The Journal of Immunology

Hematopoietic Lineage -Specific Protein 1 Functions in Concert with the Wiskott–Aldrich Syndrome Protein To Promote Podosome Array Organization and Chemotaxis in Dendritic Cells

Deborah A. Klos Dehring,*,1,2 Fiona Clarke,*,1 Brendon G. Ricart,† Yanping Huang,* Timothy S. Gomez,‡ Edward K. Williamson,* Daniel A. Hammer,† Daniel D. Billadeau,‡ Yair Argon,* and Janis K. Burkhardt*

Dendritic cells (DCs) are professional APCs that reside in peripheral tissues and survey the body for pathogens. Upon activation by inflammatory signals, DCs undergo a maturation process and migrate to lymphoid organs, where they present pathogen-derived Ags to T cells. DC migration depends on tight regulation of the to permit rapid adaptation to environmental cues. We investigated the role of hematopoietic lineage cell-specific protein 1 (HS1), the hematopoietic homolog of , in regulating the actin cytoskeleton of murine DCs. HS1 localized to lamellipodial protrusions and podosomes, actin-rich structures associated with adhesion and migration. DCs from HS12/2 mice showed aberrant lamellipodial dynamics. Moreover, although these cells formed recognizable podosomes, their podosome arrays were loosely packed and improperly localized within the cell. HS1 interacts with Wiskott–Aldrich syndrome protein (WASp), another key actin-regulatory protein, through mutual binding to WASp-interacting protein. Comparative analysis of DCs deficient for HS1, WASp or both proteins revealed unique roles for these proteins in regulating podosomes with WASp being essential for podosome formation and with HS1 ensuring efficient array organization. WASp recruitment to podosome cores was independent of HS1, whereas HS1 recruitment required Src homology 3 domain-dependent interactions with the WASp/WASp-interacting protein heterodimer. In migration assays, the phenotypes of HS1- and WASp-deficient DCs were related, but distinct. WASp2/y DCs migrating in a chemokine gradient showed a large decrease in velocity and diminished directional persistence. In contrast, HS12/2 DCs migrated faster than wild-type cells, but directional persistence was significantly reduced. These studies show that HS1 functions in concert with WASp to fine-tune DC cytoarchitecture and direct cell migration. The Journal of Immunology, 2011, 186: 4805–4818.

by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. endritic cells (DCs) are professional APCs that play riphery to naive T cells to initiate an adaptive immune response. a unique role in bridging innate and adaptive immunity DC function is critically dependent on the ability to migrate long D (reviewed in Refs. 1–3). DCs reside in peripheral tissues distances, traverse barriers, and navigate diverse tissues with and continually sample the environment for pathogens. In re- variable surface characteristics (3). DCs achieve this by mechan- sponse to pathogen-derived inflammatory molecules, these cells ical adaptation of cytoskeletal dynamics. Depending on the nature undergo a maturation program that induces their migration to of the substrate with which they are interacting, DCs can move by lymphoid organs, where they present Ags obtained in the pe- -independent amoeboid protrusion into an open space within a three-dimensional matrix, or by pushing against integrin-

http://classic.jimmunol.org *Department of Pathology and Laboratory Medicine, Children’s Hospital of Phila- based adhesive contacts with extracellular substrates (4, 5). In this delphia and University of Pennsylvania School of Medicine, Philadelphia, PA 19104; latter mode, movement is driven by the combined force of actin †Department of Chemical and Biomolecular Engineering, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104; and ‡Division of Oncology Research, polymerization and myosin contractility. This mechanism is Department of Immunology, College of Medicine, Mayo Clinic, Rochester, MN characterized by extension of an actin-rich at the 55905 front of the cell, often accompanied by the formation of adhesive 1 D.A.K.D. and F.C. contributed equally to this work. contacts termed podosomes just behind the edge of this protrusion. 2Current address: Department of Cell and Molecular Biology, Feinberg School of Podosomes are short-lived structures composed of actin-rich cores Downloaded from Medicine, Northwestern University, Chicago, IL. surrounded by adhesion molecules, including , talin, and Received for publication September 17, 2010. Accepted for publication February 11, 2011. (reviewed in Refs. 6–9). Although the exact function of Address correspondence and reprint requests to Dr. Janis K. Burkhardt, Department podosomes is still unclear, these structures serve as sites of matrix of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, 3615 metalloproteinase deposition (10, 11) and are thought to facilitate Civic Center Boulevard, 816D Abramson Research Center, Philadelphia, PA 19104. adhesion and migration through tissue barriers such as the lym- E-mail address: [email protected] phatic . In addition, podosomes may function as part The online version of this article contains supplemental material. of the mechanosensing mechanism that allows DCs and other Abbreviations used in this article: BMDC, bone marrow-derived ; DC, dendritic cell; DKO, double-knockout; FRAP, fluorescence recovery after photo- hematopoietic cells to alter their cytoskeletal dynamics in re- bleaching; HS1, hematopoietic lineage cell-specific protein 1; MMP, matrix metal- sponse to changing substrates. loproteinase; PDMS, polydimethylsiloxane; SH, Src homology; WASp, Wiskott– The plasticity of DC migration is mediated by tightly regulated Aldrich syndrome protein; WIP, WASp-interacting protein; WT, wild-type. changes in actin dynamics. Several individual actin regulatory Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 proteins have been shown to be important for controlling specific

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003102 4806 HS1 AND WASp ORGANIZE PODOSOMES IN DCs

aspects of DC migration. One key protein is WASp, the gene for from Santa Cruz Biotechnology. Anti-HA tag was purchased from which is mutated in the immunodeficiency disorder Wiskott– Roche. Aldrich syndrome. DCs deficient for WASp show an almost Recombinant HS1 was made as described previously (42). To generate recombinant cortactin, full-length human cortactin cDNA was subcloned complete lack of migratory capacity (12–15). WASp and its into pGEX-4T-2 vector (GE Healthcare) and expressed in BL21-DE3 binding partner Wiskott–Aldrich syndrome protein-interacting bacteria. The recombinant cortactin was purified using glutathione protein (WIP) colocalize with F-actin in podosome cores and Sepharose 4B (GE Healthcare). FLAG-WIP and FLAG-WASp were de- are essential for the formation of podosomes (16–19). WASp scribed previously (43). Mutations in WIP (D460 and P463A) to abrogate WASp binding were generated based on Ref. 44, and a WASp mutant functions by activating the Arp2/3 complex, a seven-subunit (D40–154) that does not bind WIP was made using standard site-directed protein complex that promotes actin polymerization by generat- mutagenesis (Stratagene). ing new actin filaments on the sides of pre-existing filaments (20). Two other proteins that have been shown to be important for DC Mice migration, CDC42 and Vav1, also function to activate Arp2/3- HS12/2 mice on the C57BL/6J background have been previously de- dependent actin polymerization (21, 22). Formation of branched scribed (45), and WASp knockout mice were purchased from The Jackson Laboratory. To generate HS1 and WASp double-knockout (DKO) mice, actin filaments is important for generating lamellipodial pro- +/2 2/y trusions as well as for generating podosome cores, which turn over HS1 and WASp mice were bred, and the F1 progeny were then in- terbred. All mice were housed under pathogen-free conditions in the rapidly and exchange actin continuously (19, 23, 24). Children’s Hospital of Philadelphia animal facility. All studies involving Another important actin regulatory protein in DCs is hemato- animals were reviewed and approved by the Children’s Hospital of Phil- poietic lineage cell-specific protein 1 (HS1, also called HCLS1 or adelphia Institutional Animal Care and Use Committee. LckBP1) (25). HS1 is the hematopoietic homolog of cortactin, BMDC culture a protein involved in adhesion, spreading, endocytosis, and mi- gration in many cell types (26–29). Cortactin is upregulated or GM-CSF was produced from the B78Hi/GMCSF.1 cell line provided by T. Laufer (University of Pennsylvania, Philadelphia, PA). Bone marrow was hyperphosphorylated in a number of metastatic and plays isolated from leg bones, cleaned of muscle tissues, and sterilized in 70% an important role in the formation of , structures that EtOH using IMDM (Life Technologies) containing 1% FBS (Atlanta resemble, but are distinct from, podosomes (30–32). Cortactin Biologicals). The cells were centrifuged at 1600 rpm and 4˚C for 10 min stabilizes branched actin filaments in vitro (33, 34) and enhances and resuspended in DC culture media (IMDM, 10% FBS, penicillin/ the persistence of actin-rich lamellipodial protrusions in fibro- streptomycin, GlutaMax, 55 mM 2-ME, and 3% supernatant GM-CSF) at a concentration of 1 3 106 cells/ml. Cell suspension (1 ml) was blasts (26). Like cortactin, HS1 is involved in the stabilization of added to wells of 24-well plates and supplemented with 1 ml of media on branched actin filaments (35). Both of these proteins have a mod- day 2. Starting on day 5, 1 ml media was replaced daily. Differentiation ular structure, with an N-terminal region that binds Arp2/3 com- into DCs was verified on day 6 by flow cytometric analysis of surface plex and actin filaments, and a C-terminal adaptor region that can CD11c levels (typically 70–80%). Cultures were used between days 6 and 9. To induce maturation, DCs between days 6 and 8 were cultured with 100 connect multiple proteins within the actin network, including Lck, ng/ml LPS for 24 h. Itk, Vav1, WASp, WIP, and Nck (36–39). HS1 promotes lamelli- podial protrusion in T cells (36, 37) and regulates adhesion and DNA constructs, retroviral production, and transduction migration in NK cells and B cells (40, 41). Thus, HS1 and cortactin A vector expressing the GFP-variant Venus (Venus/pCS2 (46)) was pro-

