed with anti- FHOD1 (B) or anti-INF2 (C) antibodies. For detection of INF2, antibodies analysis showing a clear overall shift to higher core height values specifically recognizing the CaaX (right) or non-CaaX isoforms (left) were (Fig. 3A). In reciprocal experiments, the podosome diameter was used. Arrows indicate respective bands of the expected size. (D–K) Confocal measured in macrophages overexpressing the non-autoinhibited form micrographs of primary macrophages expressing EGFP–FHOD1ΔC INF2-A149D (Fig. 3B). Overexpression of this mutant led to strongly (D, green), or EGFP–INF2-nonCaaX (H, green) and stained for F-actin using decreased average size of podosomes (0.50±0.03 µm for INF2- Alexa-Fluor-568- (E) or Alexa-Fluor-647- (I) labeled phalloidin (red), with A149D-expressing cells versus 0.95±0.03 µm for controls), which respective merges (F,J). Scale bars: 10 µm. White boxes in F,J indicate areas shown enlarged in D′–F′,H′–J′. White boxes in F′ and J′ indicate detail areas, was based on a general shift of podosome sizes to lower values with respective confocal stacks used for the generation of three dimensional (Fig. 3C), and was also accompanied by a pronounced reduction of reconstructions of single podosomes shown in D″–F″,H″–J″. Note that EGFP– podosome height (0.74±0.03 µm vs 1.02±0.03 µm for controls; FHOD1ΔC surrounds the F-actin-rich podosome core, and is also present at Fig. 3D). Furthermore, overexpression of INF2-A149D also strongly connections between individual podosomes, whereas EGFP–INF2-nonCaaX decreased overall number of podosomes per cell (Fig. 3E). Consistent is present in a cap structure on top of and partially overlapping with the with the unaltered diameter of podosomes upon FHOD1 depletion, podosome core. (G,K) Dashed lines in F,F′ and J,J′ indicate lines used for scanning of fluorescence intensity profiles shown in for single podosomes (G) podosome height was also not affected upon FHOD1 knockdown and or several podosomes (K). Dashed lines in F″,J″ indicate confocal planes used height distribution closely followed control values (Fig. 2 and not for scans. shown). Collectively, these experiments indicated that INF2 is a negative regulator of podosome core volume and podosome number. (90–96% knockdown; Fig. 2A,F). (Note: for initial testing of It should be noted that, given the limits of optical resolution by FHOD1, a pool of 4 sequences was used, which included the 2 confocal imaging, the indicated sizes for podosome diameter or height individual sequences.) Absence of endogenous formins was also cannot be accurately measured and thus represent only approximate confirmed on the single cell level, ensuring the absence also values. Also note that occasional variability in controls is based on the of residual formins at podosomes (Fig. S2I–N). Interestingly, variability between different preparations of primary macrophages. depletion of either FHOD1 or INF2 resulted in a reciprocal increase Therefore, controls of individual experiments were always performed of ∼75% of protein levels of the other formin (Fig. S3K), pointing to with cells from the same preparation, to preserve internal consistency. the existence of cross-regulatory mechanisms. Depletion of FHOD1 did not result in major changes in either the number of podosomes INF2 regulates oscillations, but not internal actin turnover, of per cell (Fig. 2B), podosome size (Fig. 2C) or podosome lifetime podosome cores (Fig. 2D), although detailed analyses showed increases in the As INF2 emerged as a regulator of podosome number and size, we number of cells containing only up to 50 podosomes (16.01±1.42%, next investigated whether it also influences podosome dynamics at compared with 7.02±0.6% in control cells; Fig. 2B) and in the the level of single podosomes. For this, we determined F-actin levels number of podosomes persisting for more than 20 min (15.24± at podosomes in a confocal plane over time, by measuring Lifeact- 5.02%, compared with 2.78±0.04% of control cells; Fig. 2D). GFP fluorescence. As reported earlier for podosomes of dendritic Fluorescence intensity measurements of FHOD1-depleted cells (van den Dries et al., 2013a), Lifeact-GFP-based fluorescence macrophages seeded on NHS–Rhodamine-labeled gelatin matrix of individual podosomes underwent constant fluctuations, showed no significant differences in matrix degradation to control indicative of the periodic actomyosin-based contractions of cells (Fig. 2E). Comparable to FHOD1 knockdown, depletion of podosomes in the z axis (Labernadie et al., 2010; Luxenburg INF2 did not result in significant changes in the number of et al., 2012), which results in varying F-actin intensities in a fixed podosomes per cell (Fig. 2G). Strikingly, however, podosome size plane of focus (Fig. 4A). Strikingly, these oscillations were was increased, in both mean values (siRNA#1: 0.84±0.06 µm; dampened in INF2-depleted cells (Fig. 4B,C), leading to a siRNA#2: 0.88±0.07 µm, compared with 0.68±0.01 µm using significant decrease in the respective coefficients of variation control siRNA) and size distribution (Fig. 2H). This was (10.68±0.72% for INF2 #1 siRNA- and 11.51±0.82% for INF2 #2 accompanied by an increase of the subgroup of long-lived siRNA-, vs 14.80±0.94% for control siRNA-treated cells) (Fig. 4D). podosomes persisting for more than 20 min (Fig. 2I), and a Addition of the myosin II inhibitor blebbistatin (20 µM) to cells significant decrease in gelatin matrix degradation (Fig. 2J). led to an even more pronounced decrease (6.41±0.28%), pointing Interestingly, depletion of INF2 did not alter the cap-like to actomyosin-based contractility as the basis for podosome localization of supervillin on top of podosome cores (Fig. S4), oscillations, consistent with previous results (Labernadie et al., indicating that although INF2 localizes to the podosome cap, it is 2010; van den Dries et al., 2013a). Journal of Cell Science

301 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 298-313 doi:10.1242/jcs.177691

Fig. 2. Effects of FHOD1 and INF2 knockdown on podosome parameters. Analysis of various podosome parameters, gained by using cells treated with control siRNA and FHOD1-specific siRNA (A–E) or INF-2 specific siRNA (F–J). (A,F) Western blot of lysates from macrophages treated with FHOD1-specific siRNA [single siRNA #1 or #2 or siRNA pool (FHOD1 p)] or INF2-specific siRNAs (single siRNA #1 or #2), with non-targeting siRNA as control. β-actin was used as a loading control. Knockdown efficiency is indicated beneath respective lanes. (B,G) Analysis of podosome numbers showing average number of podosomes per cell (left) and differential analysis of percentage of cells per size category (right). (C,H) Analysis of podosome size showing average podosome sizes (left) and differential analysis of podosome size distribution (right). (D,I) Analysis of podosome lifetime. (E,J) Analysis of podosomal matrix degradation. Left: confocal micrographs show macrophages treated with indicated siRNA and seeded on NHS–Rhodamine-labeled gelatin matrix. Matrix degradation is visible through loss of the label. Insets in upper right corners show respective F-actin staining to visualize podosome core structures. Scale bars: 10 µm. Right: quantification of gelatin degradation, as determined by fluorescence intensity measurements. Each dot represents a single measured cell (n=3 for each timepoint, from three different donors). Note that FHOD1 knockdown leads to a higher percentage of long-lived podosomes (D), with marginal influence on all other tested parameters. By contrast, INF2 knockdown leads to an overall increase in podosome size (H), a higher percentage of long-lived podosomes (I), and a reduction in gelatin matrix degradation (J). For experiments in B–E,G–J, at least 3×30 cells from three different donors were analyzed using Student’s t-test. Values are given as

mean±s.e.m. *P<0.05, **P<0.01. For specific values, see Table S1. Journal of Cell Science

