Research ArticleImmunology

Rab13 acts downstream of the kinase Mst1 to deliver the integrin LFA-1 to the cell surface for lymphocyte trafficking

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Science Signaling  29 Jul 2014:
Vol. 7, Issue 336, pp. ra72
DOI: 10.1126/scisignal.2005199

Abstract

In lymphocytes, the kinase Mst1 is required for the proper organization of integrins in the plasma membrane at the leading edge of migrating cells, which is critical for lymphocyte trafficking. We found a functional link between the small G protein Rab13 and Mst1 in lymphocyte adhesion and migration. In response to stimulation of T lymphocytes with chemokine, Mst1 promoted phosphorylation of the guanine nucleotide exchange factor DENND1C (differentially expressed in normal and neoplastic cells domain 1C), which activated Rab13. Active Rab13 associated with Mst1 to facilitate the delivery of the integrin LFA-1 (lymphocyte function–associated antigen 1) to the leading edge of lymphocytes. Delivery of LFA-1 involved the recruitment of myosin Va along actin filaments, which extended as a result of the localization of the actin regulatory protein VASP to the cell periphery through phosphorylation of VASP at Ser157 by Mst1. Inhibition of Rab13 function reduced the adhesion and migration of lymphocytes on ICAM-1 (intercellular adhesion molecule–1), the ligand for LFA-1, and inhibited the formation of a ring-like arrangement of LFA-1 at the contact sites between T cells and antigen-presenting cells. The lymphoid tissues of Rab13-deficient mice had reduced numbers of lymphocytes because of the defective trafficking capability of these cells. These results suggest that Rab13 acts with Mst1 to regulate the spatial distribution of LFA-1 and the motility and trafficking of lymphocytes.

INTRODUCTION

Lymphocyte adhesion and migration are important for the generation and execution of immune and inflammatory responses. Therefore, defining the molecular mechanisms that control cell attachment and movement is essential to understand immune responses. The β2 integrin LFA-1 (lymphocyte function–associated antigen 1, also known as CD11a/CD18 and integrin αLβ2) and its ligand ICAM-1 (intercellular adhesion molecule–1) are major adhesion molecules involved in lymphocyte trafficking (1, 2). Central to their functions is the unique ability of integrins to regulate their adhesive activity through a process termed “inside-out” signaling. The small guanosine triphosphatase (GTPase) Rap1 is rapidly activated by chemokines and cognate antigens, increases the adhesiveness of integrins to their ligands by modulating their affinity and avidity, induces polarized cell shape changes, and facilitates cell migration (3, 4). RAPL is a Rap1-GTP–binding protein that mediates the ability of Rap1 to increase adhesion through LFA-1 (5). RAPL interacts with and activates the serine and threonine kinase Mst1 (the mammalian homolog of the Drosophila Hpo protein) and forms a complex with the αL integrin subunit to modulate integrin activity through Mst1 in lymphocytes (6), although Mst1 is perhaps best known for controlling organ size through regulation of cell proliferation and apoptosis (7). Mice deficient in these effector molecules or in Rap1 exhibit diminished integrin clustering and function, as well as defective cell polarity, which leads to the impaired trafficking of lymphocytes, dendritic cells, and thymocytes, and indicates that these molecules play crucial roles in the efficient trafficking of immune cells (811).

We previously showed that the Rap1-RAPL-Mst1 signaling pathway stimulates the clustering of LFA-1 at the leading edge of lymphocytes, which is thought to generate new adhesion points at the front membrane, thereby contributing to the robust migration of lymphocytes on ICAM-1 (6, 12). This induced adhesion is dependent on the association of LFA-1 with RAPL through the cytoplasmic region of the αL chain (5). Notably, a mutant αL subunit lacking this cytoplasmic region exhibits defective chemokine-dependent clustering at the cell surface (5). Although this LFA-1 mutant is uniformly distributed over the cell body and uropod, it is absent from the leading edge of polarized cells (5). This suggests that LFA-1 clustering depends on intracellular trafficking, rather than on the lateral diffusion of LFA-1. Consistent with this notion, RAPL and Mst1 colocalize to vesicular compartments enriched for Rap1 and LFA-1, and they are dynamically translocated with LFA-1 to the leading edge of lymphocytes, suggesting that the RAPL-Mst1 complex exerts a regulatory role in the intracellular transport of LFA-1 (6).