by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. appear to carry out similar functions. Although HS1 has recently vided by A. Miyawaki (Brain Science Institute, RIKEN, Yokohama, Ja- emerged as an important actin-regulatory protein in hematopoietic pan). Human HS1 cDNA, described in Ref. 36, and Venus were amplified cells, its role in DC function has not been investigated. and ligated into the pMSCV2.1 retroviral vector (provided by W. Pear, University of Pennsylvania). GFP-actin (BD Clontech) was amplified and In this study, we show that HS1 is the sole cortactin family ligated in place of existing GFP in the MiGR retroviral vector (provided by member expressed in murine bone marrow-derived dendritic cells W. Pear). GFP-WASp (47) and GFP-Lifeact (provided by M. Sixt and (BMDCs). HS1 localizes to actin-rich structures involved in cell R. Wedlich-Solner, Max Planck Institute of Biochemistry, Martinsried, migration, including lamellipodia and podosomes, and localization Germany) (48) were amplified and ligated into MiGR and pMSCV2.1 retroviral vectors, respectively. Retrovirus was produced by calcium of HS1 to podosomes requires Src homology (SH)3 domain- phosphate cotransfection of 293T cells with 30 mg of the DNA of interest, dependent interaction with WASp through its binding partner and constructs encoding the viral envelope protein for mouse ectopic virus http://classic.jimmunol.org WIP. Comparative analysis of DCs lacking HS1, WASp, or both and the gag and pol genes. Supernatant was harvested at 24 and 30 h proteins reveals that these proteins play distinct roles: WASp is posttransfection and titered using NIH-3T3 fibroblast cells. essential for podosome formation, whereas HS1 is necessary to BMDCs were transduced by spin infection with retrovirus expressing Venus-HS1, GFP-WASp, GFP-Lifeact, or GFP-actin on day 2 of culture. organize the podosome array within the cell. Similarly, WASp is Retrovirus and 4 mg/ml Polybrene (Sigma-Aldrich) were added to the wells required for overall migration of DCs as well as for directional of a 24-well culture plate and centrifuged at 2000 rpm and 32˚C for 2 h. persistence during chemotaxis, whereas HS1 is primarily required Retrovirus-containing media were then replaced with DC culture media, and the cultures were cared for as described above. Transduction efficiency Downloaded from for directional persistence. These studies show that HS1 functions (typically $45%) was determined on day 6 by detection of Venus or GFP to fine-tune DC cytoarchitecture and direct cell migration. expression by flow cytometry. RAW/LR5 cells were a gift from D. Cox (Albert Einstein College of Medicine, New York, NY) and were cultured and retrovirally transduced as Materials and Methods described previously (19). Cells were either transduced with control virus Reagents and Abs or virus to knockdown HS1, and stable lines were selected with puromy- Rabbit anti-human HS1 (36) and rabbit anti-mouse HS1 (37) were de- cin. HS1 suppression was verified by Western blotting. scribed previously. Mouse anti-HS1 (3A3) was purchased from StressGen Western blotting Bioreagents. Anti-GAPDH was purchased from Calbiochem. Anti-cortactin (GK-18), anti-FLAG (M2), anti-vinculin, and LPS were obtained from For analysis of cortactin, HS1, and WASp expression, cells were lysed in Sigma-Aldrich. Anti-myc, anti-GFP, Alexa Fluor 594 phalloidin, anti- lysis buffer (20 mM HEPES [pH 7.5], 1% Nonidet P-40, 0.5% deoxy- mouse IgG1 Alexa Fluor 488, anti-mouse IgG Alexa Fluor 594, anti-rat cholate, 0.1% SDS, 50 mM NaCl, 5 mM EDTA, 10 mg/ml leupeptin, 500 Alexa Fluor 488, anti-rabbit IgG Alexa Fluor 488, anti-goat IgG Alexa mM AEBSF, 1 mM Na3VO4, and 5 mM NaF) on ice, cleared by centri- Fluor 488, and FITC- were obtained from Invitrogen. Anti-cortactin fugation, and protein concentration was determined by BCA assay (4F11), anti-phosphotyrosine (4G10), and anti-WASp were purchased from (Pierce). Lysates were resolved on 4–12% NuPage gels (Invitrogen) or tris- Upstate Biotechnology. Anti-b2 integrin (CD18, C71/16) was obtained glycine SDS-PAGE gels, transferred to nitrocellulose membranes, blocked from BD Pharmingen. Anti-talin (C-20) and anti–GST-HRP were obtained in 5% milk in PBS, and probed with primary Abs in TBST, followed by The Journal of Immunology 4807

secondary Abs (goat anti-mouse IgG-Alexa Fluor 680 [Invitrogen] or goat each cell was then bleached for 50 cycles, and images were captured at the anti-rabbit IgG-IR Dye 800 [Rockland]). Proteins were visualized and fastest speed for 15 s, every second for 45 s, and every 3 s for 180 s. analyzed ratiometrically using the Licor Odyssey infrared fluorescence Analysis was conducted using a single constrained exponential algorithm. system, taking care to remain within the linear range. Results are shown as t1/2, the time required for fluorescence to recover to half the original value. Immunofluorescence Kymography BMDCs were harvested and cultured on coverslips at 2 3 105 cells/well in 6-well plates overnight. The coverslips were washed in HBSS, followed by WT and HS12/2 DCs were transduced with GFP-Lifeact retrovirus on day fixation in 3% paraformaldehyde/PBS. Cells were permeabilized with 2. On days 7 or 8, DCs were harvested and cultured overnight at 1 3 105 0.3% Triton X-100 and blocked with 0.05% saponin/1.25% fish skin cells/well of a 4-well Lab-Tek chamber slide, prior to imaging. DCs were gelatin in TBS. Cells were labeled for F-actin with Alexa Fluor 594- imaged every 5 s for 15 min by confocal microscopy. Fifteen WT and 15 phalloidin and with primary Abs followed by appropriate fluorescently HS12/2 cells were analyzed. Two lines per cell were drawn through tagged secondary Abs. For endogenous HS1 staining, cells were fixed and lamellipodial projections perpendicular to the cell edge and kymographs permeabilized simultaneously using a protocol from Ref. 49. For visuali- were produced using Volocity software. Periods of protrusion, retraction or zation of Venus or GFP-tagged proteins, anti-GFP was used, followed by stationary behavior were identified and measured to determine the distance anti-rabbit IgG Alexa Fluor 488. Cells were imaged using a PerkinElmer (x) and time (y). From this, distance and velocity were calculated. Outliers Ultraview ER6 spinning disk confocal system equipped with a Zeiss (.2 SDs from the mean) were removed, and data were represented as box Axiovert 200 microscope and a 363 1.4 NA objective. Images were col- and whiskers plots with the median of each population represented. lected using an Orca ER camera (Hamamatsu) and analyzed using Volocity v.5 software (PerkinElmer). Analysis of matrix metalloproteinase secretion 3 6 Analysis of podosome array morphology For zymography, 2.5 10 BMDCs were cultured in serum-free media in bacteriological 10-cm dishes for 24 h. Supernatant was concentrated using For array analysis, images were collected without bias using spinning disk a ,30-kDa cutoff centrifugal filter device (Millipore). Proteins were sepa- confocal imaging. Cell profiles were determined using the “find object” rated on SDS-PAGE gels containing 1 mg/ml gelatin (Sigma-Aldrich). The function in Volocity, and the borders of each podosome array were drawn gel was then incubated in renaturation buffer (2.5% v/v Triton X-100, 50 by hand. The areas were calculated in Volocity. The percentage of the total mM Tris-HCl, and 0.05% NaN3) for 3 h at 37˚C and in developing buffer (50 area of individual cells covered by podosome arrays was then calculated. mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2, and 0.05% NaN3) at 37˚C To determine the number of podosomes per cell, the actin cores were overnight. Nondegraded gelatin was visualized by Coomassie brilliant blue identified and counted using the “find objects” function in Volocity, with staining and imaged on a Licor Odyssey fluorescence scanner. verification and correction by eye. FITC-gelatin degradation assays were performed as described in Ref. For analysis of array localization and packing, slides were blinded to 50. Briefly, coverslips were acid-washed, coated with 2% FITC-gelatin experimental conditions. Cell polarization was determined, based on the (Invitrogen), and quenched in serum-free media for 1 h at 37˚C. BMDCs presence of an actin-rich, spread lamellipodium. The number of arrays per were cultured on coverslips overnight, washed, and fixed. Cells were la- cell was counted and placed into one of the following groups: touching the beled for actin and imaged by spinning disk confocal microscopy. The leading edge (touch), behind the leading edge but not touching it (behind), in degraded area for each cell was quantified using Volocity and expressed as the middle of the cell not touching an edge (middle), opposite of the leading a percentage of total cell area, as defined by phalloidin labeling. edge (back), lateral to the leading edge (side), or circular rosettes (rosette). Array packing was based on the tightness of podosome packing within DC migration assays individual arrays, with cells scored as tight if most of the podosomes Ninety-six-well Transwell plates, 5-mm pore size, were from NeuroProbe. contacted one another in a regular pattern and loose if gaps were evident Immature and mature WT and HS12/2 BMDCs were harvested, pelleted at between many of the podosomes. Approximately 200 cells were analyzed 6 by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. 1500 rpm and room temperature for 5 min, and resuspended at 2 3 10 per experiment. For add-back experiments, slides were blinded to exper- cells/ml. Migration media alone (IMDM, 1% serum) or containing che- imental conditions, and cells were scored for presence or absence of mokine (200 ng/ml CXCL12 [PeproTech], 500 ng/ml CCL21, or 2.5mg/ml podosomes, whether the arrays were loosely packed, and whether HS1 or CCL19 [R&D Systems]) was added to the bottom well. Cells (5 3 104) WASp was in the podosome cores. Graphs are averages from three to five were placed on the filter, and the plate was incubated at 37˚C for 3 h. The independent experiments. filter was removed, and the cells in the bottom well were counted using a hemocytometer. The percentage of migrated cells was calculated by Analysis of podosome dynamics dividing the number of cells in the bottom well by the number of input To measure podosome lifetime, wild-type (WT) and HS12/2 DCs were cells. Chemokine receptor expression and cell maturation were verified by transduced with GFP-Lifeact retrovirus on day 2. On days 7 or 8, DCs flow cytometry of input cells. were harvested and cultured overnight at 1 3 105 cells/well of a 4-well For microfluidic migration assays, a microfluidic gradient generator was http://classic.jimmunol.org Lab-Tek chamber slide, prior to imaging. DCs were imaged every 5 s for fabricated in polydimethylsiloxane (PDMS, Sylgard 184; Dow Corning) 15 min by confocal microscopy. Fifteen WT and HS12/2 cells were an- using soft lithography as previously described (51) with modifications. alyzed, representing 4000–6000 individual podosomes per cell type. The Briefly, soft lithography was used to create an SU-8-2050 photoresist “find objects” function in Volocity was used to identify podosomes in each (MicroChem) on a silicon master. Positive replicas with embedded chan- timepoint, with visual verification. Objects were then tracked throughout nels were fabricated by molding PDMS against the master. The PDMS the video using the “Track Object” function. Tracks that contained objects replica and a glass microscope slide were activated by oxygen plasma in the first two or last two time points were excluded from analysis, as were treatment then irreversibly contact bonded. The adhesion surface was functionalized by incubation with 10 mg/ml fibronectin (Sigma-Aldrich) Downloaded from tracks that existed for less than four time points. Podosome lifetime was then calculated, based on the number of time points in any given track. for 1 h at 20˚C and blocked with 1% BSA (Sigma-Aldrich) in PBS for 2 h Outliers (.2 SDs from the mean) were removed, and data were plotted as at 20˚C. The microfluidic chemotaxis assay was performed as previously box and whiskers plots with the median of each population represented. described (52) with the following changes. BMDCs were cultured as de- Reformation of podosomes was assayed, based on a modification of Ref. scribed in Ref. 53 and matured for 24 h with 100 ng/ml LPS, harvested by 19. Briefly, BMDCs were cultured on coverslips overnight. DC culture pipetting and loaded into a syringe. The chemoattractant solution used was media containing 1 mM cytochalasin D (Calbiochem) were added for 30 CCL19 (PeproTech). min at 37˚C. The cells were then washed twice with warm DC culture media and incubated for the indicated times at 37˚C, fixed, and labeled for Results actin and vinculin. Slides were blinded, and ∼200 cells were scored for the HS1, but not cortactin, is expressed in BMDCs presence or absence of podosome arrays. To measure actin turnover within podosomes, BMDCs transduced with The structural changes associated with DC migration are orches- GFP-actin were cultured in 4-well Lab-Tek II chambered coverglasses trated by several actin-regulatory proteins, including Rho family 3 4 (Nalge Nunc) at 5 10 cells/chamber overnight. Fresh media were added , Vav1, and WASp (12, 14, 21, 22, 54). The cortactin and overlaid with mineral oil before imaging. Cells were imaged by spinning disk confocal microscopy using the Volocity v.5 fluorescence homolog HS1 functions as part of this actin regulatory complex recovery after photobleaching (FRAP) plug-in. The cells were imaged in lymphocytes and NK cells (36, 37, 40, 41), but its role in DCs every 3 s before bleaching. A 20-mm2 area within the podosome array of has not been addressed. HS1 and cortactin usually exhibit 4808 HS1 AND WASp ORGANIZE PODOSOMES IN DCs