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INF2 as a regulator of intra-podosomal contractility. However, this effect could also be achieved by INF2 influencing overall F-actin levels at podosomes, through regulating the turnover of actin itself. We therefore tested whether INF2 regulates actin turnover within podosomes, by using FRAP analysis of single podosomes in cells expressing GFP–β-actin. Measurements of normalized fluorescence intensities showed that bleaching resulted in a ∼60% decrease of GFP–β-actin-based fluorescence at single podosomes (n=3×30 podosomes, from 3 cells) in cells treated with control siRNA. Within 60 s post-bleaching, 89.8±2.1% of initial fluorescence values had been recovered, with a half time of recovery of 10.5 s±2.2 s (Fig. 4E–G). Importantly, these values were not significantly different in cells treated with INF2-specific siRNA (91.9±1.1% recovery of fluorescence levels after 60 s; half time of recovery 11.4±0.7 s) (Fig. 4F,G). The measured half times of recovery of 10 s are lower than earlier measurements of ∼30 s in osteoclasts (Destaing et al., 2003; Luxenburg et al., 2012) and dendritic cells (Gotz and Jessberger, 2013), but more similar to the 13 s value, as determined in another study using dendritic cells (Gawden-Bone et al., 2014). However, in all these previous studies, whole subcellular regions containing several podosomes were bleached, in contrast to bleaching of single podosomes in the current study. Collectively, this set of experiments showed that INF2 is a positive regulator of podosome oscillations, but has no apparent influence on the speed of actin turnover within podosomes.

INF2 regulates de novo formation of podosomes Podosomes are dynamic structures that undergo constant turnover. In order to assess the potential impact of FHOD1 and INF2 also on podosome formation, cells treated with respective siRNA were analyzed using the podosome reformation assay. This assay is based on the disruption of podosomes by the Src tyrosine kinase inhibitor PP2, with subsequent washout of the drug to monitor podosome reformation (Cervero et al., 2013; Linder et al., 2000b). ImageJ- based analysis allows the quantification of a statistically relevant number of cells and their associated podosomes (Fig. 5A) (Cervero et al., 2013). Formin-depleted macrophages were treated with 25 µM PP2, leading to almost complete disruption of podosomes, and analyzed for podosome content at timepoints 30, 60, 90 and 120 min after washout of the drug. At all timepoints, knockdown of FHOD1 by two siRNAs did not lead to discernible alterations in podosome reformation compared with controls (Fig. 5B). By contrast, INF2-depleted cells showed a significant delay in podosome reformation, leading to lower numbers of podosomes per cell at the indicated timepoints, compared with controls (Fig. 5C). (Note: regular podosome numbers were eventually reached after a prolonged washout period of 3.5 h; not shown, see Fig. 3. INF2 regulates podosome size. (A) Quantification of podosome height Fig. 2G.) in cells treated with control siRNA or with two individual siRNAs specific for INF2. Primary human macrophages show donor-dependent variations. (B) Non-autoinhibited INF2-A149D localizes to podosomes. Confocal For direct comparability between FHOD1- and IFN2-based effects, micrograph of macrophage expressing EGFP–INF2-A149D (middle), and experiments described in Fig. 5B and C were thus also conducted stained for F-actin (left), with merge (right). White box in merged image indicates in parallel, using cells from the same preparations. These detail region shown as insets. Scale bar: 10 µm. (C–E) Quantification of podosome height (C), diameter (D) or number of podosomes per cell (E) in experiments confirmed a significant, although less pronounced, macrophages overexpressing the non-autoinhibited mutant INF2-A149D. reduction of the number of reformed podosomes upon INF2 Diagrams on left in A,C–E) show overall values, diagrams on right show depletion (Fig. 5D). Moreover, podosome reformation experiments more differentiated evaluations, with podosomes or cells divided in subgroup, as in cells treated with control siRNA (to mimic transfection) and the indicated, and analyzed using Student’s t-test. Values are given as mean±s.e.m. myosin II inhibitor blebbistatin (10 µM) showed enhanced numbers *P<0.05, **P<0.01, ***P<0.001. For specific values, see Table S1. of podosomes per cell, compared with controls (Fig. 5E), pointing to a role of myosin-based contractility in the establishment of These experiments indicated that INF2 regulates podosome regular podosome numbers. Collectively, these experiments contractions in the z axis, which manifests as periodic alterations of showed that depletion of INF2 resulted in a pronounced delay in

F-actin intensity in a plane of focus, pointing to a potential role of podosome reformation. Journal of Cell Science

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Fig. 4. INF2 regulates podosome core oscillations, but not internal actin turnover. (A–D) Quantification of podosome oscillations, based on F-actin intensity. (A) Gallery of confocal micrographs from time lapse movie of macrophage expressing Lifeact-GFP to stain podosomal F-actin. Note fluctuations in F-actin intensity over time. (B) Representative graphs of Lifeact-GFP-based, normalized fluorescence intensity of single podosomes from cells treated with control siRNA or two individual INF2-specific siRNAs, or with blebbistatin (20 µM) to inhibit actomyosin contractility. (C) Mean values±s.e.m. of data represented in B. For each value, 3×5 podosomes from three cells from three donors (n=15) were evaluated. For specific values, see Table S1. (D) Coefficient of variation from data in C. (E) FRAP analysis of actin turnover in cells treated with control siRNA or INF2-specific siRNA. Gallery shows confocal micrographs taken from time lapse movie of GFP–actin-expressing macrophage. Dashed circle indicates single bleached podosome. Time before and after bleaching is given above each panel. (F) Quantification of FRAP showing GFP–actin based, normalized fluorescence intensity over time. Values are given as mean±s.e.m. For each value, 3×10 podosomes from three cells from three donors were evaluated. For specific values, see Table S1. (G) Half times of recovery for FRAP experiments presented in

