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The Journal of Immunology

The mDial Formin Is Required for Neutrophil Polarization, Migration, and Activation of the LARG/RhoA/ROCK Signaling Axis during Chemotaxis1 Yongquan Shi,2* Jinyi Zhang,*2 Michael Mullin,* Baoxia Dong,* Arthur S. Alberts,† and Katherine A. Siminovitch3* Neutrophil chemotaxis depends on actin dynamics, but the roles for specific cytoskeleton regulators in this response remain unclear. By analysis of mammalian diaphanous-related formin 1 (mDia1)-deficient mice, we have identified an essential role for this actin nucleator in neutrophil chemotaxis. Lack of mDia1 was associated with defects in chemoattractant-induced neutrophil actin polymerization, polarization, and directional migration, and also with impaired activation of RhoA, its downstream target p160-Rho-associated coil-containing protein kinase (ROCK), and the leukemia-associated RhoA guanine nucleotide exchange factor (LARG). Our data also revealed mDia1 to be associated with another cytoskeletal regulator, Wiskott-Aldrich syndrome protein (WASp), at the leading edge of chemotaxing neutrophils and revealed polarized morphology and chemotaxis to be more mildly impaired in WASⴚ/ⴚ than in mDia1ⴚ/ⴚ neutrophils, but essentially abrogated by combined mDia1/WASp deficiency. Thus, mDia1 roles in neutrophil chemotaxis appear to be subserved in concert with WASp and are realized at least in part by activation of the LARG/RhoA/ROCK signaling pathway. The Journal of Immunology, 2009, 182: 3837–3845.

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hemoattractant-driven recruitment of neutrophils to sites of injury and inflammation plays a central role in the immune response to infection, enabling the localized neutrophil microcidal activities required for host defense against invading pathogens. Neutrophil migration to these sites is initiated by the binding of such key chemoattractants as MIP-2 and fMLP to cognate G protein-coupled receptors (CXCR2 and formyl peptide receptor, respectively) at the cell surface (1, 2). Ligand binding, in turn, induces GTPase activation with consequent G protein complex dissociation and activation of tyrosine kinases, PI3K, Rho GTPases, and other effectors that link chemotaxin recognition to cytoskeletal alterations required for chemotaxis (2, 3). These alterations include cell elongation and polarization with generation of lamellipodial protrusion at the leading edge and concomitant trailing edge contraction. Such morphological changes are highly dependent on actin polymerization which enables leading edge extension to create frontness dynamics that propel the cell forward and actin complexing with myosin to generate the contractile backness forces enabling tail retraction (4, 5). Rho family GTPases play critical roles in driving the cell polarization and motility required for neutrophil chemotaxis (5– 8). *Department of Medicine, University of Toronto, Samuel Lunenfeld and Toronto Hospital Research Institutes, Toronto, Canada; and †Cell Structure and Signal Integration Laboratory, Van Andel Institute, Grand Rapids, MI 49503 Received for publication November 14, 2008. Accepted for publication January 12, 2009. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The Rac and Cdc42 GTPases, for example, promote F-actin accumulation at the leading edge, whereas Rho signaling appears particularly key to induction of the actomyosin-mediated contraction and posterior retraction required for forward propulsion. These effects of Rho family proteins involve interactions with many other signaling effectors and the creation of both positive and inhibitory feedback signaling loops enabling the spatial and temporal regulation of cytoskeletal dynamics integral to normal chemotaxic responses (8 –10). To stimulate leading edge extension and directed migration, for example, Cdc42 and Rac not only interact with phosphatidylinositol 3,4,5-triphosphate at the neutrophil anterior (4, 5), but also inhibit Rho function in this region while promoting Rho signaling at the rear (6, 8). Similarly, Rho capacity to promote actomyosin contraction and tail retraction requires its activation of the Rho-dependent kinase p160-Rho-associated coil-containing protein kinase (ROCK)4 and ROCK-driven myosin L chain phosphorylation as well as its inhibition of Rac signaling at the cell sides and back (8). Although Rho family GTPases play major roles in inducing the actin remodeling underpinning chemotaxis, cytoskeletal reorganization downstream of these enzymes requires the activities of cytoskeletal regulatory proteins that more directly link stimulatory signals to actin polymerization. Formins constitute one such class of cytoskeletal regulators, those proteins defined by a formin homology 2 domain that directly promotes actin nucleation by enhancing filament elongation so as to generate unbranched actin structures (11, 12). Among the formins implicated in cell motility, the diaphanous-related (mDia) 1, 2, and 3 formins are distinguished by their capacity to interact via an N-terminal GTPasebinding domain (GBD) with GTP-bound Rho family GTPases, an

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This work was supported by grants from the Canadian Institutes for Health Research (MOP12136) and the Leukemia and Lymphoma Society (to K.A.S.). K.A.S. is a McLaughlin Centre for Molecular Medicine Scientist and holds a Canada Research Chair in Immunogenomics.

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These authors contributed equally to this work.