mutually exclusive expression patterns, with HS1 expressed in hematopoietic lineage cells and cortactin expressed in other cell types. However, DCs have been reported to express both proteins (10, 17). Thus, we initiated our studies by carefully characterizing the expression patterns of these two proteins in BMDCs. In ad- dition to testing BMDCs from WT mice, we tested BMDCs generated from HS12/2 mice, to ask whether cortactin expression is upregulated to compensate for loss of HS1. BMDCs lacking HS1 differentiate normally in culture and upregulate costimula- tory molecules (CD80 and CD86), CD40, and MHC class I and II similarly to WT BMDCs upon maturation with LPS (data not shown). As shown in Fig. 1, a polyclonal anti-mouse HS1 Ab raised in our laboratory reacted with recombinant human HS1 but not cortactin and with a ∼70-kDa band in lysates from hemato- poietic cells (T cells and DCs) from WT mice but not HS12/2 mice. As expected, this Ab failed to interact with lysates from non-hematopoietic cell types (mouse 3T3 and human 293T). This reagent binds human HS1 weakly, as indicated by its ability to detect recombinant human HS1, but not HS1 in human Jurkat T cells. In addition, a polyclonal anti-human HS1 Ab reacts specifically with human HS1 as a recombinant protein or from Jurkat T cells, but not with mouse HS1 (DCs and T cells). Several commercially available Abs tested displayed different patterns of HS1 and cortactin rec- ognition. One monoclonal anti-cortactin Ab, 4F11, reacted with mouse and human cortactin (∼70-kDa band in 3T3 and 293T cells, respectively), but failed to detect recombinant HS1, or HS1 ex- FIGURE 2. HS1 colocalizes with F-actin in structures associated with pressed in mouse T cells or BMDCs. However, another widely used cell migration. A and B, BMDCs were cultured overnight on coverslips, Ab, GK-18, cross-reacted with HS1 and cortactin in all cell types fixed, and stained with anti-HS1 (mAb 3A3, green) and phalloidin to vi- tested and with both recombinant proteins. In keeping with these sualize F-actin structures (red). Colocalization of HS1 with F-actin in podosomes (A) and lamellipodial edges (B) is shown. C, To verify Ab biochemical data, GK-18 labeled actin-rich structures in WT, but 2/2 2/2 specificity, BMDCs from WT or HS1 mice were labeled with the in- not HS1 DCs by immunofluorescence microscopy, whereas dicated Abs (green), together with phalloidin (red). Note that the anti- 4F11 showed only background labeling in both cell types (see cortactin Ab GK-18 labels podosomes in WT but not HS12/2 cells, Fig. 2C). Taken together, these findings demonstrate that murine whereas the more specific Ab 4F11 fails to label actin-rich structures, even DCs express HS1 but not cortactin. Furthermore, they verify that in WT BMDCs. Both anti-cortactin Abs give a fine punctate pattern in WT 2/2 2/2 by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. DCs from HS1 mice lack HS1 expression and show that these and HS1 DCs, and 4F11 labels the . Given the re- cells do not exhibit compensatory upregulation of cortactin. activity of these two Abs by Western blot and the fact that these structures do not colocalize with F-actin, this is likely to be nonspecfic labeling. HS1 localizes to podosomes but is dispensable for podosome Scale bars, 10 mm. Insets, Higher magnification views of the boxed areas. formation We next investigated HS1 localization in murine BMDCs. The munofluorescence microscopy, but mAb 3A3 (StressGen Biore- polyclonal Ab raised in our laboratory did not work well for im- agents) specifically labeled WT DCs but not DCs from HS12/2 mice (Fig. 2C). Labeling with this Ab revealed that HS1 coloc- alizes with F-actin in podosome cores (Fig. 2A,2C). Similar http://classic.jimmunol.org results were obtained with cross-reacting anti-cortactin Ab GK-18 but not the more cortactin-specific Ab 4F11 (Fig. 2C). We also observed HS1 colocalization with F-actin at the edges of lamel- lipodia (Fig. 2B). This distribution is consistent with the locali- zation of cortactin in nonhematopoietic cells (55) and with the idea that HS1 functions to regulate actin-rich structures associated

Downloaded from with cell migration. To ask whether HS1 is required to organize DC cytoarchitecture, WT and HS12/2 DCs were plated onto coverslips, and the actin cytoskeleton was analyzed by immunofluorescence microscopy. Adhesion and spreading of HS12/2 DCs on both fibronectin- coated and uncoated coverslips were grossly normal, although we noted that HS12/2 DCs were somewhat more likely to exhibit multiple lamellipodial protrusions. Labeling with phalloidin and FIGURE 1. HS1 is the only cortactin family member expressed in murine 2/2 2/2 anti-vinculin revealed that both WT and HS1 DCs were able to BMDCs. BMDCs and T cells were cultured from WT or HS1 mice, and make podosomes with actin-rich cores surrounded by vinculin whole-cell lysates were analyzed by immunoblotting with anti-HS1 or anti- 2 2 rings (Fig. 3A). In some HS1 / DCs, the boundaries of actin rich cortactin Abs. Lysates from the nonhematopoietic cell lines 3T3 and 293T 2/2 were loaded as positive controls for mouse and human cortactin, re- cores and vinculin rings seemed diffuse in HS1 DCs (Sup- spectively. Lysate from Jurkat T cells was loaded as a positive control for plemental Fig. 1), but this phenotype was also observed in the human HS1. Recombinant human cortactin and HS1 were loaded as positive population of WT cells. No differences were observed in the controls for Ab specificity. GAPDH was used to verify equal loading. number of cells with podosome arrays or the number of arrays per The Journal of Immunology 4809

cell (data not shown). Moreover, other markers of podosomes, as likely to contain podosomes as WT DCs (data not shown), they including talin, b2 integrin, and phosphotyrosine (56), localized exhibited significantly fewer podosomes per cell (Fig. 3B). To normally within the podosomes of HS12/2 DCs (Supplemental determine whether HS1 promotes podosome formation, WT or Fig. 2). In addition to exhibiting normal composition, the podo- HS12/2 DCs were treated with cytochalasin D to dissolve podo- somes of HS12/2 DCs were functionally competent as sites of somes. After drug washout, the cells were allowed to recover for degradation. Supernatants from WT and varying times, and the number of cells with podosome arrays was HS12/2 DCs contained similar levels of functional matrix met- assessed. Prior to treatment, a similar number of WT and HS12/2 alloproteinases (MMPs). On the basis of their gelatinase activity DCs exhibited podosomes (Fig. 3C, points on the y-axis). In both and mobilities, the predominant bands for both cell types corre- cell types, podosomes were lost upon drug treatment, and actin spond to pro-MMP9 and pro-MMP2 (Supplemental Fig. 3A). cores were recovered within 30–60 min of drug washout (Fig. 3C). Moreover, when WT or HS12/2 DCs were cultured on FITC- However, recovery of podosome cores in HS12/2 DCs was con- gelatin-coated coverslips, there was no difference in the size, sistently delayed. In both WT and HS12/2 cells, recovery of placement or frequency of holes formed in the matrix, or in av- vinculin into rings was not observed until after actin cores were erage area of matrix degradation per cell (Supplemental Fig. 3B, formed (data not shown), consistent with the idea that a viable 3C; data not shown). We conclude that HS1 is not required for the actin core is needed for recruitment of ring proteins (15). formation of podosomes that contain many of the characteristic Because HS1 stabilizes branched actin filaments, we hypothe- proteins and function as sites of metalloproteinase release. sized that loss of HS1 would decrease the stability of podosome cores. To test this, DCs were transduced with GFP-Lifeact, which Podosome number and organization are perturbed in the selectively labels F-actin in DCs without affecting lamellipodial absence of HS1 dynamics (48), and the lifetime of individual podosomes was Although the organization of podosome arrays varies widely, even assessed by video microscopy. As shown in Fig. 3D, the median among WT cells, we observed clear differences in the podosome podosome lifetime was significantly reduced in HS12/2 DCs, but 2/2 2/2 arrays of WT and HS1 DCs. First, although HS1 DCs were this represents a minor shift in the population given the wide distribution of values. Finally, the exchange of actin molecules within podosome cores was assessed by FRAP in DCs transduced with retrovirus expressing GFP-actin. No significant differences were observed between WT and HS12/2 DCs, indicating that loss of HS1 does not affect actin turnover within pre-existing podo- somes (Supplemental Fig. 3D, Supplemental Videos 1, 2). Taken together, these data indicate that HS1 accelerates the early stages of podosome biogenesis, but is not essential for podosome for- mation or stability in DCs. HS1 is important for organizing the podosome array 2/2 by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. The most striking cytoarchitectural defects we observed in HS1 DCs involved podosome array organization. Podosomes in the HS12/2 DCs were not packed as tightly as those in WT DCs and the arrays were more randomly distributed throughout the cell (Fig. 3A, Supplemental Fig. 2). To assess differences in the po- sitioning of podosome arrays, cells were categorized into one of several groups: touching the leading edge (touch), behind the leading edge (behind), centrally located within the cell (middle), opposite the leading edge (back), lateral to the leading edge (side) http://classic.jimmunol.org or forming rosettes within the center of the cell not adjacent to any edges (rosettes). Images exemplifying each group are shown in Fig. 4C. Whereas WT DCs more frequently showed arrays that touched the leading edge, arrays in HS12/2 DCs tended to be further behind the leading edge (Fig. 4A). In addition to array 2 2 localization, array packing was affected (Fig. 4B, example images FIGURE 3. Podosome biogenesis and turnover in HS1 / BMDCs. A,

Downloaded from 2/2 shown Fig. 4D). Whereas podosomes in WT DCs tended to be WT and HS1 BMDCs were cultured on coverslips overnight, fixed, and 2/2 stained with phalloidin (red) and anti-vinculin (green) to visualize podo- tightly packed within the array, HS1 DCs showed more cells some cores and rings, respectively. DAPI (blue) was used to stain nuclei. with loosely packed podosome arrays (arrays with significant Images were captured by confocal microscopy. Arrows mark the leading space between adjacent podosomes). This qualitative finding is 2 2 edge of the cells. Scale bars, 10 mm. B, Cells were prepared and imaged as consistent with our quantitative data showing that HS1 / DCs in A. Podosome-containing cells were chosen at random, and the number have fewer podosomes than WT DCs, distributed in arrays that of podosomes per cell was counted as described in Materials and Methods. occupy a similar area (Fig. 3B, Supplemental Fig. 3E). 2/2 Each dot represents a single cell. C, WT and HS1 BMDCs cultured To verify that the phenotypes we observe do not reflect de- overnight on coverslips were treated for 30 min at 37˚C with cytochalasin velopmental changes in the HS12/2 mice and to ask whether these D, after which time the drug was washed out, and cells were allowed to results extend to other myeloid cell types, HS1 function was tested recover. At the indicated times, cells were fixed and labeled with phalloidin and anti-vinculin, and the percentage of cells containing podosome arrays in RAW/LR5 , a cell line that efficiently forms was determined. (n $ 200 cells/time point). D, Podosome lifetimes were podosomes (19, 57). Like the BMDCs, these cells express HS1 but calculated as described in Materials and Methods. Data represent .4000 not cortactin (data not shown). Fig. 5A shows that HS1 could be podosomes from 15 cells of each type with medians indicated with a line. efficiently silenced in these cells using shRNA. HS1-suppressed **p , 0.01, ****p , 0.0001. RAW/LR5 cells were able to form podosomes with normal 4810 HS1 AND WASp ORGANIZE PODOSOMES IN DCs by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd.