F. *P<0.05, **P<0.01, ****P<0.0001. Journal of Cell Science

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Fig. 5. Effects of FHOD1 and INF2 knockdown on podosome reformation. (A) Principle of podosome reformation assay. Cells are treated for 30 min with podosome-disrupting Src tyrosine kinase inhibitor PP2 (+PP2), with subsequent washout of the drug to allow podosome reformation, with cells treated with DMSO as control (−PP2). Upper row: confocal micrographs of macrophages fixed and stained for F-actin using Alexa-Fluor-488–phalloidin at indicated timepoints. Lower row: image analysis of micrographs from upper row using ImageJ. Individual cells are depicted by red outlines, podosomes are depicted as black dots. (B–E) Analysis of podosome reformation in cells treated with (B) FHOD1-specific siRNA [2 individual sequences and also a pool of 4 sequences (FHOD1 p)], (C) INF2-specific siRNA (2 individual sequences), (D) FHOD1-specific pool siRNA or INF2-specific siRNA with cells from the same donor for direct comparability, or (E) non-targeting siRNA or myosin II inhibitor blebbistatin (10 µm) as controls. Note reduction of podosome reformation in INF2-depleted cells, especially at timepoints 60, 90 and 120 min after washout. For experiments in B–E, at each timepoint at least 3×30 cells from three different donors were analyzed using Student’s t-test. Values are given as mean±s.e.m. Podosome numbers were set to 100% at the start of the experiment to account for differences in size of cells, which result in different absolute numbers of podosomes per cell. Absolute numbers of podosomes at the start or respective experiments were: (B) control siRNA, 184.0±10.3; FHOD1 siRNA#2, 181.7±17.0; FHOD1 siRNA#2, 170.5±5.8; FHOD1 pool, 206.4±20.2; (C) control siRNA, 212.0±20.2; INF2 siRNA#1, 243.0±37.4; INF2 siRNA#2, 263.3±20.0; (D) control siRNA, 180.0±17.6; FHOD1 pool, 20.2±28.0; INF2 siRNA#2, 205.2±17.4; (E) 184.0±10.3. *P<0.05, **P<0.01. For specific values, see Table S1.

In order to study the actual dynamics of podosome formation, with earlier reports showing the existence of podosome fission in macrophages treated with FHOD1- or INF2-specific siRNA were macrophages (Evans et al., 2003; Kopp et al., 2006). To better also analyzed in the podosome reformation assay using live cell visualize podosome formation over time, a temporal color code was imaging. Podosome cores were visualized by expression of Lifeact- given to time lapse videos using ImageJ software. All individual GFP. In control cells, reformation of podosomes was mostly frames of a time lapse video were thus progressively colored along detectable at early timepoints of PP2 washout (1–5 min). the spectrum, with final merges depicting all events of podosome Interestingly, two phases of podosome formation were observed, formation in the respective period (Fig. 6A″). with a first phase consisting mostly of de novo formation of For cells treated with FHOD1-specific siRNA, statistical analysis podosomes, followed by a phase in which podosome formation also showed no significant changes in the overall events observed for occurred through fission of preexisting podosomes (Fig. 6A′). As podosome de novo formation, fission and dissolution (Fig. 6D), podosomes show a typical lifetime of 2–12 min (Destaing et al., which is in line with results gained with fixed specimens (Fig. 5B). 2003), events of podosome dissolution were also detected during By contrast, macrophages depleted for INF2 showed a delay in the the course of these experiments. These observations are also in line formation of podosomes, consistent with data from fixed cells Journal of Cell Science

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Fig. 6. Live cell analysis of podosome reformation in INF2- and FHOD1-knockdown cells. (A–C) Confocal micrographs of macrophages expressing Lifeact- GFP to visualize podosome cores and treated with control siRNA (A), FHOD1-specific (B) or INF2-specific siRNA (C). White boxes indicate areas of detail images shown in (A′–C′), representing still images from time lapse videos shown in Movies 1–3. Time since start of experiment (in min) is indicated above each frame. Red and green arrowheads indicate sites of de novo podosome formation and podosome fission, respectively. (A″–C″) Rainbow analysis of podosome reformation, with each frame of respective time lapse movies colored progressively along the spectrum above A′. Images are composites of all frames taken within the first 15 min, with inset showing merges of regions depicted in A′–C′. Scale bars: 10 µm. Note that cell size is highly variable, especially during the first few minutes of PP2 washout, when cells often react with partial contraction to the addition of the washout medium. Differences in cell size are thus not necessarily typical for the respective condition. Elongated structures in the cell periphery are thus not protrusions but retraction fibers. Cells were instead chosen for their representative behavior in podosome reformation. (D–G) Statistical evaluation of podsome reformation in FHOD1- and INF2-knockdown cells. Percentages of podosomes per area formed de novo, formed by fission, or being dissolved within the course of the experiments are indicated as (D) overall rates or (E–G) in a time course, for cells treated with control siRNA (E), FHOD1-specific siRNA (F) or INF2-specific siRNA (G). Note that FHOD1-depleted cells show a reduction, although not statistically significant, of de novo formation, whereas INF2 depleted cells show significantly decreased numbers of podosome de novo formation and significantly increased numbers of podosome fission. Values are given as mean±s.e.m. using Student’s t-test. For each value, three cells from three different

donors were evaluated. *P<0.05, **P<0.01. For specific values, see Table S1. Journal of Cell Science