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Address correspondence and reprint requests to Dr. Katherine Siminovitch, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Room 778D, Toronto, Ontario, M5G 1X5 Canada. E-mail address: [email protected]

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803838

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Abbreviations used in this paper: ROCK, p160-Rho-associated coil-containing protein kinase; GBD, GTPase-binding domain; GEF, guanine nucleotide exchange factor; LARG, leukemia-associated Rho-GEF; mDia, mammalian diaphanous-related formin; MLC, myosin light chain; WASp, Wiskott-Aldrich syndrome protein; pMLC, phosphorylated MLC; LPA, lysophosphatidic; DIC, differential interference contrast. Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

3838 interaction that triggers mDia activation by disrupting autoinhibitory structural constraints imposed by interaction of a C-terminal diaphanous autoregulatory domain with mDia1 N-terminal sequences (12, 13). The mDias thus serve as Rho family GTPase effectors, but their influence on cytoskeletally driven cell processes appear mechanistically complex, achieved through induction of both actin and microtubule remodeling and via associations with other effectors such as Src and the Dia-interacting protein and microtubule plus-end binding protein adaptors (14 –16). Among the mDias, mDia1 is of particular potential relevance to neutrophil chemotaxis because it is activated by RhoA and has been shown to play key roles in migration of several cell types, including T lymphocytes (16 –20). We have therefore capitalized on the availability of mDia1-deficient mice to investigate the roles for this formin in neutrophil chemotaxis. Here we show that mDia1-deficient neutrophils have impaired capacity to polymerize actin, polarize, and migrate with directionality in response to chemoattractants. These functional defects are associated with reduced activation of RhoA-ROCK signaling and are significantly more severe in mDia1⫺/⫺ neutrophils rendered deficient for a second cytoskeletal regulator, Wiskott-Aldrich syndrome protein (WASp). These data identify essential roles for mDia1 in neutrophil chemotaxis and reveal that mDia1 subserves this role in conjunction with WASp and via modulation of actin polymerization, polarization and RhoA-ROCK signaling.

Materials and Methods Mice mDia1⫺/⫺ and WASp⫺/⫺ mice were generated as previously described (21, 22) and bred with one another to derive mDia1⫺/⫺ and WAS⫺/⫺ mice. All mouse strains were maintained on the C57BL/6 background and under pathogen-free conditions at the Samuel Lunenfeld Animal Facility.

Reagents The Abs used in this study included anti-myosin L chain (MLC) and antiphospho-MLC Abs purchased from Cell Signaling Technology; antiROCK, anti-leukemia-associated Rho- guanine nucleotide exchange factor (GEF; LARG) and anti-mDia2 Abs purchased from Santa Cruz Biotechnologies; anti-Gr-1, anti-Mac-1, anti-mDia1, anti-CD62L, anti-VLA-4, and anti-LFA-1 Abs purchased from BD Biosciences; anti-CXCR2 Ab purchased from R&D Systems; anti-WASp Ab provided by Millipore; and anti-mDia2 and mDia3 Abs derived by A. Alberts. Fluorescently labeled secondary Abs (FITC, Cy3, and Cy5) were purchased from Jackson ImmunoResearch Laboratories; and HRP-conjugated goat anti-rabbit and goat anti-mouse Abs were obtained from BioRad. FITC-conjugated formylated peptide (FNLPNTL) and FITC- and Alexa 635-conjugated phalloidin were purchased from Molecular Probes, and MIP-2 and fMLP were purchased from Sigma-Aldrich.

Histochemistry For H&E staining, tissue sections were fixed in 4% (v/v) paraformaldehyde in PBS, embedded in paraffin, and sectioned at 5 mm thickness. For immunohistochemistry, frozen tissue sections were fixed in 4% paraformaldehyde in PBS (w/v) and blocked with donkey serum; the sections were incubated with rat anti-mouse Gr-1, developed with diaminobenidine, and counterstained with hematoxylin.

Isolation of bone marrow neutrophils Bone marrow from 6- to 8-wk-old mice was flushed from femurs using chemotaxis buffer (HBSS, HEPES (pH 7.4), 0.1% BSA) and RBCs removed by cell lysis. After resuspension in calcium/magnesium-containing chemotaxis buffer, neutrophils were purified by centrifugation through discontinuous Percoll density gradients of 52%/65%/75%, retrieved from the 65–75% interface, and incubated with anti-B220-coated magnetic beads (Miltenyi Biotech) to remove B cells. Purity was determined to be at least 90% by forward and side scatter and Gr-1 staining.

Flow cytometry Bone marrow neutrophils resuspended in PBS (1 ⫻ 106/ml) were incubated for 30 min on ice with Gr-1-PE and biotinylated Mac-1, or Abs to selected

mDia1 IS REQUIRED FOR NEUTROPHIL CHEMOTAXIS integrin or chemokine receptors. After staining, cells were washed with PBS, incubated for 30 min on ice with streptavidin-allophycocyanin or secondary Ab, washed, and analyzed using a FACSCalibur (BD Biosciences).

Actin polymerization assay Bone marrow neutrophils were stimulated with MIP-2 (25 ng/ml) for 30 s, fixed with 4% paraformaldehyde for 10 min at 37°C, and then permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature. The cells were washed and incubated for 30 min with 2 U/ml Alexa 635-phalloidin in 1% BSA-PBS containing 0.05% saponin and staining analyzed using a FACSCalibur (BD Biosciences).