FIGURE 4. HS1 is required for efficient localization and organization of podosome arrays. WT and HS12/2 BMDCs were cultured on coverslips

http://classic.jimmunol.org overnight, fixed, and stained with phalloidin (red) and anti-vinculin (green) to visualize podosome cores and rings, respectively. DAPI (blue) was used to stain nuclei. Images were captured by confocal microscopy. A and B, Cells were scored for position and organization of the podosome array as described in Materials and Methods. C, Examples of podosome array localization as quantified in A. D, Examples of tight and loose packing of podosome arrays as quantified in B. C and D, Original magnification 3300.

frequency (data not shown), but the podosome array in these cells the effects of cortactin on lamellipodial dynamics in fibroblasts

Downloaded from became more loosely packed (Fig. 5B,5C). We conclude that HS1 (26). is not required for podosome formation or stability in myeloid HS1 interacts with WIP and WASp cells, but is required for proper organization of podosome arrays. 2/2 Cortactin interacts via its SH3 domain with the WASp/WIP het- HS1 DCs exhibit increased lamellipodial dynamics erodimer (59, 60), but the ability of HS1 to interact with these Because podosome formation and dissolution are mechanistically proteins has not been directly addressed. To test this, lysates from linked to lamellipodial dynamics (15, 17, 58), and HS1 is present cells expressing FLAG-tagged WASp or WIP were subjected to at the edges of lamellipodial protrusions (Fig. 2B), we asked pulldown assays using the GST-tagged HS1 SH3 domain. As whether lamellipodial dynamics are altered in HS12/2 DCs. DCs shown in Fig. 7A, both WASp and WIP interacted with the HS1 were transduced with GFP-Lifeact and monitored by video mi- SH3 domain. To ask whether these proteins can interact in intact croscopy and kymographic analysis. As shown in Fig. 6A, cells, we overexpressed FLAG-tagged HS1 with WASp and WIP lamellipodial protrusion and retraction events occurred over lon- (tagged with HA and myc, respectively) in 293T cells. As shown ger distances in HS12/2 DCs. The velocity of these events was in Fig. 7B, immunoprecipitation of HS1 led to coimmunoprecipi- also increased (Fig. 6B). These results are in good agreement with tation of both WIP and WASP, and mutation of the critical tryp- the effects of HS1 on lamellipodial dynamics in T cells (36) and tophan residue in the HS1 SH3 domain abolished this interaction. The Journal of Immunology 4811

FIGURE 6. HS12/2 DCs exhibit increased lamellipodial dynamics. WT and HS12/2 BMDCs were transduced with GFP-Lifeact to facilitate identification of lamellipodial boundaries, and lamellipodial dynamics were monitored by video microscopy as detailed in Materials and Meth- ods. On the basis of the kymographic analysis, the distance (A) and ve- locity (B) of lamellipodial protrusions and retractions was determined. Box and whiskers plots represent data from 30 lamellipodial protrusions for each cell type with horizontal lines representing median values. *p , 0.05, **p , 0.01, ***p , 0.005.

suggests that the actin regulatory portion of HS1 may limit ac- cessibility of the SH3 domain in the full-length molecule.

by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. WASp and WIP exist as a heterodimer in cells. To ask whether HS1 can bind WASp independently of WIP, cells were transfected with FLAG-tagged WASp or with a WASp deletion mutant that lacks the WIP binding region (D40–154). Because of its inability to bind WIP, this WASp mutant is unstable (not shown), but it is expressed at reasonable levels using this overexpression system. As shown in Fig. 7C, WT WASp binds well to the HS1 SH3 domain, but the WIP binding mutant does not. To test WIP-HS1 binding, similar studies were performed in cells transfected with http://classic.jimmunol.org FLAG-tagged WIP or WIP mutants that fail to bind WASp (P436A or D460). The two WIP mutants failed to bind efficiently to WASp, but interacted strongly with the HS1 SH3 domain, in- dicating that WIP binding to HS1 is independent of WASp (Fig. 7D). Finally, cell lysates from FLAG-WIP expressing cells were FIGURE 5. Suppression of HS1 perturbs the organization of podosome probed with recombinant WT HS1 SH3 domain or the inactive

Downloaded from arrays in RAW macrophages. RAW/LR5 cells were untransduced or tryptophan mutant using a gel overlay approach. As shown in Fig. transduced with control or shHS1 retrovirus. A, Whole-cell lysates were 7E, the WT SH3 domain, but not the mutant, bound specifically to immunoblotted with anti-HS1 Ab and with anti-GAPDH to verify equal WIP. This confirms direct interaction between HS1 and WIP. loading. B and C, Cells were cultured on coverslips overnight, fixed, and Taken together, these studies indicate that WIP binds directly to stained with phalloidin (red) and anti-vinculin (green) to visualize podo- the HS1 SH3 domain and mediates indirect interactions between some cores and rings, respectively. DAPI (blue) was used to stain the HS1 and WASp. nucleus. Images were captured by confocal microscopy, and the organi- zation of the podosome arrays was determined as described in Materials HS1 and WASp carry out distinct roles in podosome formation and Methods. Original magnification 31000. *p , 0.05. and organization Like HS1, WASp is involved in podosome formation and lamel- Interestingly, deletion of the N-terminal half of HS1 enhanced lipodial protrusion (12, 16, 18, 19, 58), and HS1 is thought to binding to both WASp and WIP. Because this region contains the stabilize branched actin filaments generated by WASp (35, 36). To binding sites for Arp2/3 complex and F-actin, this demonstrates investigate the functional relationship between HS1 and WASp in that binding of HS1 to WASp and WIP does not require mutual DCs, we compared the phenotypes of DCs cultured from mice binding to F-actin. Moreover, the enhanced binding to this mutant lacking HS1 alone, WASp alone, or both proteins. DCs cultured 4812 HS1 AND WASp ORGANIZE PODOSOMES IN DCs

FIGURE 7. The SH3 domain of HS1 binds to the WIP/WASp heterodimer. A, WASp and WIP interact with the HS1 SH3 domain. Cells were transfected with empty vector or FLAG-tagged WIP (F.WIP) or WASP (F.WASP), and lysates were incubated with GST-tagged HS1 SH3 domain (SH3) or GST alone. Bound WIP and WASP were detected via anti-FLAG immunoblot. Bottom panel, Coomassie stain. B, WASp and WIP coimmunoprecipitate with HS1. Cells were cotransfected with myc-WIP and HA-WASp, together with FLAG-tagged full length HS1 (F.HS1), the C- terminal half of HS1 (F.HS1DN), or the corresponding SH3 domain mutants (F.HS1 W→Y or F.HS1DN W→Y). Lysates were immunoprecipitated with anti- FLAG and blotted with the indicated Abs. C, HS1 does not interact with a WASp mutant that cannot bind WIP. Cells were transfected with FLAG-tagged WASp or with a mutant that fails to bind WIP (D40–154). Lysates were incubated with GST-tagged HS1 SH3 domain and blotted with anti-FLAG. Bottom panel, Coomassie stain. D, HS1 binds to WIP mutants that cannot bind WASp. Cells were transfected with FLAG- tagged WIP or with mutants that fail to bind WASp (P436A or D460). Lysates were incubated with GST- tagged HS1 SH3 domain and blotted with anti-FLAG or immunoprecipitated with anti-FLAG and blotted with the indicated Abs. Arrow, FLAG-WIPD460; HC, IgH. E, WIP interacts directly with the HS1 SH3 do- main. Cells were transfected with empty vector or FLAG-tagged WIP, and lysates were probed by gel overlay with recombinant GST alone, WT GST-HS1 SH3 domain, or the SH3 domain mutant.

by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. from these mice exhibited loss of the appropriate proteins, and WASp also significantly increases the number of double-deficient knockout of one had no effect on the expression levels of the other DCs displaying podosomes. In contrast, expression of HS1 (gray (Fig. 8A). As shown in the black bars in Fig. 8B, HS12/2 and bars) in double-deficient DCs does not rescue this defect, sup- WASp2/y DCs differed with respect to the proportion of cells porting the idea that WASp, but not HS1, is essential for efficient exhibiting podosomes. Although significantly fewer WASp- podosome formation. deficient cells displayed podosomes, HS1-deficiency had no ef- When transduced DCs were analyzed with respect to podosome fect on this parameter (the slight increase relative to WT cells in packing, reciprocal results were obtained (Fig. 8C). The abnor- this experiment was not reproducible). The defect in podosome mally loose packing observed in HS12/2 DCs and DKO cells was formation in WASp2/y DCs is consistent with previous reports rescued by ectopic expression of HS1 (gray bars). Expression of http://classic.jimmunol.org (61), although the magnitude of the defect is somewhat less severe GFP-WASp (hatched bars) in DKO DCs did not rescue this defect, in our hands. It has been proposed that HS1 may contribute to supporting the idea that HS1, but not WASp, is needed to organize residual podosome formation in WASP2/y DCs (62); however, we a closely packed podosome array. Interestingly, the HS1 SH3 found that DCs deficient for both HS1 and WASp were in- domain mutant (open bar) was unable to rescue podosome orga- distinguishable from cells deficient for WASp alone. We next nization in HS12/2 DCs, suggesting that interactions mediated by compared the effects of loss of HS1 or WASp with respect to the SH3 domain are required for HS1 function.