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(Fig. 6D, Fig. 5C). In line with these results, statistical analyses podosome-covered area of cells were measured. Strikingly, we showed significantly decreased levels of de novo generated found elevated levels of p-MLC during podosome reformation in podosomes (31.42±9.14% for INF2 siRNA- vs 54.36±2.38% for control cells, compared with non-PP2-treated cells, indicative of control siRNA-treated cells), which was accompanied by a increased actomyosin contractility. This was especially pronounced significant increase in the levels of fission-generated podosomes at the 90 min timepoint of reformation (Fig. 7L). By contrast, (68.59±5.68% for INF2 siRNA- vs 45.64±2.91% for control FHOD1-depleted cells showed no significant alteration in p-MLC siRNA-treated cells). Respective time courses of cells treated with levels during the whole reformation period (Fig. 7L), whereas cells control siRNA (Fig. 6E), FHOD1-specific siRNA (Fig. 6F) or overexpressing the EGFP-FHOD1ΔC construct showed enhanced INF2-specific siRNA (Fig. 6G) showed that, in addition to the p-MLC levels (Fig. 7M). We conclude that FHOD1 localizes effects described above for fixed cells in 30 min intervals, the onset together with myosin IIA around podosomes and at podosome- of both de novo formation and fission of podosomes are delayed in connecting cables, and that FHOD1 activity is necessary for the cells depleted for either FHOD1 or INF2, compared with controls. regulation of actomyosin contractility at and between podosomes, This was especially pronounced in INF2 depletion (Fig. 6G). which probably helps to establish the regular pattern of podosome Reduced de novo formation of podosomes can also be appreciated in groups. the color-coded merges of INF2-knockdown cells, showing In order to assess the impact of FHOD1 and INF2 on podosome podosome formation mostly in clusters, indicative of podosome connecting cables in more detail, we imaged respective single and formation in the vicinity of preexisting structures, through fission double knockdown cells, stained for F-actin by Alexa-Fluor-488– (Fig. 6C″). phallodin, using structured illumination microscopy (SIM). In cells Collectively, these experiments show that podosome reformation treated with control siRNA, F-actin-rich podosome cores were occurs in two phases: first, predominantly by de novo formation, clearly visible, whereas connecting cables appeared often only as and second, by a phase also including fission of daughter faint structures that connect individual podosomes (Fig. 8A). By podosomes from preexisting structures. Overall values for de novo contrast, cells depleted for FHOD1 showed a more diffuse formation and fission are not significantly influenced by FHOD1. arrangement of cable-like structures surrounding and connecting However, a time course analysis revealed a delayed onset of both podosome cores (Fig. 8B). Similar observations were made for phases in case of FHOD1 depletion. By contrast, INF2 could be knockdown of INF2 (Fig. 8C) or knockdown of both FHOD1 and identified as a positive regulator of the de novo phase of podosome INF2 (Fig. 8D). In some cases, we also observed an alteration of reformation, and also as a negative regulator of podosome fission. the radially symmetric shape of podosomes to a more dash-like appearance. To assess the impact of FHOD1 and INF2, respective FHOD1 regulates myosin-based contractility at podosome- specimens were grouped according to the severity of the phenotype connecting cables in double-blinded experiments (Fig. 8E). Depletion of either formin FHOD1 shows a prominent localization at podosomes and led to a more pronounced phenotype compared with controls, with podosome-connecting cables (Fig. 1D−F). However, the data so depletion of FHOD1 causing the most severe cases (Fig. 8E). far showed no significant impact of FHOD1 depletion on podosome Collectively, these data indicate that FHOD1 and INF2 are involved parameters. We therefore focused next on a potential role of FHOD1 in the upkeep of regular podosome connecting cables, and that at connecting cables. Notably, podosome-connecting cables also depletion of either formin results in phenotypic aberrancies. contain myosin, which is important for regulating actomyosin- based contractility between podosomes (Bhuwania et al., 2012). DISCUSSION Consistent with previous results (Bhuwania et al., 2012), In this study, we identify the formins INF2 and FHOD1 as novel endogenous myosin IIA, the predominant myosin II isoform in components and regulators of macrophage podosomes. INF2 macrophages (Maupin et al., 1994), was found to localize to localizes to the podosome cap structure, regulating podosome podosome groups in macrophages expressing EGFP–FHOD1 de novo formation, size and oscillation, as well as matrix constructs, in clusters surrounding individual podosomes, and degradation, whereas FHOD1 localizes around podosome cores and also between podosomes (Fig. 7A–D). Note that connecting cables at podosome-connecting cables, regulating podosome connectivity. are hard to visualize using fluorescently labeled phalloidin Recent research has revealed an increasing complexity of both (presence of connecting cables was checked by overexpression of podosome sub- and superstructures (Veillat et al., 2015). Unbranched Lifeact-GFP constructs; see also Fig. S1J). However, especially in actin filaments were found to surround the podosome core, cells expressing non-autoinhibited EGFP-FHOD1ΔC(∼50% of connecting the top of the podosome to the ring of adhesion plaque cells), F-actin-based connecting cables were more prominent, and proteins, thus providing additional anchoring to the plasma their decoration by myosin IIA was clearly discernible (Fig. 7E–H). membrane (Luxenburg et al., 2007). Moreover, radial fibers Moreover, podosome-connecting cables in these cells were often emerging from the podosome cores connect individual podosomes aligned in parallel, indicative of increased tension. This is in line into higher-ordered groups (Akisaka et al., 2008; Luxenburg et al., with earlier results showing that increased actomyosin-based 2007). The identification of a cap structure on top of the podosome contractility results in a symmetry break in the uniform podosome core is the most recent addition (Linder et al., 2011; Mersich et al., pattern and parallel alignment of podosomes and podosome- 2010), although the potential function of this substructure is currently connecting cables (Bhuwania et al., 2012). unexplored. These observations indicated that FHOD1 activity could regulate Of note, the existence of several subsets of unbranched actin actomyosin-based contractility at podosome-connecting cables. To filaments at or between podosomes (Akisaka et al., 2008; Luxenburg test this possibility, macrophages with established knockdown of et al., 2007) pointed to the presence of regulators also of unbranched FHOD1 were submitted to the podosome reformation assay, fixed at actin filaments, in addition to the well-known influence of Arp2/3 the indicated timepoints, stained for phosphorylated myosin light complex (Kaverina et al., 2003; Linder et al., 2000a). We thus chain (p-MLC), a reporter of myosin contractility (Matsumura, screened for the potential localization of formins at macrophage

2005; Vicente-Manzanares et al., 2009), and p-MLC levels in the podosomes and detected prominent enrichment of both INF2 and Journal of Cell Science

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Fig. 7. FHOD1 enhances myosin-based contractility at podosomes. (A–H) Confocal fluorescence micrographs of macrophages expressing EGFP–FHOD1 (green, A–D) or EGFP–FHOD1ΔC (green, E–H), stained for myosin IIA (red; B,F), with respective merges (C,G), and costained for F-actin using Alexa-Fluor-647– phalloidin (white; D,G). White boxes in D,H indicate areas magnified in right-hand panels. Note presence of myosin IIA around and between podosomes (B,C), which is especially prominent with expression of the non-autoinhibited construct (F,G). (I–K) Confocal fluorescence micrographs of macrophages treated with control siRNA, and stained for F-actin using Alexa-Fluor-568–phalloidin (I, red in merge), and for phospho-myosin light chain using phosphospecific antibody (J, green in merge), with merge in (K). White box in K indicates area shown as insets in I–K. (L,M) Fluorescence intensity measurement of phospho-myosin light chain during podosome reformation in macrophages treated with FHOD1-specific siRNA or control siRNA (L) or expressing EGFP–FHOD1ΔC (M). Macrophages were treated with podsosome-disrupting PP2, and fixed and stained at the indicated timepoints during podosome reformation. Values are given as mean±s.e.m., using Student’s t-test. *P<0.05, ***P<0.001. For specific values, see Table S1. Scale bars: 10 μm.

FHOD1. In particular, we identify INF2 as a novel component of the identified at the podosome cap. Interestingly, INF2 does not seem to podosome cap. Beside FMNL1 (Mersich et al., 2010) and supervillin be involved in upkeep of the cap structure, as supervillin localization

(Bhuwania et al., 2012), INF2 is one of the few proteins currently at the cap is not altered in INF2-knockdown cells. Journal of Cell Science

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Fig. 8. Knockdown of FHOD1 and INF2 leads to aberrancies in podosomal F-actin. (A–D) Structured illumination micrographs of macrophages treated with control siRNA (A) or siRNA specific for FHOD1 (B), INF2 (C) or both (D) and stained for F-actin using Alexa-Fluor-488–phalloidin. White boxes indicate area of detail shown in A′–D′. Note irregular F-actin, especially between podosomes, in case of FHOD1- and/or INF2- knockdown, compared with control. (E) Evaluation of F-actin phenotypes at podosomes and connecting cables. Panels on right depict aberrant phenotypes categorized according to their severity, ranked as + to ++++.