Chemotaxis Following resuspension in chemotaxis buffer, neutrophils (1–2 ⫻ 105 cells/ ml) were adhered to a glass coverslip for 10 min, mounted onto a Zigmond chamber loaded with 10 ␮M fMLP or 25 ng/ml MIP-2, and then placed on a heated stage at 37°C for analysis. Differential interference contrast (DIC) images of cells on a chamber bridge were acquired with an Axiovert 200M at 10-s intervals for 20 min using a 75-min exposure time and recorded with a CoolSnap HQ CCD camera (Roper Scientific). Image ProPlus (Media Cybernetics) was used to determine morphology parameters and generate time lapse videos by compiling each successive time point obtained in sequence files. These video images were used to plot cell tracks in reference to the starting point of each cell and to determine cell migration speed. Migration parameters for wild-type and mutant cells were calculated by compiling data on 30 –50 cells in 4 independent experiments and determining the mean value across all 4 experiments. Different mice were used as a source of cells for each of the four experiments. Cells migrating more than 30 ␮m in 20 min were scored to determine straightness and directionality of cell movement. Speed was defined as the centroid movement of the cell in micrometers per minute along the total path length and speed in direction as the distance moved in the direction of the chemoattractant source divided by time. Straightness was defined at the net distance traveled divided by total linear distance traveled and time interval between directional changes was calculated as time between directional changes (angle/turn) ⬎45 degrees relative to a straight line connecting the first and final cell positions. Traces of neutrophil boundaries were generated with images selected at 50-s intervals from the time lapse videos. Each successive time frame was layered using Photoshop software.

Mouse peritonitis model WT or mDia1⫺/⫺ mice (n ⫽ 6) were injected i.p. with 1 ml of PBS alone or with 2 ␮g of fMLP. Mice were sacrificed 4 h later, and peritoneal exudate cells were harvested by successive PBS washes. Exudate and differential cell counts were determined by hemacytometer and microscopic analysis of Wright-Giemsa-stained cytospins.

Immunofluorescence microscopy Purified neutrophils were adhered to glass coverslips and stimulated with a uniform concentration of MIP-2 (25 ng/ml) at 37°C for 3 min. Cells were fixed in cytoskeleton buffer containing 3.7% paraformaldehyde for 10 min, then permeabilized using 0.2% Triton X-100, washed, and blocked with 3% BSA-PBS for 20 min. Cells were incubated with primary and fluorescently labeled secondary Abs, and stained samples were mounted in antifade mounting medium (DakoCytomation). Images were collected and analyzed using the Leica SP2 scanning confocal microscope.

Phagocytosis For Fc␥R-mediated phagocytosis, SRBCs (20%) in PBS (v/v) were opsonized with rabbit anti-sheep IgG at 37°C for 1 h and washed in PBS, and the opsonized SRBCs (50 ⫻ 106) were then incubated with 10 ⫻ 106 neutrophils in the presence of fMLP (1 ␮M). Cells were pelleted to promote binding and resuspended in PBS; and after 1 h of 37°C incubation, excess cells were lysed in H2O, and neutrophils were resuspended in cold PBS. For CR3-mediated phagocytosis, 100 ␮l of SRBCs in PBS were opsonized with 50 ␮l of rabbit anti-sheep IgM for 1 h at room temperature and washed in PBS; the opsonized cells incubated at 37°C for 20 min with C5-deficient serum and then for 40 min with fMLP (1 ␮M)-stimulated neutrophils. Phase contrast microscopy was used to count the number of RBCs internalized in 50 randomly chosen neutrophils.

Immunoprecipitation and immunoblotting Unstimulated or fMLP (1 ␮M)-stimulated bone marrow neutrophils (2 ⫻ 107) were incubated for 15 min on ice in lysis buffer as previously described (21), and the lysates were then purified by centrifugation at


3839 anthraniloyl provided in Rho GEF Exchange Assay kits (Cytoskeleton). Relative fluorescence intensity was spectrometrically monitored every 30 s over 30 min, and the specific exchange rate was calculated per the manufacturer’s formula. To assay ROCK2 activity, ROCK immunoprecipitates from fMLP-stimulated neutrophils were washed in kinase buffer (20 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1 mM DTT, 5 mM MgCl2, 1 mM EDTA, 1 mM ATP), and the complexes were then incubated for 30 min at 30°C in kinase buffer containing 10␮Ci of [␥-32P]ATP (Amersham) with 6 ␮g of MLC 20 (Sigma-Aldrich). The kinase reactions were resolved over SDS-PAGE and transferred to the nitrocellulose; the phosphorylated proteins were visualized by autoradiography. The membrane was then incubated with anti-ROCK Ab followed by peroxidase-conjugated goat antirabbit IgG and ROCK loading visualized by chemiluminescence.

Results mDia1⫺/⫺ mice manifest expansion of bone marrow and splenic neutrophil populations and impairment of neutrophil chemotaxis FIGURE 1. Expansion of the bone marrow neutrophil population in mDia1⫺/⫺ mice. A, Western blot of wild-type (WT) and mDia1⫺/⫺ bone marrow neutrophil lysates using anti-mDia1, mDia2, mDia3, and ␤-actin Abs. B, Staining of bone marrow and peripheral blood of 6- to 8-wk-old mice with H&E and Wright-Giemsa, respectively. Arrows identify mature neutrophils. Scale bar, 50 ␮M. C and D, Flow cytometric analyses showing mean (⫾SEM) numbers of Gr-1⫹ (C) or Gr-1 and Mac-1 (D) staining bone marrow cells. Data and images are representative of three to five independent experiments. 12,000 ⫻ g for 20 min at 4°C. For immunoprecipitation, lysates were precleared by incubation with protein A-Sepharose 4B beads for 1 h at 4°C followed by overnight incubation with specific Ab. Immunoprecipitated proteins were then collected over protein A beads and eluted by boiling in Laemmli sample buffer. Lysate or immunoprecipitated proteins were resolved on 10% SDS-PAGE and transferred to nitrocellulose (Schleicher & Schuell); the blocked membranes were sequentially incubated with the appropriate Ab and peroxidase-conjugated goat anti-rabbit Ig as previously described (21).