Downloaded from podosome organization. As shown in the black bars in Fig. 8C, HS12/2 DCs showed defective packing of the podosome array, Recruitment of HS1 to podosomes is dependent on interactions a phenotype that was not observed in WASp-deficient cells. with WASp Podosome packing in DCs deficient in both HS1 and WASp was To complement our functional analysis of HS1 and WASp inter- indistinguishable from packing in cells lacking HS1 alone. Taken actions, we asked whether these proteins depend on one another for together, these results show that WASp is required for efficient recruitment to podosomes. As shown in Fig. 9A and 9C, Venus- formation of podosomes, whereas HS1 is important for organizing HS1 localized efficiently to podosome cores when expressed in the podosome array. HS12/2 DCs. Costaining with anti-phosphotyrosine or anti- To confirm these findings and to ask whether HS1 and WASp vinculin showed that Venus-HS1 exhibits podosome core locali- show interdependent function, DCs lacking these proteins in- zation similar to that observed with endogenous HS1 (Supple- dividually or together were transduced with WASp, HS1, or with mental Fig. 4). In DKO DCs, however, Venus-HS1 localization to the HS1 SH3 domain mutant that abrogates interaction of HS1 with podosome cores was faint or nonexistent, and the protein instead the WIP/WASp heterodimer. As shown in Fig. 8B, hatched bars, exhibited a diffuse cytoplasmic distribution (Fig. 9A,9C). In- ectopic expression of WASp restores the number of WASP2/y DCs terestingly, HS1 was sometimes apparent at lamellipodial pro- cells displaying podosomes to WT frequency. Transfection with trusions in these cells (compare Fig. 9C, HS1 in DKO), suggesting The Journal of Immunology 4813

FIGURE 9. HS1 requires WASp for localization to podosome cores, but WASp localizes independently of HS1. HS12/2, WASp2/y, or DKO cells were untransduced or transduced with Venus-HS1 (HS12/2 and DKO), GFP-WASp (WASp2/y and DKO), or Venus-HS1W465Y (HS12/2). Cells were prepared as in Fig. 3A but stained with anti-GFP to visualize the transduced proteins. A, Cells were scored for the presence of HS1 in podosome cores as described in Materials and Methods (**p , 0.01). B, Cells were scored for the presence of WASp in podosome cores as de- by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. scribed in Materials and Methods. C, Colocalization of HS1 or WASp with FIGURE 8. HS1 and WASp cooperate to form organized podosome phalloidin staining in podosomes cores was visualized by confocal mi- 2/2 2/y 2/2 arrays. A, BMDCs were cultured from WT, HS1 , WASp , or HS1 croscopy. Insets, Enlargements of the indicated regions. Scale bars, 10 mm. WASp2/y (DKO) mice, and whole-cell lysates were analyzed by immu- noblotting with anti-HS1 or anti-WASp Abs. GAPDH was used to verify 2 2 equal loading. B and C, WT, HS12/2, WASp2/y, or DKO cells were (MIP3b), respectively (Fig. 10A,10B). HS1 / DCs migrated 2 2 untransduced or transduced with Venus-HS1 (HS1 / and DKO, gray), with the same efficiency as DCs from WT mice and showed the 2/y W465Y 2/2 GFP-WASp (WASp and DKO, hatched), or Venus-HS1 (HS1 , same switch in chemokine sensitivity with maturation. Although white) and prepared as in Fig. 3A. Cells were scored for the presence of immature DCs migrated slightly less well to CXCL12 in the assay podosomes (B) and array organization in cells containing podosomes (C) http://classic.jimmunol.org shown Fig. 10A, this difference was not statistically significant and as described in Materials and Methods.*p , 0.05, **p , 0.01 compared was not reproducible over multiple assays. Migration of WT and with WT cells. HS12/2 DCs toward CCL21 (SLC) was very similar to migration toward CCL19 (data not shown). Furthermore, WT and HS12/2 that the requirements for podosome localization and lamellipodial DCs exhibited similar sensitivity in chemokine dose-response localization may differ. Similar results were obtained when the studies, and flow cytometry analysis showed that these two pop- 2/2 Downloaded from SH3 domain mutant of HS1 was expressed in HS1 DCs. As ulations express similar surface levels of chemokine receptors showninFig.9B and 9C, WASp localized efficiently to the (data not shown). podosome core when expressed in either WASp2/y or DKO cells. Transwell assays measure only end-point effects and can fail to Taken together, these results indicate that WASp localization to detect defects in directional migration. Thus, we used video mi- 2/2 2/y podosome cores is independent of HS1, but HS1 localization to croscopy to compare the ability of mature WT, HS1 , WASp podosome cores depends on SH3 domain-mediated interactions and DKO DCs to undergo chemotaxis in a gradient of CCL19. with the WIP/WASp heterodimer. Only mature DCs were studied using this assay, because immature 2/2 cells were tightly adherent to the microfluidic chamber and largely HS1 DCs exhibit defects in directional migration immotile. As shown in Fig. 10C and 10D and Supplemental 2 2 Podosomes are thought to promote cell migration in some settings, Videos 3–6, HS1 / DCs moved significantly faster than WT 2 and WASp2/y DCs are defective in migration (13, 14, 63). We DCs, although WASp /y DCs and DKO cells moved significantly 2 2 therefore asked whether HS1 is also required for DC migration. slower. Analysis of directionality revealed HS1 / DCs, like Initial studies were performed using transwell assays. As reported WASp-/y DCs, exhibit diminished directional persistence (che- previously (64), immature and mature BMDCs from WT mice motactic index) (Fig. 10C,10E). The magnitude of this defect was 2 preferentially migrated toward CXCL12 (SDF1a) and CCL19 greater in WASp /y and DKO DCs, but all three mutants were 4814 HS1 AND WASp ORGANIZE PODOSOMES IN DCs by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. http://classic.jimmunol.org

FIGURE 10. HS12/2 BMDCs show altered migration in a chemokine gradient. A and B, WT and HS12/2 BMDCs were treated with 100 ng/ml LPS (mature) or left untreated (immature). Migration toward media alone or media containing CXCL12 (A) or CCL19 (B) was assessed by Transwell assay. The percentage of cells that migrated was determined by dividing the number of cells in the bottom well by the number of input cells. Data are means 6 SD of replicates from one representative experiment (out of four). C–E, WT, HS12/2, WASp2/y, or DKO BMDCs were injected into a fibronectin-coated microfluidic chamber with a gradient of CCL19 (0–20 nM). Cells were allowed to settle and loosely adhered cells were cleared from the chamber. Phase

Downloaded from contrast images were collected every minute for 1 h, migration was analyzed, and trajectory plots of 100 cells were analyzed with positive movement along the y-axis corresponding to movement up the gradient (C). The average velocity of motile cells in C was determined (D). The chemotactic index, defined as the distance migrated toward the chemokine source divided by the absolute distance traveled for each motile cell in C, was calculated (E). Data represent averages of multiple cells 6 SEM. ***p , 0.00001.

significantly less persistent than WT DCs. Taken together, these express cortactin; however, megakaryocytes, platelets, and osteo- data show that although WASp is required for overall DC mi- clasts express both proteins (65, 66). Thus, an important starting gration as well as directional persistence, the primary role of HS1 question was whether these cells express HS1, cortactin, or both. is to promote persistent directional migration. Using carefully controlled non–cross-reactive Abs, we find that HS1 is the only cortactin family member present in murine BMDCs. Discussion Because cortactin expression has been reported in murine splenic This study addresses for the first time, to our knowledge, the function DCs (10, 17), this raises the possibility that these related, but dis- of the cortactin family member HS1 in DCs. In general, hemato- tinct, cortactin family members are differentially expressed in DC poietic lineage cells express HS1, whereas nonhematopoietic cells subsets. The Journal of Immunology 4815

Our analysis of BMDCs from HS12/2 mice revealed three re- 76). Function of HS1 in podosomes and at the lamellipodial edge lated defects: disorganization of the podosome array, altered need not be mutually exclusive processes. On the contrary, HS1 is lamellipodial dynamics, and diminished directional migration. likely to function at both sites, with outcomes that are functionally WASp, which interacts indirectly with HS1, also affects these intertwined via a feedback process. Because forward movement of processes, but our analysis shows that the roles of these two the DC lamellipodium is closely linked to the cycle of podosome proteins are distinct. It is well established that WASp and its ob- formation and dissolution (12, 15, 24, 68, 71), erratic leading edge ligate binding partner, WIP, are essential for formation of the dynamics in HS1-deficient DCs could result in disorganization of actin-rich cores that nucleate podosome biogenesis (10, 12, 15–17, the podosome array. Indeed, this seems the likeliest mechanism to 19, 24, 61, 67–71). Depending on the experimental system, create the observed mislocalization of the array with respect to the WASp-deficient DCs and macrophages either lack podosomes leading edge of the cell. Conversely, it is thought that the podo- altogether or show severe reductions in podosome numbers, and some array stabilizes the dominant leading edge of the cell, such our data are consistent with this. In contrast, we find that HS1- that a disordered or misplaced podosome array may lead to deficient DCs and macrophages can form podosomes containing lamellipodial instability, and diminished directional persistence many, if not all, of the characteristic components. However, HS1- during migration. deficient cells show disordered podosome array packing and The importance of HS1 function in podosomes and at the mislocalization of the arrays with respect to the leading edge of leading edge of the cell is demonstrated by the diminished ability the cell. Similar effects were noted in a recent study in which of HS12/2 DCs to undergo directional chemotaxis. In this study, WIP2/2 DCs were reconstituted with a WIP mutant lacking the too, the phenotypes of HS12/2 and WASp2/y DCs are related but HS1/cortactin binding site (10). Thus, we conclude that HS1 is not distinct. As reported previously (13, 14, 63), we found that WASp required for podosome formation, but rather for organization of expression is essential for DC migration. WASp2/y DCs migrating the podosome array. in a chemokine gradient showed a large decrease in velocity, and The mechanisms through which HS1 controls podosome array those cells that did migrate exhibited diminished directional per- organization are unclear. Because HS1 stabilizes branched-actin sistence. In contrast, HS1-deficient DCs actually migrated faster filaments generated by WASp and other Arp2/3 complex activa- than WT cells, but directional persistence was significantly re- tors, it seems likely that it stabilizes actin filaments within duced. The defects in directional persistence in HS12/2 cells may podosomes. Although our FRAP data show that loss of HS1 does reflect the increased lamellipodial instability in these cells. Al- not affect actin exchange in mature podosomes, it does delay ternatively, the defects in directional persistence may reflect the podosome reformation. Thus, HS1 may stabilize newly formed role of podosomes in stabilizing a dominant leading edge (15, 17, actin cores, such that the diminished numbers and loose packing of 58). In this scenario, HS1 would function to fine-tune the packing podosomes in HS1-deficient cells could result from stochastic and localization of podosomes formed by WASp to aid the sta- disassembly of some newly formed podosome cores within the bilization of the leading edge, promoting efficient directional cell array. This interpretation is consistent with the modest decrease in migration. These two possibilities are not mutually exclusive and, average podosome lifetime in HS12/2 DCs; this may represent in fact, are likely to represent intertwined aspects of HS1 function. increased instability of a small number of podosomes. Another Our data point to a hierarchical relationship between WASp and

by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. appealing possibility is that HS1 aids in stabilizing the long actin HS1 in controlling DC actin dynamics. HS1 interaction with WASp filaments that form connections between adjacent podosomes (72). is indirect and involves binding of the HS1 SH3 domain to WIP. Interestingly, these interconnecting filaments are frequently dec- Using add-back experiments, we found that WASp can localize to orated with clathrin coated endocytic pits (72), and the HS1 ho- podosomes independently of HS1, but HS1 is not recruited effi- molog cortactin associates with coated vesicle components (27). ciently to podosomes in the absence of WASp or if its WIP-binding Finally, it should be noted that in addition to directly regulating SH3 domain is mutated. This indicates that HS1 is recruited to actin dynamics, HS1 functions as an adaptor molecule and can podosomes through SH3-domain-dependent interactions with the recruit other signaling molecules to sites of actin polymerization WIP/WASp complex. These results are consistent with a recent (36, 37). We show in this study that HS1 is not needed for re- study showing that cortactin fails to localize to podosomes in 2/2 http://classic.jimmunol.org cruitment of WASp to podosomes, but HS1 could recruit Vav1 WIP DCs and that the proline rich region of WIP responsible or phospholipase Cg, proteins that are important regulators of for binding to cortactin’s SH3 domain is important for podosome podosome dynamics and directional persistence in DCs (22, 73). architechture (10). Indeed, WIP seems to play a key role in In addition to podosomes, HS1 is enriched at lamellipodial edges podosome assembly, because WASp also fails to localize to in DCs. HS1 was sometimes enriched in lamellipodial protrusions podosomes in WIP2/2 DCs (17). Given our data showing that in the absence of WASp (see Fig. 9C, second row), but we were HS1 binds directly to WIP rather than to WASp, it would be in- 2/Y