Rab proteins are members of the Ras superfamily of GTPases, and they function as molecular switches to control diverse stages of vesicular traffic (13). More than 60 mammalian Rab proteins localize to different cellular compartments and control distinct transport systems. Integrin trafficking involves transit through specific Rab-containing compartments in the cell. The Rab5 family member Rab21 and the Rab11 family member Rab25 directly interact with β1 integrins and play important roles in the intracellular distribution of integrins (1416). Although LFA-1 (αLβ2) is endocytosed through a Rab5-independent, cholesterol-sensitive pathway and recycles in a Rab11-dependent manner through a YXXΦ motif in the cytoplasmic region of the β2 subunit (1719), little is known about specific regulators of the αL-dependent activation of LFA-1. Rab13, Rab10, and Rab8 constitute a subfamily of closely related Rab proteins, but the greatest degree of conservation in these proteins is found in the first 100 N-terminal amino acid residues, which encompass the GTPase-activating protein (GAP) and guanine nucleotide exchange factor (GEF) interaction regions. The amino acid determinants of specific effector regions in the C-terminal regions of these proteins are divergent, which suggests that each molecule has distinct functions (20). Rab13 and its effector protein MICAL-L2 (molecule interacting with CasL-like 2) are involved in the polarized transport of cell adhesion molecules to tight junctions in epithelial cells (21).

Here, we demonstrated that the previously uncharacterized Mst1–DENND1C (differentially expressed in normal and neoplastic cells domain 1C)–Rab13 signaling pathway is crucial for LFA-1– and ICAM-1–dependent adhesion and migration in lymphocytes in response to chemokine. We found that lymphocyte trafficking in Rab13-deficient mice was impaired similarly to that in RAPL- or Mst1-deficient mice (9, 22), revealing an indispensable function of Rab13 in vivo. Furthermore, Mst1 played roles in the polymerization of F-actin through the phosphorylation at Ser157 of VASP (vasodilator-stimulated phosphorylation), an important regulator of actin network architecture, which was required for the Rab13-dependent delivery of LFA-1 to the leading edge of lymphocytes. Thus, Mst1 and Rab13 cooperatively regulate lymphocyte trafficking through the distribution of integrin.

RESULTS

Mst1 and Rab13 physically interact in lymphocytes

To explore the possible involvement of Rab proteins in LFA-1 trafficking mediated by Mst1, we examined the interactions between dominant-active (DA) and dominant-negative (DN) forms of 58 Rab proteins with Mst1 through yeast two-hybrid screening (20). Rab13 interacted with Mst1 in a GTP-dependent manner (table S1), and Rab13 DA colocalized with Mst1 when expressed in COS cells. To confirm the physical interaction between Rab13 and Mst1, we transfected COS cells with plasmids encoding green fluorescent protein (GFP)–tagged DA (Q69L) or DN (T22N) mutants of Rab13 and V5-tagged Mst1 and examined their association through immunoprecipitation experiments (Fig. 1A). More Mst1 coimmunoprecipitated with Rab13 DA than with Rab13 DN (Fig. 1A). Rab13 DA also physically interacted with endogenous Mst1 in BAF/LFA-1 cells, mouse pro-B cells (BAF/3 cells) that express human LFA-1 (3) (Fig. 1B). Mutant forms of Mst1 lacking either the N-terminal or C-terminal regions showed a reduced ability to interact with Rab13 DA (Fig. 1C), suggesting that the three-dimensional structure of Mst1 is required for binding to Rab13. These results suggest that the binding of Rab13 to Mst1 is increased by Rab13 activation.

Fig. 1 Mst1 physically interacts with Rab13.

(A to C) Physical associations between (A) V5-Mst1 and GFP-Rab13 (both DA and DN forms) in COS cells, (B) endogenous Mst1 and Rab13 DA in BAF/LFA-1 cells, and (C) V5-tagged wild-type (WT) Mst1 or C-terminal (ΔC) or N-terminal (ΔN) deletion mutants of Mst1 and GFP-Rab13 in COS cells were analyzed by immunoprecipitation (IP) followed by Western blotting analysis with the indicated antibodies. (D) Left: Lysates of BAF/LFA-1 cells transfected with plasmids encoding GFP-Rab13 DN, DA, or WT (upper) and of BAF/LFA-1 cells stimulated with 100 nM CXCL12 for the indicated times (lower) were subjected to pull-down with GST–MICAL-L2-C. Right: BAF/LFA-1 cells transfected with plasmids encoding V5-Mst1 and GFP-Rab13 were stimulated with CXCL12 for 10 min before being subjected to immunoprecipitation and Western blotting analysis with the indicated antibodies. (E) Control or Mst1 KD BAF/LFA-1 cells were stimulated with CXCL12 for 5 min, and GTP-bound Rab13 was analyzed as described in (D). In Mst1 KD cells, the abundance of Mst1 protein was reduced to 15 ± 2.2% of that of control cells. Data are means ± SD from three independent experiments. (F) Left: The physical association between V5-MST and T7-DENND1C in COS cells was analyzed by immunoprecipitation and Western blotting analysis with the indicated antibodies. Right: COS cells transfected with plasmid encoding T7-DENND1C, together with or without plasmid encoding V5-Mst1, were lysed and immunoprecipitated with anti-T7 antibody. Phosphorylation of DENND1C was detected with Phos-tag-biotin. (G) COS cells transfected with plasmids encoding FLAG-Rab13, T7-DENND1C, and V5-Mst1 were analyzed as described in (D). The abundance of Rab13-GTP in the presence of T7-DENND1C was increased 5 ± 0.7– and 9.8 ± 2.4–fold, respectively, in the absence or presence of Mst1. Data are means ± SD from three independent experiments. (H) Left: Control or Mst1 KD BAF/LFA-1 cells transfected with plasmid encoding T7-DENND1C were stimulated with CXCL12 for the indicated times. Phosphorylation of DENND1C was detected with Phos-tag-biotin. Right: Control or DENND1C KD BAF/LFA-1 cells transfected with plasmid encoding FLAG-Rab13 were stimulated with CXCL12 for the indicated times and then subjected to pull-down with GST–MICAL-L2-C. In DENND1C KD cells, the abundance of DENND1C protein was reduced to 14 ± 2.8% of that of control cells. Data are means ± SD from three independent experiments. For each panel, a representative blot of at least three experiments is shown.