Rho-GEF and ROCK in vitro kinase assays LARG immunoprecipitates were suspended in 100 ␮l of exchange buffer containing 2 ␮M GDP-loaded RhoA-His protein and 0.75 ␮M N-methyl-

FIGURE 2. mDia1 is required for neutrophil chemotaxis. Wild-type (WT) and mDia1⫺/⫺ neutrophils were allowed to migrate for 20 min through MIP-2 or fMLP gradients in a Zigmond chamber, and their trajectories were tracked from frames taken every 10 s. A, Plots of migration tracts for each cell relative to its initial position (⬎30 cells scored in each of 4 independent experiments; arrows indicate direction of chemoattractant source). B–E, Comparisons of chemotactic parameters between wild-type and mDia1⫺/⫺ neutrophils as determined from time lapse movies (supplemental videos 1 and 2) and calculated as described in Materials and Methods. F, Neutrophil recruitment to sites of inflammation. Total neutrophil content in peritoneal lavage fluid collected 4 h post-i.p. PBS or PBS-fMLP injection was determined by analysis of Wright-Giemsa-stained cytospins. Data are representative of at least four independent experiments and are expressed as means ⫾ SEM. ⴱ, p ⬍ 0.01 vs wild-type neutrophils.

The ability of mDias to couple Rho GTPases to actin remodeling suggests an important role for these formins in actin-based neutrophil functions. On the basis of immunoblotting analyses identifying mDia1 as the predominant mDia expressed in bone marrow neutrophils (Fig. 1A), we investigated the roles of mDia1 in neutrophil physiology using mDia1-deficient mice generated by targeted disruption of the Drf1 gene (22). Histological and cytological examination of 8-wkold mDial⫺/⫺ mice revealed progressive bone marrow hypercellularity, reflecting an almost 2-fold increase in marrow neutrophil numbers (Fig. 1, B and C). Splenomegaly with marked expansion of splenic neutrophil numbers was also observed although peripheral blood neutrophil number was normal (data not shown). These findings are in keeping with recent data suggesting that aging mDia1⫺/⫺ mice develop a myeloproliferative phenotype associated with increases in myeloid cell numbers and dysplasia (22). In the younger mDia1⫺/⫺ mice studied here, however, mature neutrophil morphology appeared normal (Fig. 1B), and almost all Gr-1-positive bone marrow cells coexpressed the Mac-1 differentiation Ag (Fig. 1D), thus showing an immunophenotype (Gr-1highMac-1high) characteristic of mature neutrophils (23).

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mDia1 IS REQUIRED FOR NEUTROPHIL CHEMOTAXIS

FIGURE 3. mDia1 is required for neutrophil polarization and actin polarization. A–D, Wild-type (WT) and mDia1⫺/⫺ neutrophils seeded in a Zigmond Chamber were monitored by videomicroscopy for chemotaxic responses to fMLP. A, DIC images showing morphology of resting and chemotaxing neutrophils. Bar, 5 ␮m. B, Outlines of single migrating cells observed at 50-s intervals (indicated by colors) over a 250-s period. Each color dot represents the center of the leading edge at the selected time point (see supplementary videos). C and D, The indicated parameters of neutrophil morphology were determined (see Materials and Methods) at selected times after fMLP stimulation; ⴱ, p ⬍ 0.01 vs wild-type cells. E, Factin staining before and after MIP-2 stimulation of Alexa 635-phalloidinstained wild-type and mDia1⫺/⫺ cells and expressed as mean (⫾SEM) fluorescence intensity. F, F-actin and mDia1 staining of MIP-2-stimulated (Stim) neutrophils adhered to glass coverslips (left). Polarized cells (numbers of neutrophils/100-cell field) were scored for F-actin accumulation restricted to a single leading lamellipodium (right). G, CR3 and Fc␥Rmediated phagocytosis in fMLP-stimulated neutrophils incubated with C3bi (left)- or IgG (right)-opsonized SRBCs. All data and images are representative of three to five independent experiments. Unstim, Unstimulated; max, maximum; min, minimum.