Downloaded from unable to quantify the frequency of this localization pattern. The teresting to ask whether WASP DCs fail to correctly localize mechanism through which HS1 is localized to lamellipodial pro- WIP. trustions is not known. In addition to binding to the WIP/WASp Our functional studies also support the view that the WASp/WIP heterodimer, the SH3 domain of HS1 can interact with other heterodimer serves a central role in podosome biogenesis, whereas proteins, including Src kinases and Dynamin 2 (Ref. 25 and D.A. HS1 fine-tunes podosome array organization. In cells lacking both Klos Dehring, unpublished observations). In other systems, in- WASp and HS1, the defects in both cell morphology and che- teraction of phosphotyrosines with Src kinases and other SH2 motaxis were indistinguishable from cells deficient for WASp domain-containing molecules has been shown to mediate plasma alone. One finding, however, suggests that HS1 function may be at membrane targeting (36, 41, 74, 75). Finally, actin binding could least partially independent of WASp. In analyzing the 30–40% of be involved. HS12/2 DCs show increased protrusion and re- DCs that generated podosomes in the absence of WASp, we ob- traction distance and velocity, indicating that HS1 functions to served loose packing of podosome arrays if HS1 was also absent, stabilize lamellipodial dynamics in DCs. This finding is consistent but tight packing of podosome arrays if HS1 was expressed (either with our previous work showing that HS1 stabilizes lamellipodial WASp single knockout cells or DKO cells transduced with HS1). protrusions in T cells (36) and with studies showing that cortactin This result is particularly surprising given our finding that HS1 stabilizes lamellipodial protrusion in nonhematopoietic cells (26, fails to localize to podosomes efficiently in the absence of WASp. 4816 HS1 AND WASp ORGANIZE PODOSOMES IN DCs

This apparent discrepancy may reflect WASp-independent HS1 Acknowledgments function at the leading edge. Alternatively, it may reflect the We thank Dr. D. Cox for providing materials and guidance for studies in ability of HS1 to interact weakly with podosomes by binding to F- RAW/LR5 macrophages. We also thank Drs. T. Laufer, S. Gallucci, and actin. Evidence that such binding occurs is shown by the higher P. Oliver and members of the Oliver and Burkhardt laboratories for helpful frequency of podosome localization of the HS1 SH3 domain discussions and critical reading of the manuscript. mutant in WASp-sufficient cells as compared with WT HS1 in WASp2/y DCs (see Fig. 9A). Disclosures 2 2 The mild podosome phenotype we observe in HS1 / DCs is The authors have no financial conflicts of interest. somewhat surprising given that cortactin is essential for formation of invadopodia in metastatic tumor cells (77, 78). Although we cannot exclude the possibility that HS12/2 mice undergo com- References 1. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu, pensatory developmental changes that blunt the DC phenotype, we B. Pulendran, and K. Palucka. 2000. Immunobiology of dendritic cells. Annu. deem this unlikely because we found no upregulation of WASp or Rev. Immunol. 18: 767–811. cortactin, and because similar defects were observed with HS1 2. Trombetta, E. S., and I. Mellman. 2005. Cell biology of antigen processing in vitro and in vivo. Annu. Rev. Immunol. 23: 975–1028. shRNA in a cell line that also lacks cortactin. A more 3. Alvarez, D., E. H. Vollmann, and U. H. von Andrian. 2008. Mechanisms and likely possibility is that HS1 and cortactin are functionally dis- consequences of dendritic cell migration. Immunity 29: 325–342. 4. La¨mmermann, T., B. L. Bader, S. J. Monkley, T. Worbs, R. Wedlich-So¨ldner, tinct. Because HS1 is expressed in hematopoietic cells that gen- K. Hirsch, M. Keller, R. Fo¨rster, D. R. Critchley, R. Fa¨ssler, and M. Sixt. 2008. erate podosomes, whereas cortactin is typically expressed in Rapid leukocyte migration by integrin-independent flowing and squeezing. nonhematopoietic cells that generate invadopodia, it will be in- Nature 453: 51–55. 5. Renkawitz, J., K. Schumann, M. Weber, T. La¨mmermann, H. Pflicke, M. Piel, teresting to explore the differential role of these proteins in gen- J. Polleux, J. P. Spatz, and M. Sixt. 2009. Adaptive force transmission in erating podosomes versus invadopodia. Interestingly, cortactin is amoeboid cell migration. Nat. Cell Biol. 11: 1438–1443. required for formation of podosomes in , hematopoietic 6. Gimona, M., R. Buccione, S. A. Courtneidge, and S. Linder. 2008. Assembly and biological role of podosomes and invadopodia. Curr. Opin. Cell Biol. 20: 235–241. lineage cells that express both HS1 and cortactin (66). Although 7. Linder, S., and P. Kopp. 2005. Podosomes at a glance. J. Cell Sci. 118: 2079– this may represent an exception to the podosome/invadopodium 2082. 8. Linder, S., and M. Aepfelbacher. 2003. Podosomes: adhesion hot-spots of in- distinction, podosomes resemble invadopodia in that vasive cells. Trends Cell Biol. 13: 376–385. they are key sites of matrix degradation. This points to another 9. Albiges-Rizo, C., O. Destaing, B. Fourcade, E. Planus, and M. R. Block. 2009. interesting difference between HS1 and cortactin. Although cor- Actin machinery and mechanosensitivity in invadopodia, podosomes and focal adhesions. J. Cell Sci. 122: 3037–3049. tactin is required for matrix metalloproteinase release at invado- 10. Banon-Rodriguez, I., J. Monypenny, C. Ragazzini, A. Franco, Y. Calle, podia in nonhematopoietic cells (79) and at podosomes in splenic G. E. Jones, and I. M. Anton. 2011. The cortactin-binding domain of WIP is DCs (10), we found no requirement for HS1 in metalloproteinase essential for podosome formation and extracellular matrix degradation by mu- rine dendritic cells. Eur. J. Cell Biol. release in BMDCs. It will be interesting to explore these dis- 11. Gawden-Bone, C., Z. Zhou, E. King, A. Prescott, C. Watts, and J. Lucocq. 2010. 2 2 tinctions by asking whether it is possible to rescue HS1 / cells Dendritic cell podosomes are protrusive and invade the extracellular matrix with cortactin and vice versa. using metalloproteinase MMP-14. J. Cell Sci. 123: 1427–1437. 12. Burns, S., A. J. Thrasher, M. P. Blundell, L. Machesky, and G. E. Jones. 2001. The relationship between HS1 and WASp defined in this study is Configuration of human dendritic cell cytoskeleton by Rho GTPases, the WAS

by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. also somewhat different from the relationship between cortactin protein, and differentiation. Blood 98: 1142–1149. 13. Snapper, S. B., P. Meelu, D. Nguyen, B. M. Stockton, P. Bozza, F. W. Alt, and N-WASp in other cell types. Although we find that WASp F. S. Rosen, U. H. von Andrian, and C. Klein. 2005. WASP deficiency leads to recruits HS1 to podosomes, cortactin has been shown to recruit and global defects of directed leukocyte migration in vitro and in vivo. J. Leukoc. promote N-WASp activity at sites of actin polymerization (80, 81). Biol. 77: 993–998. 14. de Noronha, S., S. Hardy, J. Sinclair, M. P. Blundell, J. Strid, O. Schulz, Moreover, we find that the WIP-binding SH3 domain of HS1 is J. Zwirner, G. E. Jones, D. R. Katz, C. Kinnon, and A. J. Thrasher. 2005. Im- needed for recruitment to podosomes in DCs, but this domain of paired dendritic-cell homing in vivo in the absence of Wiskott-Aldrich syndrome cortactin has been shown to be dispensible for podosome targeting protein. Blood 105: 1590–1597. 15. Monypenny, J., H. C. Chou, I. Banon-Rodriguez, A. J. Thrasher, I. M. Anton, in osteoclasts (66). Phosphorylation of cortactin has been shown to G. E. Jones, and Y. Calle. 2011. Role of WASP in cell polarity and podosome play an important role in its ability to regulate N-WASP (38, 39, dynamics of myeloid cells. Eur. J. Cell Biol. 16. Calle, Y., H. C. Chou, A. J. Thrasher, and G. E. Jones. 2004. Wiskott-Aldrich http://classic.jimmunol.org 77, 82). Phosphorylation of HS1 is important for its actin- syndrome protein and the cytoskeletal dynamics of dendritic cells. J. Pathol. regulatory function in T cells and NK cells (36, 41), but its role 204: 460–469. in DCs remains to be explored. 17. Chou, H. C., I. M. Anto´n, M. R. Holt, C. Curcio, S. Lanzardo, A. Worth, S. Burns, A. J. Thrasher, G. E. Jones, and Y. Calle. 2006. WIP regulates the An important open question in this field is the extent to which 2 stability and localization of WASP to podosomes in migrating dendritic cells. podosomes are important for DC function in vivo. WASP /y DCs Curr. Biol. 16: 2337–2344. have significant migration defects in vivo, but it is unclear to 18. Linder, S., D. Nelson, M. Weiss, and M. Aepfelbacher. 1999. Wiskott-Aldrich syndrome protein regulates podosomes in primary human macrophages. Proc.