We next examined whether Rab13 was activated and interacted with Mst1 in response to chemokine. To pull down Rab13-GTP, we used glutathione S-transferase (GST) fused to the Rab13-binding domain of MICAL-L2 (MICAL-L2-C), which was identified as a Rab13-GTP–binding protein by yeast two-hybrid screening (21). Whereas Rab13 DA was readily precipitated by GST–MICAL-L2-C, reduced amounts of Rab13 DN or wild-type Rab13 were precipitated, indicating that GST–MICAL-L2-C preferably bound to Rab13-GTP (Fig. 1D). With this GST fusion protein, we observed a substantial increase in the abundance of Rab13-GTP in BAF/LFA-1 cells within 0.5 to 10 min after stimulation with the CXC chemokine CXCL12 (Fig. 1D). About threefold more Mst1 interacted with Rab13 in CXCL12-stimulated BAF/LFA-1 cells compared to that in unstimulated cells (Fig. 1D).

We next examined whether Mst1 and Rab13 were required for their mutual activation. We found that inhibiting Rab13 activation did not affect the kinase activity of Mst1 (fig. S1A). On the other hand, knockdown of Mst1 reduced the extent of CXCL12-dependent activation of Rab13 to about 10% of that in control cells (Fig. 1E), suggesting the involvement of Mst1 in the activation of Rab13 in response to chemokine. DENN domain proteins are a class of Rab GEFs, and they activate individual Rab GTPases (23). Because DENND1C is thought to have GEF activity toward Rab13 (23), we examined whether DENND1C could mediate chemokine-induced Rab13 activation in an Mst1-dependent manner. We found that DENND1C associated with Mst1, which promoted the phosphorylation of DENND1C (Fig. 1F). Expression of exogenous DENND1C in transfected COS cells activated Rab13, and the amount of Rab13-GTP increased twofold when Mst1 was overexpressed (Fig. 1G), suggesting that the Mst1-dependent phosphorylation of DENND1C increased its GEF activity for Rab13. When BAF/LFA-1 cells were stimulated with CXCL12, the abundance of phosphorylated DENND1C increased fivefold, an increase that was prevented by the knockdown of Mst1, indicating that DENND1C was phosphorylated in response to chemokine in an Mst1-dependent manner (Fig. 1H). Furthermore, knockdown of DENND1C in BAF/LFA-1 cells abrogated the activation of Rab13 in response to CXCL12 (Fig. 1H). These results suggest that DENND1C is a substrate of Mst1, and that it links Mst1 to Rab13.

The distribution of Mst1 is regulated by Rab13 activation, which enables LFA-1–dependent adhesion and migration

To examine the subcellular localization of Rab13 and Mst1, we transfected BAF/LFA-1 cells with plasmids encoding GFP-Rab13 and Mst1 fused to mRFP (Mst1-mRFP). Wild-type Rab13 was present in the plasma membrane and a region around the Golgi in unstimulated cells (Fig. 2A and fig. S1B). As previously reported (6), Mst1 was present in a region around the Golgi and cytoplasm (Fig. 2A). When the cells were stimulated with CXCL12, wild-type Rab13 and Mst1 moved to the leading edge where they colocalized (Fig. 2A and fig. S1B). However, Rab13 DN mutant showed a diffuse distribution in the cytoplasm, and upon CXCL12 stimulation, Mst1 did not translocate to the leading edge in cells expressing Rab13 DN cells (Fig. 2A). Rab13 DA also localized at the plasma membrane and around Golgi, and was distributed in the cytoplasm, which was adjacent to areas in which Mst1 was localized (fig. S1, C and D). However, in cells expressing Rab13 DA, Mst1 did not spontaneously translocate to the front membrane, although translocation of Mst1 was more efficient in cells expressing Rab13 DA after stimulation with chemokine (fig. S1D), indicating that Rab13 activation was required, but not sufficient, for the redistribution of Rab13 and Mst1 to the leading edge of polarized cells.