In view of the key role for mDia1 in cytoskeletal regulation, in vitro chemotaxic responses of mDia1⫺/⫺ neutrophils were examined using Zigmond chamber assays. Whereas ⬎90% of wild-type neutrophils migrated toward the MIP-2 chemokine or fMLP formyl peptide, ⬍60% of mutant cells moved toward chemoattractant (Fig. 2, A and B). Speed of movement, straightness of migration toward the chemoattractant and time interval between directional changes were also markedly reduced in mDia1⫺/⫺ cells (Fig. 2, C–E). Thus, impaired chemotaxis in the mutant cells reflects defects in both speed and directionality, a conclusion supported by their profoundly reduced speed in direction, a measure of both rate and directional persistence of migration (Fig. 2C). These defects are not ascribable to changes in CXCR2 or formyl peptide receptor expression, given that surface levels of these and key

integrin proteins appear normal in the mutant cells (supplemental Fig. 1).5 To ascertain whether the chemotaxic defect engendered by mDia1⫺/⫺ deficiency impedes neutrophil recruitment to sites of inflammation, neutrophil numbers were evaluated in peritoneal lavage fluid obtained 4 h after fMLP i.p. injection of live mice. Whereas fMLP evoked a substantial increase in peritoneal exudate neutrophil numbers (2 ⫻ 106) in wild-type mice (Fig. 2F), significantly fewer neutrophils were recruited to the mDia1⫺/⫺ peritoneal cavity. Thus, mDia1 effects on neutrophil chemotaxis appear

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The online version of this article contains supplemental material.


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FIGURE 4. Impairment of RhoA/ROCK/LARG signaling in mDia1⫺/⫺ neutrophils. A, Western blot using Abs to RhoA, cdc42, and Rac1 to detect GTP-bound GTPase precipitated from neutrophils at the indicated times after fMLP-stimulation using GST-rhotekin or P21-activated kinase cdc42/RAC interactive binding domain protein. Total amount of GTPase in cell lysates shown in lower panel of each blot pair. B, Neutrophils purified from wild-type (WT) and mDia1⫺/⫺ mouse bone marrow were plated on coverslips coated with 60 ␮g/ml collagen in the presence of fMLP, fixed at the indicated time points, and stained with GST-rhotekin (provided by Dr. T. Pawson, Samuel Lunenfeld Research Institute, Ontario, Canada), anti-GST Ab, FITC-conjugated secondary Ab to detect RhoA-GTP, Alexa 647-phalloidin to detect F-actin, and anti-RhoA and Cy3-conjugated secondary Ab to detect endogenous RhoA. Images are representative of at least three independent experiments. C, Wild-type and mDia1⫺/⫺ neutrophils were stimulated with fMLP for the indicated times, subjected to immunoprecipitation with anti-ROCK Ab and the immunoprecipitates (IP), and then incubated with MLC and [␥-32P]ATP in an in vitro kinase assay. ROCK (bottom) and pMLC (top) were visualized by immunoblotting analysis and autoradiography. D, Immunoblotting analysis of anti-MLC Ab immunoprecipitates from fMLP-stimulated wild-type and mDia1⫺/⫺ neutrophils. Membranes were sequentially probed for pMLC and MLC. E, F-actin and pMLC staining of MIP-2-stimulated WT and mDia1⫺/⫺ neutrophils. F, Anti-mDial Western blot of LARG immunoprecipitates from fMLP-stimulated neutrophils. G, Histograms showing ratios of mDia1/LARG band intensities as determined by densitometric analysis of the immunoblot in F. H, LARG and mDia1 staining was assessed before and after cell stimulation with MIP-2. I, LARG immunoprecipitates from fMLP-stimulated neutrophils were incubated with GDP-RhoA and N-methylanthraniloyl (mant)-GTP and GEF activity measured by spectrometry. Shown are the mean (⫾SEM) exchange rates representative of three independent experiments. ⴱ, p ⬍ 0.01 vs wild-type neutrophils.

to be physiologically relevant, lack of this effector impairing neutrophil capacity to efficiently reach peripheral inflammatory regions. mDia1 regulates neutrophil polarization Polarization is an integral facet of the neutrophil migratory response and was thus examined at the single cell level in the mDia1⫺/⫺ cells using time lapse videomicroscopy. As revealed by the representative examples shown in Fig. 3A, polarized morphology was rapidly induced by fMLP in wild-type neutrophils, each cell acquiring elongated shape with a well-defined single leading edge and tail and clearly reorienting toward the chemoattractant source. By contrast, mDia1⫺/⫺ neutrophils show more diverse morphologies, the cells failing to form a well-demarcated leading edge or tail and instead appearing flattened and poorly polarized. Similarly, evaluation of cell migration tracks (Fig. 3B and supplemental videos 1 and 2) revealed that the mutant cells form multiple, unstable pseudopods rather than a discrete leading edge, fail to sustain directional migration, and have reduced capacity for tail retraction, with many cells appearing immobilized at the rear. These findings are in keeping with the reduced eccentricity and radius ratio (i.e., deviation from circular shape) of fMLP-exposed mDia1⫺/⫺ cells (Fig. 3,

C and D), morphological differences again indicative of inefficient polarization. Neutrophil polarizing responses to chemoattractants require that polymerized actin accumulate anteriorly to form a dominant Factin-rich pseudopodium. However, evaluation of F-actin generation in phalloidin-stained neutrophils revealed F-actin levels to be equivalent in mDia1⫺/⫺ and control neutrophils at baseline, but to increase only 2-fold in mDia1⫺/⫺ vs almost 5-fold in wild-type cells at 15 s after MIP-2 stimulation and to remain comparatively low (close to baseline levels) in the mutant cells (Fig. 3E). Also in contrast to wild-type cells in which MIP-2 induced intense F-actin as well as mDia1 accumulation at the leading edge and anterior, less than one-third of mDia1⫺/⫺ cells showed restricted F-actin localization at the cell anterior, and the intensity of F-actin staining was generally reduced (Fig. 3F). As was also apparent from the time lapse videos (supplemental videos 1 and 2), a high frequency (⬃50%) of mDia1⫺/⫺ cells developed multiple pseudopodia rather than a single dominant pseudopod, an abnormality observed in neutrophils deficient for other polarity modulators (24) and in keeping with a critical role for mDia1 in regulating the anterior spatial restriction of F-actin required for neutrophil polarization.