Podosomes display several levels of dynamics, including of FRAP experiments showed that INF2 depletion has no detectable de novo formation, fission, fusion, lateral mobility and dissolution effect on actin turnover within podosomes, which seems to point to a (Destaing et al., 2005; Evans et al., 2003; Linder et al., 2000b), with more structural role of INF2. Still, it should be kept in mind that most concomitant turnover of core actin (Destaing et al., 2003). In of the actin filaments in podosomes are Arp2/3-generated, and major addition, podosomes undergo periodic oscillations, as demonstrated fluctuations as a result of INF2 depletion are thus not to be expected. in macrophages (Labernadie et al., 2010) and dendritic cells (van den Furthermore, INF2 emerged as a crucial regulator of podosome Dries et al., 2013a), and this phenomenon is coupled to their size. Knockdown of INF2 led to an overall increase of ∼25% in mechanosensing ability (Luxenburg et al., 2012). In macrophages, podosome diameter. This was accompanied by a ∼20% increase podosome cores show periodic cycles of stiffness, with a period of in podosome height, thus corresponding to a true net increase 40–60 s (Labernadie et al., 2010). This is based on cycles of both in size (as opposed to the theoretical case of wider but more flat myosin II activity and actin polymerization. Moreover, the protrusive podosomes). Consistently, expression of a non-autoinhibited mutant, force of podosomes, which is in the 102–103 nN range, shows a INF2-A149D, led to a reciprocal effect, resulting in a ∼50% similar periodicity, with cycles of 40–50 s (Labernadie et al., 2014). reduction of podosome diameter and a ∼30% decrease of podosome The current model thus pictures podosomes as a contractile system, height. Beside PAK4 (Gringel et al., 2006) and calpain (Chou et al., constantly balancing forces generated by actin polymerization in the 2006), INF2 is thus one of the few identified regulators of podosome core and by the lateral actin cables that function as springs size, pointing to an important modulatory role of INF2 in podosome (Labernadie et al., 2014), ultimately enabling podosomes to architecture. It is unlikely that INF2 at the cap directly inhibits the function as mechanosensory devices (Linder and Wiesner, 2015). growth of Arp2/3-generated actin filaments, as formins track the We now show that siRNA-mediated knockdown of INF2 leads to a barbed ends of actin filaments, and barbed ends within the core are ∼25% dampening of podosome oscillations, pointing to a role of expected to localize towards the plasma membrane (Linder, 2007). In INF2 in the mechanosensing ability of these structures. Involvement an alternative scenario, INF2-regulated lateral cables, embedded into of the cap in podosomal substrate sensing is particularly novel, as no the cap, could act as a ‘corsage’ that enwraps the podosome, thus function of the cap structure has been identified so far. Furthermore, limiting growth of the core structure. Further analyses of podosome Journal of Cell Science

309 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 298-313 doi:10.1242/jcs.177691 dynamics showed that INF2 depletion led to a delay in podosome by phospho-myosin light chain levels, at and between podosomes reformation, which is based on reduced de novo formation of during podosome reformation. Control cells showed increasing podosomes and is accompanied by increased fission of podosomes. pMLC levels during the reformation phase, which probably reflects This points to a common requirement of both Arp2/3-dependent the not-yet balanced forces between podosomes during the polymerization of core material (Linder et al., 2000a) and INF2- establishment of the equidistant podosome pattern. These results dependent lateral fibers in the initial stages of podosome formation, fit well with the observed preference of both myosin IIA and also seems to be in line with the proposed model of INF2- (Verkhovsky and Borisy, 1993) and FHOD1 (Schulze et al., dependent cables limiting podosome core growth. It is also 2014) to bind to anti-parallel, i.e. potentially contractile, actin noteworthy that, although INF2 depletion leads to an upregulation filaments. In contrast, FHOD1-depleted cells showed no alterations of protein levels of FHOD1, and vice versa (Fig. S3K), FHOD1 is in pMLC levels, pointing to defects in actomyosin contractility. In a apparently not able to compensate for INF2 in these functions. reciprocal experiment, expression of a non-autoinhibited form of Considering the potential biochemical basis for these effects, two FHOD1, lacking the C-terminal DAD domain, led to pronounced points should be mentioned. First, INF2 displays both actin formation of connecting cables, their prominent decoration with polymerizing and depolymerizing activity (Chhabra and Higgs, myosin IIA, and to increased levels of p-MLC at podosomes. These 2006), and this twofold ability can lead to the formation of transient results are also in line with current literature showing an impact of actin filaments, a mechanism that has been shown to be involved in FHOD1 in the formation of actin cables in several cell types, as mitochondrial fission (Korobova et al., 2013). Second, in order to expression of a dominant active form of the Drosophila homolog achieve oscillation of podosomes, the lateral actin filaments have to Knittrig led to stress fiber formation in macrophages and endothelial be modeled as springs (Labernadie et al., 2014), and the constant cells (Lammel et al., 2014), and expression of non-autoinhibited polymerization and depolymerization of actin by INF2 might FHOD1-V228E led to increased formation of stress fibers in U2OS provide the necessary flexibility for this system. This seems to be osteosarcoma cells (Schulze et al., 2014). counterintuitive, as we did not detect changes in actin turnover in Further analysis using superresolution microscopy showed FRAP experiments of INF2-knockdown cells. However, it should disorganized connecting cables in cells depleted for FHOD1, and be kept in mind that the vast majority of podosomal F-actin is also for INF2. Although FHOD1 knockdown gave the severest localized in the core and thus generated by Arp2/3 complex, phenotype, the apparent participation of INF2 might point to the cap whereas the lateral cables constitute only a minor amount of the structure as an organizer of these cables, as hypothesized earlier podosomal F-actin pool. (Linder and Wiesner, 2015). It is currently unclear whether these The impact of INF2 on podosome formation, size and oscillation disorganized filaments reflect a net increase in inter-podosomal is probably linked to a regulation of podosome architecture itself. In F-actin, or whether, following formin depletion, connecting cables contrast, the observed impact of INF2 on podosomal matrix are more loosely bundled and thus more accessible for phalloidin degradation is probably more indirect. Upon INF2 depletion, most staining. In fact, both scenarios could be envisioned, considering the podosomes show normal lifetime, and also their number is unaltered currently known biochemical activities of FHOD1. In vitro, FHOD1 under these conditions. The ∼30% decrease in matrix degradation is shows no actin polymerization or depolymerization activities, thus clearly not based on a reduction of available podosome but acts as capping and bundling protein for actin filaments structures, but more likely based on defects in the recruitment or (Schonichen et al., 2013). The disordered connecting cables in release of matrix-lytic enzymes. Indeed, it has been speculated that FHOD1-knockdown cells could thus result from a lack of bundling. the podosome cap could function as a hub for incoming vesicles In addition, the ability of FHOD1 to inhibit the growth of parallel, (Linder et al., 2011). Moreover, INF2 activity has been implicated in i.e. non-contractile actin filaments (Schulze et al., 2014), might help the secretory pathway, and the importance of INF2-regulated short to ensure that only anti-parallel bundles of actin are formed between actin filaments was shown for vesicle trafficking during transcytosis podosomes. Depletion of FHOD1 might thus also lead to enhanced in hepatocytes (Madrid et al., 2010), and also for targeting of Lck formation of parallel actin filaments between podosomes. Taken tyrosine kinase-containing vesicles to the plasma membrane in together, our results point to FHOD1 as a regulator of inter- Jurkat T cells (Andres-Delgado et al., 2010). Clearly, the possibility podosomal contractility. FHOD1 regulates podosome-connecting of INF2-regulated vesicle transport at podosomes is a field that cables, probably by bundling anti-parallel actin filaments, thus needs further investigation in the future. Collectively, our data forming the basis for proper levels of actomyosin contractility, identify INF2 as a novel important regulator of multiple aspects of which ensures efficient force transduction between podosomes. podosome architecture, dynamics and function. INF2 is a positive Collectively, our results identify the formins INF2 and FHOD1 regulator of podosome de novo formation, and a negative regulator as novel components of macrophage podosomes that regulate of podosome size. It is important for the generation of podosome different aspects of podosome-associated contractility. INF2 oscillations and thus for mechanosensing, and it is also involved in emerged as a novel component of the recently identified the matrix degrading ability of these structures. podosome cap structure, and is involved in regulating podosome In contrast to the multiple aspects influenced by INF2, the impact size, de novo formation, fission, and oscillations. Especially the of FHOD1 on podosomes has been more elusive. FHOD1 localizes latter fact points to a potential role of the cap structure and its around podosomes and also at podosome-connecting cables, in both components in the regulation of intrastructural contractility, and overexpressed and endogenous forms. As FHOD1 depletion did not thus of mechanosensing by podosomes. In contrast, FHOD1 influence a variety of tested parameters associated with individual regulates actomyosin-based contractility of podosome-connecting podosomes, we focused on the potential role of FHOD1 on the cables, thus regulating the connectivity of podosomes in higher- regulation of podosome-connecting cables. These fibers, consisting ordered clusters. Podosomes thus present as one of the few of actomyosin, are contractile (Bhuwania et al., 2012), and thus currently identified structures, which depend on the concerted ideally placed to mediate force propagation between podosomes and activity of both Arp2/3 complex and specific formins as their to regulate coherence within podosome groups (Proag et al., 2015). integral components, and might serve as a model system for the