Downloaded from what extent this reflects a requirement for podosome formation. It Natl. Acad. Sci. USA 96: 9648–9653. has long been assumed that podosomes are sites for integrin- 19. Dovas, A., J. C. Gevrey, A. Grossi, H. Park, W. Abou-Kheir, and D. Cox. 2009. Regulation of podosome dynamics by WASp phosphorylation: implication in dependent adhesion to the extracellular matrix, but the impor- matrix degradation and chemotaxis in macrophages. J. Cell Sci. 122: 3873–3882. tance of integrins in regulating DC migration is complex and 20. Goley, E. D., and M. D. Welch. 2006. The ARP2/3 complex: an actin nucleator highly dependent on environmental cues (4, 5, 83, 84). In this comes of age. Nat. Rev. Mol. Cell Biol. 7: 713–726. 21. La¨mmermann, T., J. Renkawitz, X. Wu, K. Hirsch, C. Brakebusch, and M. Sixt. context, an appealing possibility is that these structures are im- 2009. Cdc42-dependent leading edge coordination is essential for interstitial portant as mechanosensors, to allow DCs to adapt to movement dendritic cell migration. Blood 113: 5703–5710. along variable surfaces (9, 21, 85). Finally, because podosomes 22. Spurrell, D. R., N. A. Luckashenak, D. C. Minney, A. Chaplin, J. M. Penninger, R. S. Liwski, J. L. Clements, and K. A. West. 2009. Vav1 regulates the migration are most prominent in immature DCs, these structures may a play and adhesion of dendritic cells. J. Immunol. 183: 310–318. an important role in maintaining cell anchorage and/or dynamics 23. Destaing, O., F. Saltel, J. C. Ge´minard, P. Jurdic, and F. Bard. 2003. Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing of dendritic processes in peripheral tissues. By identifying and actin-green fluorescent protein. Mol. Biol. Cell 14: 407–416. characterizing individual proteins that control distinct aspects 24. Calle, Y., N. O. Carragher, A. J. Thrasher, and G. E. Jones. 2006. Inhibition of of podosome function, we will have a better understanding of calpain stabilises podosomes and impairs dendritic cell motility. J. Cell Sci. 119: 2375–2385. whether and how these structures contribute to the regulation of 25. Takemoto, Y., M. Sato, M. Furuta, and Y. Hashimoto. 1996. Distinct binding DC movements during an in vivo immune response. patterns of HS1 to the Src SH2 and SH3 domains reflect possible mechanisms of The Journal of Immunology 4817

recruitment and activation of downstream molecules. Int. Immunol. 8: 1699– 52. Jannat, R. A., M. Dembo, and D. A. Hammer. 2010. Neutrophil adhesion and 1705. chemotaxis depend on substrate mechanics. J. Phys. Condens. Matter 22: 26. Bryce, N. S., E. S. Clark, J. L. Leysath, J. D. Currie, D. J. Webb, and 194117. A. M. Weaver. 2005. Cortactin promotes cell motility by enhancing lamellipo- 53. Matheu, M. P., D. Sen, M. D. Cahalan, and I. Parker. 2008. Generation of bone dial persistence. Curr. Biol. 15: 1276–1285. marrow derived murine dendritic cells for use in 2-photon imaging. J. Vis. Exp. 27. Cao, H., J. D. Orth, J. Chen, S. G. Weller, J. E. Heuser, and M. A. McNiven. 17: e773 . 2003. Cortactin is a component of clathrin-coated pits and participates in 54. Quast, T., B. Tappertzhofen, C. Schild, J. Grell, N. Czeloth, R. Fo¨rster, R. Alon, receptor-mediated endocytosis. Mol. Cell. Biol. 23: 2162–2170. L. Fraemohs, K. Dreck, C. Weber, et al. 2009. Cytohesin-1 controls the acti- 28. Lai, F. P., M. Szczodrak, J. M. Oelkers, M. Ladwein, F. Acconcia, S. Benesch, vation of RhoA and modulates integrin-dependent adhesion and migration of S. Auinger, J. Faix, J. V. Small, S. Polo, et al. 2009. Cortactin promotes mi- dendritic cells. Blood 113: 5801–5810. gration and platelet-derived growth factor-induced actin reorganization by sig- 55. Ammer, A. G., and S. A. Weed. 2008. Cortactin branches out: roles in regulating naling to Rho-GTPases. Mol. Biol. Cell 20: 3209–3223. protrusive actin dynamics. Cell Motil. Cytoskeleton 65: 687–707. 29. van Rossum, A. G., W. H. Moolenaar, and E. Schuuring. 2006. Cortactin affects 56. Linder, S. 2007. The matrix corroded: podosomes and invadopodia in extra- cell migration by regulating intercellular adhesion and cell spreading. Exp. Cell cellular matrix degradation. Trends Cell Biol. 17: 107–117. Res. 312: 1658–1670. 57. Isaac, B. M., D. Ishihara, L. M. Nusblat, J. C. Gevrey, A. Dovas, J. Condeelis, 30. Weaver, A. M. 2008. Cortactin in tumor invasiveness. Lett. 265: 157– and D. Cox. 2010. N-WASP has the ability to compensate for the loss of WASP 166. in macrophage podosome formation and chemotaxis. Exp. Cell Res. 316: 3406– 31. Buday, L., and J. Downward. 2007. Roles of cortactin in tumor pathogenesis. 3416. Biochim. Biophys. Acta 1775: 263–273. 58. Burns, S., S. J. Hardy, J. Buddle, K. L. Yong, G. E. Jones, and A. J. Thrasher. 32. Artym, V. V., K. Matsumoto, S. C. Mueller, and K. M. Yamada. 2011. Dynamic 2004. Maturation of DC is associated with changes in motile characteristics and membrane remodeling at invadopodia differentiates invadopodia from podo- adherence. Cell Motil. Cytoskeleton 57: 118–132. somes. Eur. J. Cell Biol. 59. Mizutani, K., H. Miki, H. He, H. Maruta, and T. Takenawa. 2002. Essential role 33. Weed, S. A., A. V. Karginov, D. A. Schafer, A. M. Weaver, A. W. Kinley, of neural Wiskott-Aldrich syndrome protein in podosome formation and deg- J. A. Cooper, and J. T. Parsons. 2000. Cortactin localization to sites of actin radation of extracellular matrix in src-transformed fibroblasts. Cancer Res. 62: assembly in lamellipodia requires interactions with F-actin and the Arp2/3 669–674. complex. J. Cell Biol. 151: 29–40. 60. Kinley, A. W., S. A. Weed, A. M. Weaver, A. V. Karginov, E. Bissonette, 34. Higgs, H. 2001. Branching out: cortactin stabilizes actin networks generated by J. A. Cooper, and J. T. Parsons. 2003. Cortactin interacts with WIP in regulating the Arp2/3 complex. Trends Biochem. Sci. 26: 219. Arp2/3 activation and membrane protrusion. Curr. Biol. 13: 384–393. 35. Uruno, T., P. Zhang, J. Liu, J. J. Hao, and X. Zhan. 2003. Haematopoietic lineage 61. Olivier, A., L. Jeanson-Leh, G. Bouma, D. Compagno, J. Blondeau, K. Seye, cell-specific protein 1 (HS1) promotes actin-related protein (Arp) 2/3 complex- S. Charrier, S. Burns, A. J. Thrasher, O. Danos, et al. 2006. A partial down- mediated actin polymerization. Biochem. J. 371: 485–493. regulation of WASP is sufficient to inhibit podosome formation in dendritic cells. 36. Gomez, T. S., S. D. McCarney, E. Carrizosa, C. M. Labno, E. O. Comiskey, Mol. Ther. 13: 729–737. J. C. Nolz, P. Zhu, B. D. Freedman, M. R. Clark, D. J. Rawlings, et al. 2006. HS1 62. Calle, Y., I. M. Anto´n, A. J. Thrasher, and G. E. Jones. 2008. WASP and WIP functions as an essential actin-regulatory adaptor protein at the immune synapse. regulate podosomes in migrating leukocytes. J. Microsc. 231: 494–505. Immunity 24: 741–752. 63. Bouma, G., S. Burns, and A. J. Thrasher. 2007. Impaired T-cell priming in vivo 37. Carrizosa, E., T. S. Gomez, C. M. Labno, D. A. Klos Dehring, X. Liu, resulting from dysfunction of WASp-deficient dendritic cells. Blood 110: 4278– B. D. Freedman, D. D. Billadeau, and J. K. Burkhardt. 2009. Hematopoietic 4284. lineage cell-specific protein 1 is recruited to the immunological synapse by IL-2– 64. Vecchi, A., L. Massimiliano, S. Ramponi, W. Luini, S. Bernasconi, R. Bonecchi, inducible T cell kinase and regulates phospholipase Cg1 Microcluster dynamics P. Allavena, M. Parmentier, A. Mantovani, and S. Sozzani. 1999. Differential during T cell spreading. J. Immunol. 183: 7352–7361. responsiveness to constitutive vs. inducible chemokines of immature and mature 38. Martinez-Quiles, N., H. Y. Ho, M. W. Kirschner, N. Ramesh, and R. S. Geha. mouse dendritic cells. J. Leukoc. Biol. 66: 489–494. 2004. Erk/Src phosphorylation of cortactin acts as a switch on-switch off 65. Zhan, X., C. C. Haudenschild, Y. Ni, E. Smith, and C. Huang. 1997. Upregu- mechanism that controls its ability to activate N-WASP. Mol. Cell. Biol. 24: lation of cortactin expression during the maturation of megakaryocytes. Blood 5269–5280. 89: 457–464. 39. Tehrani, S., N. Tomasevic, S. Weed, R. Sakowicz, and J. A. Cooper. 2007. Src 66. Tehrani, S., R. Faccio, I. Chandrasekar, F. P. Ross, and J. A. Cooper. 2006. phosphorylation of cortactin enhances actin assembly. Proc. Natl. Acad. Sci. Cortactin has an essential and specific role in osteoclast actin assembly. Mol.

by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. USA 104: 11933–11938. Biol. Cell 17: 2882–2895. 40. Scielzo, C., M. T. Bertilaccio, G. Simonetti, A. Dagklis, E. ten Hacken, C. Fazi, 67. Charrier, S., D. Stockholm, K. Seye, P. Opolon, M. Taveau, D. A. Gross, M. Muzio, V. Caiolfa, D. Kitamura, U. Restuccia, et al. 2010. HS1 has a central S. Bucher-Laurent, C. Delenda, W. Vainchenker, O. Danos, and A. Galy. 2005. A role in the trafficking and homing of leukemic B cells. Blood 116: 3537–3546. lentiviral vector encoding the human Wiskott-Aldrich syndrome protein corrects 41. Butler, B., D. H. Kastendieck, and J. A. Cooper. 2008. Differently phosphory- immune and cytoskeletal defects in WASP knockout mice. Gene Ther. 12: 597– lated forms of the cortactin homolog HS1 mediate distinct functions in natural 606. killer cells. Nat. Immunol. 9: 887–897. 68. Jones, G. E., D. Zicha, G. A. Dunn, M. Blundell, and A. Thrasher. 2002. Res- 42. Huang, Y., E. O. Comiskey, R. S. Dupree, S. Li, A. J. Koleske, and toration of podosomes and chemotaxis in Wiskott-Aldrich syndrome macro- J. K. Burkhardt. 2008. The c-Abl tyrosine kinase regulates actin remodeling at phages following induced expression of WASp. Int. J. Biochem. Cell Biol. 34: the immune synapse. Blood 112: 111–119. 806–815. 43. Savoy, D. N., D. D. Billadeau, and P. J. Leibson. 2000. Cutting edge: WIP, 69. Tsuboi, S. 2007. Requirement for a complex of Wiskott-Aldrich syndrome a binding partner for Wiskott-Aldrich syndrome protein, cooperates with Vav in protein (WASP) with WASP interacting protein in podosome formation in http://classic.jimmunol.org the regulation of T cell activation. J. Immunol. 164: 2866–2870. macrophages. J. Immunol. 178: 2987–2995. 44. Volkman, B. F., K. E. Prehoda, J. A. Scott, F. C. Peterson, and W. A. Lim. 2002. 70. Calle, Y., G. E. Jones, C. Jagger, K. Fuller, M. P. Blundell, J. Chow, T. Chambers, Structure of the N-WASP EVH1 domain-WIP complex: insight into the mo- and A. J. Thrasher. 2004. WASp deficiency in mice results in failure to form lecular basis of Wiskott-Aldrich Syndrome. Cell 111: 565–576. osteoclast sealing zones and defects in . Blood 103: 3552–3561. 45. Taniuchi, I., D. Kitamura, Y. Maekawa, T. Fukuda, H. Kishi, and T. Watanabe. 71. Linder, S., H. Higgs, K. Hu¨fner, K. Schwarz, U. Pannicke, and M. Aepfelbacher. 1995. Antigen-receptor induced clonal expansion and deletion of lymphocytes 2000. The polarization defect of Wiskott-Aldrich syndrome macrophages is are impaired in mice lacking HS1 protein, a substrate of the antigen-receptor- linked to dislocalization of the Arp2/3 complex. J. Immunol. 165: 221–225. coupled tyrosine kinases. EMBO J. 14: 3664–3678. 72. Akisaka, T., H. Yoshida, R. Suzuki, and K. Takama. 2008. Adhesion structures