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FIGURE 5. mDia1 and WASp are both required for neutrophil chemotaxis. Neutrophils from WT and mutant mice were allowed to migrate for 20 min through a MIP-2 or fMLP gradient in a Zigmond chamber, and their trajectories were tracked at 10-s intervals. A, All tracks are plotted for each cell relative to its starting position and aligned with direction (black arrow) of fMLP (⬎30 cells scored/experiment). B, Speeds of cell movement were calculated from data collected over 10-min intervals on 70 cells/genotype. C, Outlines showing shape and direction of individual control and mutant neutrophils in an fMLP gradient followed at 50-s intervals (denoted by individual colors). D, F-actin, mDia1, and WASp staining of MIP-2-stimulated wild-type (WT) and mutant neutrophils. Data and images are representative of at least four independent experiments. ⴱ, p ⬍ 0.01 vs wild-type neutrophils.

Because actin polymerization is also needed for CR3 and Fc␥Rmediated phagocytosis in neutrophils (25, 26), enabling assembly of the phagocytic cup required for particle engulfment, the phagocytic properties of mDia1⫺/⫺ neutrophils were also studied. As shown in Fig. 3G, the capacity of mDia1⫺/⫺ neutrophils to ingest either IgM/C3 or IgG-opsonized SRBC was substantially reduced (by ⬃70% and 50%, respectively) compared with wild-type cells. Thus, mDia1 is required for both CR3- and Fc␥R-mediated phagocytosis in neutrophils, in keeping with the requirement of both phagocytic pathways for actin polymerization and with the dysregulation of actin remodeling imbued by mDia1 deficiency. mDia1 promotes LARG induction of RhoA-ROCK signaling The importance of Rho GTPases to diaphanous formin activation is well established, but recent data suggest that mDia1 also modulates cell responses by regulating RhoA activation. To determine whether altered Rho family GTPase activity contributes to the chemotactic/polarity defects manifested by mDia1⫺/⫺ neutrophils, we compared mDia1⫺/⫺ and wild-type neutrophils with respect to levels of activated cdc42, Rac, and Rho induced by fMLP stimulation. As shown in Fig. 4A, the amounts of activated cdc42 and Rac precipitated with GST-PAK1 GBD fusion protein were equivalent in the mutant and wild-type cells. However, the amounts of RhoAGTP precipitated by rhotekin GBD fusion protein were markedly reduced in the mDia1⫺/⫺ cells, although total RhoA levels were comparable with those of wild-type neutrophils. Thus, chemoattractant-induced induction of RhoA activation appears to be impaired in the context of mDia1 deficiency. In keeping with these findings, evaluation of RhoA and RhoA-GTP subcellular distribution revealed accumulation of rhotekin-bound activated RhoA at the trailing edge of wild-type cells to be substantially disrupted in mDia1⫺/⫺ neutrophils (Fig. 4B). RhoA effects on neutrophil chemotaxis are mediated in part via induction of ROCK, a RhoA-binding kinase that promotes MLC activation via its serine phosphorylation and posterior translocation in the cell (27, 28). As shown in Fig. 4, C and D, fMLPinduced ROCK activation and induction of MLC phosphorylation were strongly reduced in mDia1⫺/⫺ neutrophils. Also in contrast to wild-type cells, in which stimulation induced phosphorylated MLC (pMLC) relocalization to the uropod and sides, mDia1⫺/⫺

cells showed almost random pMLC distribution (Fig. 4E). These data confirm that Rho signaling to ROCK/MLC is impaired in mDia1⫺/⫺ cells, as is in keeping with their tail retraction defect and lateral pseudopodia formation, processes normally regulated by pMLC. The impairment in RhoA signaling in mDia1⫺/⫺ cells implies that mDia1 serves as both a RhoA effector and regulator. mDia1 capacity to regulate RhoA has been recently described in relation to lysophosphatidic (LPA)-triggered tumor cell invasion and linked to mDia1 induction of the Rho-GEF, LARG (29). To ascertain whether mDia1 effects on RhoA signaling during chemotaxis also reflect LARG modulation, mDia1 association with and effects on LARG were studied in fMLP- and MIP-2-stimulated neutrophils. As shown in Fig. 4, F and G, mDia1 and LARG coimmunoprecipitated from wild-type neutrophils and their association increased after stimulation and localized predominantly to the leading edge. By contrast, LARG distribution was diffuse and no longer anteriorly concentrated in mDia1⫺/⫺ cells (Fig. 4H). LARG immunoprecipitates from mDia1⫺/⫺ cells were also markedly impaired in capacity to induce GDP release from RhoA in an in vitro GEF assay (Fig. 4I). These findings are consistent with reported capacity of the mDia1 formin homology 2 domain to activate LARG in vitro (29) and reveal a complex functional relationship between mDia1 and RhoA, with mDia1 serving not only to couple chemoattractant-triggered RhoA activation to the neutrophil cytoskeleton but also, once activated, to further stimulate RhoAROCK signaling via a LARG-dependent mechanism. mDia1 and WASp colocalize at the leading edge of polarized neutrophils WASp family proteins are another class of cytoskeletal regulators activated by Rho family GTPases (30). Among these proteins, WASp has been implicated in T cell and macrophage chemotaxis, but its roles in neutrophil migration remain uncertain (31–36). To address this issue and to explore the respective contributions of WASp and mDia1 to neutrophil chemotaxis, we compared mDia1⫺/⫺ neutrophil chemotactic responses with those of WAS⫺/⫺ and mDia1⫺/⫺WAS⫺/⫺ neutrophils. This analysis revealed migratory responses to both fMLP (Fig. 5A) and MIP-2 (not shown) as well as migration speeds (Fig. 5B) to be impaired in