Indeed, we found marked differences in myosin activity, as detected analysis of complex actin architectures in cells. Journal of Cell Science

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MATERIALS AND METHODS Basel, Switzerland)]. After 30 min on ice, lysates were centrifuged (10 min, Isolation, culture and transfection of cells 10,000 g, 4°C) and supernatants were examined by standard immunoblotting Human peripheral blood monocytes were isolated from buffy coats (kindly procedure, with actin as a loading control, using the above-mentioned primary provided by Frank Bentzien, University Medical Center Eppendorf, Hamburg, antibodies, and HRP-coupled anti-mouse or anti-rabbit IgG as secondary Germany) and differentiated into macrophages, as described previously antibodies. Protein bands were visualized by using Super Signal Pico kit (Wiesner et al., 2013). Approval for the analysis of anonymized blood (Pierce, Rockford, IL) and X Omat AR films (Kodak, Stuttgart, Germany). donations was obtained by the Ethical Committee of the Ärztekammer Hamburg (Germany). Cells were cultured in RPMI-1640 (containing 100 Immunofluorescence and microscopy units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine and 20% Cells were fixed for 10 min in 3.7% formaldehyde and permeabilized for autologous serum) at 37°C, 5% CO2 and 90% humidity. Monocytes were 10 min in (0.5% Triton X-100, PBS, pH 7.5). After staining the cells with differentiated in culture for at least seven days, under addition of 20% human antibodies or labelling reagents mentioned above, the coverslips were autologous serum. For transfection experiments, differentiated macrophages, mounted in Mowiol (Calbiochem, Darmstadt, Germany) containing at days 7–10 of culture, were transiently transfected using the Neon® DABCO (25 mg/ml; Sigma-Aldrich) as anti-fading reagent. Transfection System (Thermo Fisher Scientific, Waltham, MA), an Images of fixed samples were acquired with confocal laser-scanning electroporation-based system, with standard settings (1000 V, 40 ms, 2 microscopes (Leica DM IRE2 with a Leica TCS SP2 AOBS confocal point pulses) with siRNA (1 µM) and plasmids (0.5 µg/1×105 cells), respectively. scanner equipped with an oil-immersion HCX PL APO 63× NA 1.4 λblue objective and 3× PMT detectors or Leica DMI 6000 with a Leica TCS SP5 Expression constructs and siRNA AOBS confocal point scanner equipped with an oil-immersion HCX PL APO EGFP–FHOD1 and EGFP–FHOD1ΔC [amino acids (aa) 1–1010] were CS 63× NA 1.4 objective and 2× HyD, 2× PMT detectors). Acquisition and kindly provided by Oliver Fackler (University Hospital Heidelberg, Germany) processing of images were performed with Leica confocal software (Leica, (Gasteier et al., 2005). EGFP–INF2-nonCaaX (aa 1–1240) and EGFP–INF2- Wetzlar, Germany) and/or Volocity 6.1.1 software (Perkin Elmer, Waltham, nonCaaX A149D were kindly provided by Henry N. Higgs (Dartmouth MA) and Photoshop CS5 (Adobe, Dublin, Ireland). For analyzing podosome Medical School) (Ramabhadran et al., 2011). EGFP–FMNL2B and EGFP– number and size, primary human macrophages were transfected with control FMNL2BΔDAD were kindly provided by Klemens Rottner (Technische siRNA and siRNA against FHOD1 and INF2 and seeded on glass coverslips at Universität Braunschweig, Germany). mRFP–SV was kindly provided by a density of 1×105 cells. After 72 h of incubation, the cells were fixed, Elisabeth J. Luna (University of Massachusetts) (Fang et al., 2010). EGFP- permeabilized and stained with Alexa-Fluor-488–phalloidin for highlighting actin was generated as described previously (Osiak et al., 2005). Lifeact-GFP podosome cores. Images of fixed samples were taken as mentioned above. was purchased from Ibidi (Martinsried, Germany). For knock down of Podosome numbers (3 donors, 100 cells per condition) were measured using FHOD1, an ON-TARGETplus SMARTpool (FHOD1 p) was used (Thermo an ImageJ (NIH, Bethesda, MD) macro (see Table S2). Podosome diameters Fisher Scientific), as well as two individual siRNAs present in the pool: #1, 5′- were measured with ImageJ (3 donors, 8 cells per condition). The core height GCGCUUGAGUAUCGGACUU-3′,and#2,5′-GAUACUACCUGGACA- was measured using an adapted ‘Analyze Particle’ macro and an additional CCGA-3′. siRNAs specific for human INF2 were purchased from Ambion macro for ImageJ (see Table S2) to measure the intensity of individual ROIs. (Life Technologies, Carlsbad, CA) with the following sequences: #1, 5′-C- The core height for INF2-nonCaaX-A149D-expressing cells (3 donors, 80 CAUGAAGGCUUUCCGGGA-3′;#2,5′-CAUCCAACGUGAUGGUGA- podosomes from 8 cells) was measured using the line tool of Volocity 6.1.1 in A-3′. Non-targeting siRNA No. 2 (Thermo Fisher Scientific) was used as xz mode. To detect pMLC levels at podosomes, pMLC-based fluorescence negative control. Onset and persistence of respective knockdowns was intensity of confocal micrographs of cells stained with pMLC antibody and checked by western blots of respective cell lysates. labeled with Alexa-Fluor-568 anti-rabbit and Alexa-Fluor-488–phalloidin was measured using Volocity 6.1.1. The ROI tool was used to restrict the Antibodies and staining reagents measurement to the podosome area. All data were processed using Excel 2013 F-actin was stained using Alexa-Fluor-488–, Alexa-Fluor-568– or Alexa- (Microsoft, Redmond, WA) and GraphPad Prism 6 (La Jolla, CA). Fluor-647–phalloidin, as indicated (Molecular Probes, Eugene, OR). Mouse monoclonal anti-actin antibody MAB1501 (clone C4) was purchased from Live-cell imaging Millipore (Billerica, MA) and used at 1:5000 on western blots. FHOD1 was Live-cell imaging was performed using a spinning disk microscope [Nikon detected using mouse polyclonal antibody (pAb) FM3521 (ECM Bioscience, Eclipse Ti with a UltraVIEW VoX system (Perkin Elmer)] and a small Versailles, KY) at 1:50 in immunofluorescence and mouse monoclonal temperature- and CO2-controllable environmental chamber combined with an antibody (mAb) D-6 (sc-365437; Santa Cruz Biotechnology, Dallas, TX) at objective lens heater [(Tokai Hit INU-F1, Japan), equipped with a Yokogawa 1:200 on western blots. Rabbit pAbs specific for INF2-nonCaaX and INF2- CSU X1 spinning disk, an oil-immersion 60× Apo TIRF (corr.) objective, a CaaX, and a general anti-INF2 polyclonal, were kindly provided by Henry 527 nm (W55) emission filterand a Hamamatsu EM-CCD C9100-50 camera], N. Higgs (Dartmouth Medical School) (Ramabhadran et al., 2011) and were or the above mentioned confocal laser-scanning microscope (Leica DMI used at 1.5 ng/ml on western blots or at 1:50 in immunofluorescence. FMNL2 6000). Acquisition and processing of images were performed with Leica was detected using rabbit pAb 72105 from Abcam (Cambridge, UK) at 1:500 confocal software and/or Volocity 6.1.1 software. To evaluate podosome on western blots. Rabbit anti-myosin IIA pAb M8064 was purchased from lifetime and oscillations, cells from three individual donors were transfected Sigma-Aldrich (St Louis, MO), used at 1:100 in immunofluorescence, and a with control siRNA and siRNA against FHOD1 and INF2. After 48 h, 1×105 phospho-specific (pS20) anti-myosin II light chain rabbit pAb ab2480 from cells were re-transfected with siRNAs, as well as Lifeact-GFP and seeded on Abcam, used at 1:100 in immunofluorescence. Mouse anti-vinculin mAb glass bottom live cell dishes. After further incubation for 24 h, cells were V9264 was purchased from Sigma-Aldrich and used at 1:500 in imaged for a period of 45 min with image acquisition rate of 4× min−1. immunofluorescence. All fluorochrome-coupled secondary antibodies Podosome lifetime of FHOD1-knockdown cells was evaluated by tracking the (Alexa-Fluor-488 goat anti-mouse and Alexa-Fluor-568 goat anti-rabbit) podosomes with Imaris software (Bitplane, Zurich, Switzerland). Each track were purchased from Molecular Probes (Eugene, OR), and were used at 1:200 resembles the lifetime of an individual podosome. All track data were in immunofluorescence, and all horseradish peroxidase (HRP)-coupled ones processed in Excel 2013 and GraphPad Prism 6. Podosome lifetime of INF2- (HRP goat-anti mouse, HRP goat-anti-rabbit) from GE Healthcare (Chalfont knockdown cells was evaluated using Volocity (Perkin Elmer). xy-drift was St Giles, UK), and were used at 1:5000 on western blots. To stain the whole corrected with the Rigid body transformation of the StackReg plugin in cytoplasm, HCS CellMask™ Red Stain (Thermo Fisher Scientific; 2 µg/ml) ImageJ. To analyze podosome oscillations, the intensities of five individual was used. podosomes per cell for three different donors per condition were measured at every timepoint using ImageJ. All data were processed with Excel 2013 and Immunoblotting GraphPad Prism 6. For FRAP experiments, cells were treated with INF2 Cells were scraped from dishes in buffer [150 mM NaCl, 1% Triton X-100, siRNA#1 or control siRNA. After 48 h of incubation, 1×105 cell were re-

