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Chemotaxis in Dendritic Cells Podosome Array Organization And Hematopoietic Lineage Cell-Specific Protein 1 Functions in Concert with the Wiskott− Aldrich Syndrome Protein To Promote Podosome Array Organization and This information is current as Chemotaxis in Dendritic Cells of September 29, 2021. Deborah A. Klos Dehring, Fiona Clarke, Brendon G. Ricart, Yanping Huang, Timothy S. Gomez, Edward K. Williamson, Daniel A. Hammer, Daniel D. Billadeau, Yair Argon and Janis K. Burkhardt Downloaded from J Immunol 2011; 186:4805-4818; Prepublished online 11 March 2011; doi: 10.4049/jimmunol.1003102 http://www.jimmunol.org/content/186/8/4805 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2011/03/11/jimmunol.100310 Material 2.DC1 References This article cites 81 articles, 38 of which you can access for free at: http://www.jimmunol.org/content/186/8/4805.full#ref-list-1 by guest on September 29, 2021 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Hematopoietic Lineage Cell-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 Downloaded from Ags to T cells. DC migration depends on tight regulation of the actin cytoskeleton to permit rapid adaptation to environmental cues. We investigated the role of hematopoietic lineage cell-specific protein 1 (HS1), the hematopoietic homolog of cortactin, 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 http://www.jimmunol.org/ 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 September 29, 2021 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- integrin-independent amoeboid protrusion into an open space within a three-dimensional matrix, or by pushing against integrin- *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 lamellipodium 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 Medicine, Northwestern University, Chicago, IL. surrounded by adhesion molecules, including vinculin, talin, and Received for publication September 17, 2010. Accepted for publication February 11, 2011. integrins (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 endothelium. 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 dendritic cell; 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
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