The Journal of Immunology WAS⫺/⫺ neutrophils, but less severely than in mDia1⫺/⫺- or mDia1/WASp-deficient cells, the latter of which failed to move up the gradient. As revealed by time-lapse videomicroscopy (Fig. 5C and supplemental videos 3 and 4), multiple pseudopods rather than a single leading edge develop in many WAS⫺/⫺ cells, but these structures persist longer and the polarity defect is less severe than that of mDia1⫺/⫺ cells. As is in keeping with the complete loss of chemotactic responses in the mDia1⫺/⫺WAS⫺/⫺neutrophils, these mutant cells show almost no polarization, form only occasional, highly unstable pseudopods, and appear immobilized at the rear, failing to retract their tails and thus lacking capacity to detach and move forward. Although mDia1⫺/⫺ and some WAS⫺/⫺ cells also show impaired tail retraction, this defect was far more pronounced and almost universally observed in mDia1/WASp-deficient cells. Thus, the combined absence of mDia1 and WASp activities appears to engender a loss of both front end polarization and back end retraction capabilities. To further evaluate mDia1 and WASp contributions to neutrophil chemotactic responses, the subcellular location of WASp relative to that of mDia1 was also examined in MIP-2-stimulated cells. As shown in Fig. 5D, stimulation of wild-type neutrophil induced the translocation of both WASp and mDia1 to the F-actinconcentrated region at the leading edge. However, anterior polarization of each effector was essentially disrupted in the absence of the other protein, with WASp, for example, concentrating not only in pseudopodia, but also at the relatively actin-deficient cell posterior in mDia1⫺/⫺ cells and mDia1 showing similar displacement in WAS⫺/⫺ cells. These images also reveal anterior F-actin accumulation to be essentially abrogated in mDia1⫺/⫺WAS⫺/⫺ cells. Thus, neutrophil chemotaxic responses require the activities of both mDia1 and WASp, which appear to colocalize at the leading edge so as to promote F-actin accumulation, polarization, and directed migration.

Discussion Cytoskeletal remodeling plays a central role in neutrophil chemotactic responses, but the contributions of specific actin assembly effectors to such responses are not well defined. Here we show that neutrophils lacking the mDia1 actin regulator manifest significant defects in chemotaxis, these cells displaying severely reduced directional sensing and rates of directed migration in response to fMLP or MIP-2 and impaired capacity to accumulate in the peritoneum in response to inflammatory stimuli. Chemoattractant-induced F-actin generation and development of polarized morphology are also defective in mDia1⫺/⫺ neutrophils, the cells failing to polarize F-actin interiorly, developing multiple, short-lived pseudopodia rather than a dominant leading edge and showing reduced tail retraction capacity. Chemoattractant-evoked activation of the Rho GEF LARG and its Rho substrate were also impaired in the mDia1⫺/⫺ cells, and these defects were in turn associated with marked reduction in ROCK activation and in phosphorylation and posterior redistribution of the ROCK target, MLC. Whereas deficiency of the WASp actin regulator was associated with chemotactic defects similar, but milder than those observed in mDia1⫺/⫺ neutrophils, combined mDia1 and WASp deficiency completely abrogated chemotactic responses, the mDia1⫺/⫺WAS⫺/⫺ neutrophils showing virtually no polarization or directed migration in response to chemoattractants. These data identify an important role for mDia1 in neutrophil chemotaxis and suggest that this role is subserved in part via modulation of LARG/Rho signaling and in conjunction with WASp, the combined activities of mDia1 and WASp being essential for neutrophil chemotactic responses. An important contribution of mDia1 to neutrophil chemotaxis is consistent with data identifying key roles for diaphanous formins