50 mM Tris-HCl, pH 8.0, with protease inhibitor cocktail (Roche, transfected with the individual siRNAs, as well as GFP–actin and seeded on Journal of Cell Science

311 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 298-313 doi:10.1242/jcs.177691 glass bottom live cell dishes. After additional incubation for 24 h, cells were Author contributions imaged using the Leica DMI 6000. Images of single cells were taken every L.P., L.T. and M.K. designed and performed experiments, B.J., J.S. and A.C. 1.29 s. After collecting five pre-bleach images, two circular ROIs with performed SIM, and S.L. designed experiments and wrote the manuscript. individual podosomes were bleached for five timepoints using 78% of a 405 nm laser. Recovery images were taken for additional 60 s. FRAP analysis Funding was performed with the FRAP profiler (ImageJ). The data was processed with This work was supported by the Deutsche Forschungsgemeinschaft [grant number LI925/3-1 and 3/-2, to S.L.] as part of SPP 1464; by Wilhelm Sander- Excel 2013 and GraphPad Prism 6. Fluorescence recovery was measured for Stiftung [grant number 2014.135.1, to S.L.]; and by Human Frontiers [grant 10 individual podosomes at each timepoint for three cells from three donors. RGP0027/2012, to A.C.].

2D gelatin degradation assay Supplementary information Gelatin (from swine; Roth, Karlsruhe, Germany) was fluorescently labeled Supplementary information available online at with NHS–Rhodamine (Thermo Fisher Scientific), according to Chen et al. http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.177691/-/DC1 (1994). Coverslips were coated with labeled matrix solution, fixed in 0.5% glutaraldehyde (Roth) and washed with 70% ethanol, RPMI and culture References medium. Cells were re-seeded on coated coverslips with a density of 1×105 Akisaka, T., Yoshida, H., Suzuki, R. and Takama, K. (2008). Adhesion structures and their cytoskeleton-membrane interactions at podosomes of osteoclasts in cells 72 h after siRNA transfection, fixed and permeabilized 6 h post – culture. Cell Tissue Res. 331, 625-641. seeding and stained with Alexa-Fluor-647 phalloidin. Values of matrix Andres-Delgado, L., Anton, O. M., Madrid, R., Byrne, J. A. and Alonso, M. A. degradation were determined by a loss of fluorescence intensity relatively to (2010). Formin INF2 regulates MAL-mediated transport of Lck to the plasma non-degraded matrix, using ImageJ software. For comparability, laser membrane of human T lymphocytes. Blood 116, 5919-5929. intensity was not changed between measurements. For each value, 3×30 Bechtold, M., Schultz, J. and Bogdan, S. (2014). FHOD proteins in actin cells were evaluated. Values were analyzed using Excel 2013 and statistical dynamics–a formin’ class of its own. Small GTPases 5, e973765. analysis was performed with GraphPad Prism software. Differences Bhuwania, R., Cornfine, S., Fang, Z., Kruger, M., Luna, E. J. and Linder, S. between mean values were analyzed using Student’s t-test. (2012). Supervillin couples myosin-dependent contractility to podosomes and enables their turnover. J. Cell Sci. 125, 2300-2314. Boyer, O., Nevo, F., Plaisier, E., Funalot, B., Gribouval, O., Benoit, G., Huynh Podosome reformation assay Cong, E., Arrondel, C., Tete, M.-J., Montjean, R. et al. (2011). INF2 mutations in This assay was performed and analyzed in control and knockdown, fixed and Charcot-Marie-Tooth disease with glomerulopathy. N. Engl. J. Med. 365, living cells 72 h after siRNA transfection as published previously (Cervero 2377-2388. et al., 2013). For live cell imaging, siRNA transfected cells were re-transfected Brown, E. J., Schlöndorff, J. S., Becker, D. J., Tsukaguchi, H., Tonna, S. J., after 48 h with both siRNA and Lifeact-GFP and seeded on glass bottom live Uscinski, A. L., Higgs, H. N., Henderson, J. M. and Pollak, M. R. (2010). cell dishes foranother 24 h before imaging. The podosome reformation process Mutations in the formin gene INF2 cause focal segmental glomerulosclerosis. Nat. was further analyzed in live cell movies, counting the events of podosome de Genet. 42, 72-76. novo formation, cluster fission and dissolution in defined areas of control and Burgstaller, G. and Gimona, M. (2005). Podosome-mediated matrix resorption and cell motility in vascular smooth muscle cells. Am. J. Physiol. Heart Circ. Physiol. knockdown cells. Numbers were processed in Excel 2013 and GraphPad Prism 288, H3001-H3005. 