Downloaded from 46. Nagai, T., K. Ibata, E. S. Park, M. Kubota, K. Mikoshiba, and A. Miyawaki. and their cytoskeleton-membrane interactions at podosomes of osteoclasts in 2002. A variant of yellow fluorescent protein with fast and efficient maturation culture. Cell Tissue Res. 331: 625–641. for cell-biological applications. Nat. Biotechnol. 20: 87–90. 73. Cremasco, V., E. Benasciutti, M. Cella, M. Kisseleva, M. Croke, and R. Faccio. 47. Labno, C. M., C. M. Lewis, D. You, D. W. Leung, A. Takesono, N. Kamberos, 2010. Phospholipase Cg2 is critical for development of a murine model of in- A. Seth, L. D. Finkelstein, M. K. Rosen, P. L. Schwartzberg, and J. K. Burkhardt. flammatory arthritis by affecting actin dynamics in dendritic cells. PLoS ONE 5: 2003. Itk functions to control actin polymerization at the immune synapse e8909. through localized activation of Cdc42 and WASP. Curr. Biol. 13: 1619–1624. 74. Brunati, A. M., R. Deana, A. Folda, M. L. Massimino, O. Marin, S. Ledro, 48. Riedl, J., A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, L. A. Pinna, and A. Donella-Deana. 2005. Thrombin-induced tyrosine phos- F. Bradke, D. Jenne, T. A. Holak, Z. Werb, et al. 2008. Lifeact: a versatile marker phorylation of HS1 in human platelets is sequentially catalyzed by Syk and Lyn to visualize F-actin. Nat. Methods 5: 605–607. tyrosine kinases and associated with the cellular migration of the protein. J. Biol. 49. Herzog, M., A. Draeger, E. Ehler, and V. J. Small. 1994. Immunofluorescence Chem. 280: 21029–21035. microscopy of the cytoskeleton: double and triple immunofluorescence. In Cell 75. Hao, J. J., G. B. Carey, and X. Zhan. 2004. Syk-mediated tyrosine phosphory- Biology: A Laboratory Handbook. J. E. Celis, ed. Harcourt Brace and Company, lation is required for the association of hematopoietic lineage cell-specific pro- San Diego, CA, p. 355–360. tein 1 with lipid rafts and B cell antigen receptor signalosome complex. J. Biol. 50. Bowden, E. T., P. J. Coopman, and S. C. Mueller. 2001. Invadopodia: unique Chem. 279: 33413–33420. methods for measurement of extracellular matrix degradation in vitro. Methods 76. Krueger, E. W., J. D. Orth, H. Cao, and M. A. McNiven. 2003. A dynamin- Cell Biol. 63: 613–627. cortactin-Arp2/3 complex mediates actin reorganization in growth factor- 51. Ricart, B. G., B. John, D. Lee, C. A. Hunter, and D. A. Hammer. 2011. Dendritic stimulated cells. Mol. Biol. Cell 14: 1085–1096. cells distinguish individual chemokine signals through CCR7 and CXCR4. J. 77. Oser, M., H. Yamaguchi, C. C. Mader, J. J. Bravo-Cordero, M. Arias, X. Chen, Immunol. 186: 53–61. V. Desmarais, J. van Rheenen, A. J. Koleske, and J. Condeelis. 2009. Cortactin 4818 HS1 AND WASp ORGANIZE PODOSOMES IN DCs

regulates cofilin and N-WASp activities to control the stages of invadopodium activation of actin polymerization that is regulated by cortactin. J. Biol. assembly and maturation. J. Cell Biol. 186: 571–587. Chem. 280: 5836–5842. 78. Artym, V. V., Y. Zhang, F. Seillier-Moiseiwitsch, K. M. Yamada, and 82. Grassart, A., A. Dujeancourt, P. B. Lazarow, A. Dautry-Varsat, and S. C. Mueller. 2006. Dynamic interactions of cortactin and membrane type 1 N. Sauvonnet. 2008. Clathrin-independent endocytosis used by the IL-2 receptor matrix metalloproteinase at invadopodia: defining the stages of invadopodia is regulated by Rac1, Pak1 and Pak2. EMBO Rep. 9: 356–362. formation and function. Cancer Res. 66: 3034–3043. 83. Jain, P., C. Coisne, G. Enzmann, R. Rottapel, and B. Engelhardt. 2010. a4b1 Integrin 79. Clark, E. S., A. S. Whigham, W. G. Yarbrough, and A. M. Weaver. 2007. mediates the recruitment of immature dendritic cells across the blood-brain barrier Cortactin is an essential regulator of matrix metalloproteinase secretion and during experimental autoimmune encephalomyelitis. J. Immunol. 184: 7196–7206. extracellular matrix degradation in invadopodia. Cancer Res. 67: 4227–4235. 84. Schumann, K., T. La¨mmermann, M. Bruckner, D. F. Legler, J. Polleux, 80. Kowalski, J. R., C. Egile, S. Gil, S. B. Snapper, R. Li, and S. M. Thomas. 2005. J. P. Spatz, G. Schuler, R. Fo¨rster, M. B. Lutz, L. Sorokin, and M. Sixt. 2010. Cortactin regulates cell migration through activation of N-WASP. J. Cell Sci. Immobilized chemokine fields and soluble chemokine gradients cooperatively 118: 79–87. shape migration patterns of dendritic cells. Immunity 32: 703–713. 81. Kempiak, S. J., H. Yamaguchi, C. Sarmiento, M. Sidani, M. Ghosh, 85. Collin, O., S. Na, F. Chowdhury, M. Hong, M. E. Shin, F. Wang, and N. Wang. R. J. Eddy, V. Desmarais, M. Way, J. Condeelis, and J. E. Segall. 2005. A 2008. Self-organized podosomes are dynamic mechanosensors. Curr. Biol. 18: neural Wiskott-Aldrich Syndrome protein-mediated pathway for localized 1288–1294. by guest on October 1, 2021. Copyright 2011 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from Supplemental Figure 1: Podosome structure in HS1-/- BMDCs. WT and HS1-/- BMDCs were cultured on coverslips, fixed and stained with phalloidin (red) to label F-actin and anti-vinculin (green). Lower panels show high magnification images of the boxed regions. Recognizable podosomes with F-actin rich cores and vinculin rings were present in both WT and HS1-/- DCs. The HS1-/- DC shown here exhibits blurred boundaries between these regions. This phenotype was observed in both HS1-/- and WT cells, but was more frequent in the HS1-/- population.

Supplemental Figure 2: Podosomes of HS1-/- BMDCs are comprised of characteristic proteins. WT and HS1-/- BMDCs were cultured on coverslips, fixed and stained with phalloidin (red) to label F-actin and with anti-2-integrin (green, A), anti-talin (green, B) or anti-phosphotyrosine (green, C). Scale bars equal 10 m.

Supplemental Figure 3: Analysis of podosomes in HS1-/- BMDCs. A. Culture supernatants from WT and HS1-/- BMDCs were harvested. Proteins were concentrated and separated on an SDS-PAGE gel supplemented with 1 mg/ml gelatin. The gel was renatured and stained with Coomassie Blue to determine regions of degraded gelatin. B. WT and HS1-/- BMDCs were cultured on coverslips coated with gelatin containing 2% FITC-labeled gelatin (green) overnight. Cells were fixed and stained with phalloidin (red) to visualize podosomes. Note that hole size varied from cell to cell; while this pair of images shows a smaller hole shown for the HS1-/- DC, this was not consistently observed. Scale bars equal 10 m. C. Cells treated as in B were analyzed for area of hole in gelatin, as a function of cell footprint area. Each dot represents a single cell. D. WT and HS1-/- BMDCs were transduced with GFP-actin, cultured overnight on chambered coverglasses and imaged live by spinning disk confocal microscopy. A region of interest (ROI) within the podosome array was bleached and monitored for recovery of fluorescence, and the half- life (1/2) for fluorescence recovery within the ROI was measured. Each dot represents a single cell. E. Cells were prepared as in figure 3A. Podosome-containing cells were chosen at random and the percent of cell area covered by podosomes was determined as described in Materials and Methods; each dot represents a single cell.

Supplemental Figure 4: HS1 colocalizes with phosphotyrosine and is surrounded by vinculin in podosomes. HS1-/- DCs were transduced with Venus HS1 and cultured overnight on coverslips. Cells were then fixed and stained with anti-GFP to detect Venus-HS1 (green) and phalloidin (red, A), anti-vinculin (red, B) or anti-phosphotyrosine (red, C). DAPI (blue) was used to identify nuclei. Scale bars equal 10 m.

Video 1: FRAP sequence showing GFP-actin recovery in WT BMDCs. WT DCs were transduced with retrovirus expressing GFP-actin and cultured on chambered coverglasses overnight. Cells with distinct podosome arrays were chosen. A region of interest within the array was bleached, and recovery monitored as detailed in Materials and Methods. Images are played back at 20X capture speed.

Vdieo 2: FRAP of GFP-actin recovery in HS1-/- BMDCs. HS1-/- DCs were transduced with retrovirus expressing GFP-actin and cultured on chambered coverglasses overnight. Cells with distinct podosome arrays were chosen. A region of interest within the array was bleached, and recovery monitored as detailed in Materials and Methods. Images are played back at 20X capture speed.

Video 3: WT BMDCs migrating in a chemokine gradient. WT DCs were matured for 24 hours with 100 ng/ml LPS, then injected into a microfluidic chamber coated with fibronectin and a gradient of CCL19 (0-20 nM). Phase contrast images were collected every minute for 1 hour. Images are played back at 10 frames/second.

Video 4: HS1-/- BMDCs migrating in a chemokine gradient. HS1-/- DCs were matured for 24 hours with 100 ng/ml LPS, then injected into a microfluidic chamber coated with fibronectin and a gradient of CCL19 (0-20 nM). Phase contrast images were collected every minute for 1 hour. Images are played back at 10 frames/second.

Video 5: WASp-/y BMDCs migrating in a chemokine gradient. WASp-/y DCs were matured for 24 hours with 100 ng/ml LPS, then injected into a microfluidic chamber coated with fibronectin and a gradient of CCL19 (0-20 nM). Phase contrast images were collected every minute for 1 hour. Images are played back at 10 frames/second.

Video 6: DKO BMDCs migrating in a chemokine gradient. DKO DCs were matured for 24 hours with 100 ng/ml LPS, then injected into a microfluidic chamber coated with fibronectin and a gradient of CCL19 (0-20 nM). Phase contrast images were collected every minute for 1 hour. Images are played back at 10 frames/second.