3843 in cell mobility. The mDia2 formin, for example, appears to control filopodial dynamics required for cell movement and the formin homology domain-containing protein promotes migration of melanoma cells and fibroblasts (37, 38). mDia1 activity also appears key to fibroblast capacity to migrate in scratch wound assays and to the chemotactic and trafficking abilities of T lymphocytes (19, 20, 39). In view of the roles of these formins in actin nucleation, it appears likely that their influence on cell mobility is mediated at least in part via effects on actin organization. However, mDias also play essential roles in regulating microtubule organization and dynamics. The mDia1 protein, for example, has been shown to link T cell Ag receptor engagement to microtubule-organizing center polarization (40) and G protein-coupled receptor stimulation to the microtubule stabilization required for fibroblast migration (39). Thus, mDia1 effects on chemotaxis may reflect its modulation of both actin and microtubule components, a functional role already ascribed to the related formin homology domain-containing protein in relation to its regulation of HeLa cell elongation (41). Drfs such as mDia1 also control activities of several transcriptional activators (such as serum response factor and its cofactor, MAL) that regulate cytoskeletal-modulatory genes and, by extension, cytoskeletally-dependent functions, such as cell migration (14, 42, 43). The extent to which each of these functions enables mDia1 effects on chemotaxis requires further investigation, but the impaired capacity of mDia1⫺/⫺ neutrophils to generate and anteriorly polarize F-actin in response to chemoattractant stimuli suggests that the influence of mDia1 on neutrophil chemotaxis relates at least in part to its actin-nucleating function. Although the role for Rho in activating mDia1 is well established, our data indicate that in migrating neutrophils, mDia1 also, conversely, regulates the activation and localization of Rho. These findings are in keeping with recent data revealing an essential role for mDia1 in LPA-mediated induction of RhoA/ROCK activation and tumor cell invasion (29). In these latter studies, mDia1 effects on Rho signaling were linked to its capacity to bind and stimulate LARG, a Rho GEF known to connect G protein-coupled LPA receptors to Rho activation (44, 45). The current data reveal that mDia1 also links chemoattractant stimuli to LARG in neutrophils, mDia1 associating with this GEF at the leading edge of chemotaxing cells and inducing LARG activation and downstream Rho/ ROCK induction. These findings indicate a critical role for LARG in neutrophil chemotaxis, a conclusion supported by a previous report of neutrophil chemotaxic defects in mice lacking another Rho GEF, Lsc (44). As observed in the mDia1⫺/⫺ cells, neutrophils from Lsc-deficient mice manifest significantly impaired directional migration as well as supernumerary pseudopodia and other morphological defects when stimulated with fMLP. However, in contrast to mDia1⫺/⫺ cells, Lsc⫺/⫺ neutrophils migrate with increased speed in response to fMLP and are recruited normally to Escherichia coli-inflamed peritoneum. These data imply at least some distinct roles for Lsc and LARG in relation to neutrophil chemotaxis, roles that may be Rho dependent or potentially independent and also suggest that LARG activity partially compensates for the absence of Lsc in modulating some aspects of neutrophil chemotaxis. These possibilities and other details of the mechanisms whereby LARG and other Rho GEFs influence neutrophil chemotaxis require further investigation, but recent data implicating mDia1/LARG signaling in the regulation of fibroblast microtubule-organizing center polarization (45), are again suggestive of the importance of both actin and microtubule cytoskeletal dynamics to the effects of mDia1 on chemotaxis. The capacity of mDia1 to regulate LARG activation downstream of chemoattractant receptor stimulation is also consistent with a bidirectional functional relationship between mDia1 and the Rho GTPase

3844


wherein Rho activation induces mDia1 activity and the latter potentially enables a positive feedback regulatory circuit by promoting LARG-driven activation of Rho and Rho targets such as ROCK. Although mDia1⫺/⫺ neutrophils manifest significantly altered chemotaxic responses, these responses are even more impaired and essentially abrogated in cells lacking both mDia1 and WASp. Impaired neutrophil chemotactic capacity has been previously reported in some but not all studies of WAS-deficient patients, the disparate findings possibly relating to variation in WASp expression levels among different WAS patients (33, 34) and the relative mildness of the defect as revealed in this study. WASp-deficient mice, however, have been shown in a previous study to manifest impaired neutrophil chemotactic responses (35), and WASp relevance to such responses is also implied by data implicating WASp in lymphocyte, monocyte and dendritic cell chemotaxis (31–34), by the polarization and motility defects observed in the current study of WAS⫺/⫺ neutrophils, and by the significant worsening of neutrophil polarizing and migratory responses conferred by superimposing WASp deficiency onto the mDia1⫺/⫺ background. Together, these data are in keeping with involvement of both mDia1 and WASp in neutrophil chemotaxic responses and suggest that such responses depend upon the combined activities of these effectors. The loss of polarized morphology, leading edge formation, and actomyosin-mediated tail retraction in mDia1⫺/⫺WAS⫺/⫺ neutrophils implies a central role for altered actin dynamics in their chemotactic defect. This conclusion is consistent with the impairment of CR3- and Fc␥R-mediated phagocytosis, both actin-dependent pathways (46), in mDia1⫺/⫺ neutrophils and with prior findings of a phagocytic defect in WAS⫺/⫺ neutrophils (21). WASp and mDia1 may cooperate in regulating neutrophil actin remodeling, given that these effectors play distinct but potentially complementary roles in actin remodeling, the former inducing actin filament linear extension and the latter promoting actin branching (47). Whether mDia1 and WASp activities are spatially and/or temporally coordinated in the context of neutrophil chemotaxis remains to be determined, but colocalization of these effectors at the neutrophil leading edge, their dependence on one another for such localization, and the more severe chemotactic defects imbued by combined compared with individual mDia1 or WASp deficiency, suggest that mDia1 and WASp, like their upstream Rho GTPase inducers, act not only in concert, but also coordinately to mediate the cytoskeletal rearrangements driving chemotaxis in neutrophils. Thus, interactions with both the WASp and the LARG/Rho signaling pathways appear key to the effects of mDia1 on the polarizing and migratory dynamics underpinning neutrophil chemotaxic responses.

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Disclosures The authors have no financial conflict of interest.

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