6. For rainbow analysis, the movies were processed with ImageJ, adding a Cervero, P., Himmel, M., Krüger, M. and Linder, S. (2012). Proteomic analysis of temporal color code for each timepoint (LUT: Rainbow Smooth). podosome fractions from macrophages reveals similarities to spreading initiation centres. Eur. J. Cell Biol. 91, 908-922. SIM sample preparation and microscopy Cervero, P., Panzer, L. and Linder, S. (2013). Podosome reformation in Cells were fixed for 1 min at 37°C with 0.25% PFA/0.05% Triton X-100 in macrophages: assays and analysis. Methods Mol. Biol. 1046, 97-121. CB buffer (10 mM MES, 138 mM KCl, 3 mM MgCl, 2 mM EGTA) and for Chabadel, A., Banon-Rodriguez, I., Cluet, D., Rudkin, B. B., Wehrle-Haller, B., 30 min at room temperature with 4% PFA in CB buffer. Permeabilization Genot, E., Jurdic, P., Anton, I. M. and Saltel, F. (2007). CD44 and beta3 integrin was done with 0.5% Triton X-100 in CB buffer for 5 min at room organize two functionally distinct actin-based domains in osteoclasts. Mol. Biol. Cell 18, 4899-4910. temperature. Cells were washed once with CB/0.1 M Glycine for 10 min and Chellaiah, M., Kizer, N., Silva, M., Alvarez, U., Kwiatkowski, D. and Hruska, K. A. twice with TBS-T (0.1% Tween) for 10 min before staining with 132 nM (2000). Gelsolin deficiency blocks podosome assembly and produces increased Alexa-Fluor-488–Phalloidin in TBS-T+5% NGS+5% NHS for 60 min. bone mass and strength. J. Cell Biol. 148, 665-678. Samples were washed 3×5 min with TBS-T and 2×2 min with TBS. 250 µl Chen, W. T., Yeh, Y., and Nakahara, H. (1994). An in vitro cell invasion assay: 0.1 µm Tetraspec beads (Life Technologies, Darmstadt, Germany) diluted determination of cell surface proteolytic activity that degrades extracellular matrix. 1:2000 were added per sample for 15 min at room temperature. After J. Tissue Culture Meth. 16, 177-181. washing 2×2 min with TBS coverslips were mounted in Mowiol containing Chhabra, E. S. and Higgs, H. N. (2006). INF2 is a WASP homology 2 motif- DABCO (25 mg/ml; Sigma-Aldrich) as anti-fading reagent. Structured containing formin that severs actin filaments and accelerates both polymerization and depolymerization. J. Biol. Chem. 281, 26754-26767. illumination imaging was performed using a Zeiss Elyra PS1 system. 3D- Chhabra, E. S. and Higgs, H. N. (2007). The many faces of actin: matching SIM data was acquired using a 63×1.4 NA oil objective. 405, 488, 561, 642 assembly factors with cellular structures. Nat. Cell Biol. 9, 1110-1121. 100 mW diode lasers were used to excite the fluorophores together with a Chhabra, E. S., Ramabhadran, V., Gerber, S. A. and Higgs, H. N. (2009). INF2 is BP 420-480+LP 750, BP 495-575+LP 750, BP 570-650+LP 750 or LP 655 an endoplasmic reticulum-associated formin protein. J. Cell Sci. 122, 1430-1440. excitation filter, respectively. For 3D-SIM imaging the recommended Chou, H.-C., Antón, I. M., Holt, M. R., Curcio, C., Lanzardo, S., Worth, A., Burns, S., grating was present in the light path. The grating was modulated in five Thrasher, A. J., Jones, G. E. and Calle, Y. (2006). WIP regulates the stability and phases and five rotations, and multiple z-slices with an interval of 110 nm localization of WASP to podosomes in migrating dendritic cells. Curr. Biol. 16, were recorded on an Andor iXon DU 885, 1002×1004 EMCCD camera. 2337-2344. Destaing, O., Saltel, F., Geminard, J.-C., Jurdic, P. and Bard, F. (2003). Raw images were reconstructed using the Zeiss Zen 2012 software. Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein. Mol. Biol. Cell 14, 407-416. Acknowledgements Destaing, O., Saltel, F., Gilquin, B., Chabadel, A., Khochbin, S., Ory, S. and Jurdic, We thank Frank Bentzien for buffy coats, Oliver Fackler for FHOD1 constructs, Henry P. (2005). A novel Rho-mDia2-HDAC6 pathway controls podosome patterning – N. Higgs for INF2 antibodies and constructs, Elizabeth Luna for mRFP supervillin, through microtubule acetylation in osteoclasts. J. Cell Sci. 118, 2901-2911. Andrea Mordhorst for expert technical assistance, Klemens Rottner for FMNL2B Evans, J. G., Correia, I., Krasavina, O., Watson, N. and Matsudaira, P. (2003). constructs, the UKE microscope facility (umif) for help with microscopy and image Macrophage podosomes assemble at the leading lamella by growth and analysis, Adriaan Houtsmuller for use of the SIM microscope, and Martin Aepfelbacher fragmentation. J. Cell Biol. 161, 697-705. for continuous support. This work is part of the doctoral theses of L.P. and L.T. Fang, Z., Takizawa, N., Wilson, K. A., Smith, T. C., Delprato, A., Davidson, M. W., Lambright, D. G. and Luna, E. J. (2010). The membrane-associated protein, Competing interests supervillin, accelerates F-actin-dependent rapid integrin recycling and cell motility.

The authors declare no competing or financial interests. Traffic 11, 782-799. Journal of Cell Science

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