Fig. 2 The physical association of active Rab13 with Mst1 is required for their translocation to the front membrane and for LFA-1 activation.

(A) Left: Confocal microscopic analysis of the localization of GFP-Rab13 WT or GFP-Rab13 DN and Mst1-mRFP in transfected BAF/LFA-1 cells that were left untreated (−) or were treated with 100 nM CXCL12 for 10 min (+). DIC, differential interference contrast. Line profiles of Mst1 (red) and Rab13 (green) intensity along the arrows (X-Y) are shown. Right: Frequency of cells showing Mst1 patches in front in the absence or presence of CXCL12 (n = 40 cells of each group from three independent experiments). *P < 0.002. (B) Left: Confocal microscopic analysis of the localization of mCherry-Rab13 in BAF/LFA-1 cells that were transfected with either scrambled control siRNA or Mst1-specific siRNA and then were incubated in the absence (None) or presence of CXCL12. Right: Frequency of cells showing Rab13 patches in front (n = 30 cells of each group). *P < 0.005. (C) Left: Confocal microscopic analysis of the localization of GFP-Rab13 WT or GFP-Rab13 DN and surface LFA-1 in control or Mst1 KD BAF/LFA-1 cells that were stimulated with CXCL12. LFA-1 was immunostained with an anti-human LFA-1 antibody after the cells were stimulated. Line profiles of LFA-1 (red) and GFP-Rab13 (green) intensity along the arrows (X-Y) are shown. Right: Frequency of cells showing LFA-1 patches in front (n = 50 cells of each group). *P < 0.002. (D) After BAF/LFA-1 cells were incubated on ICAM-1 in the absence or presence of CXCL12, and their shear stress–resistant adhesion was measured as described in Materials and Methods (n = 50 cells of each group). *P < 0.02 compared with control cells expressing Rab13 WT. (E) Control or DENND1C KD BAF/LFA-1 cells were stimulated and stained as described in (C). Line profiles of LFA-1 staining intensity along the arrows (X-Y) are shown. Center: Ratio of cells showing LFA-1 patches in front (n = 35 cells of each group). *P < 0.007, compared with control. Right: After BAF/LFA-1 cells were incubated on ICAM-1 in the absence or presence of CXCL12, their shear stress–resistant adhesion was measured (n = 40 cells of each group). *P < 0.02. For all images, two or three cells from a single experiment that is representative of at least three independent experiments are shown. Scale bars, 5 μm. For each bar graph, data are means ± SEM of triplicate experiments.

Subcellular fractionation by continuous sucrose density gradient centrifugation showed that Rab13 and DENND1C were present together in fractions containing light vesicles (fractions 2 to 4) containing endosomal marker proteins (EEA1 and Rab11), as well as Mst1 and LFA-1, in addition to being present together in the heavier fractions, which included Golgi matrix protein of 130 kD (GM130) and transferrin receptor (TfR), indicative of the Golgi complex (fractions 8 and 9) and the plasma membrane (fractions 10 to 12) (fig. S2A). Extracellular signal–regulated kinase (ERK), which was mainly present in the cytosol, was also found in the heavier fractions (8 to 12) (fig. S2A). Rab13 DA was more abundant in the vesicle-containing fractions than was wild-type Rab13, suggesting that Rab13 activation promotes its redistribution and association with Mst1 in vesicles.

We examined the distribution of Rab13 in Mst1-knockdown (Mst1 KD) cells. As described earlier, after stimulation of cells with CXCL12, Rab13 accumulated at the leading edge in control cells, but Rab13 clustering was not evident in the Mst1 KD cells (Fig. 2B), consistent with impaired Rab13 activation in these cells (Fig. 1E). Although the cellular localization of Rab13 was normal, the number of Rab13-rich membrane protrusions was reduced in Mst1 KD cells compared to that in control cells (Fig. 2B), suggesting that Mst1 might promote the cytoskeletal assembly required for vesicular transport.

Mst1 translocates with LFA-1 to the leading edge of cells by forming a complex with RAPL in response to chemokine (6). We found that the accumulation of Rab13 at the leading edge was also coincident with LFA-1 clustering (Fig. 2C and fig. S1E). Expression of Rab13 DN in BAF/LFA-1 cells inhibited LFA-1 clustering at the leading edge similarly to that in Mst1 KD cells (Fig. 2C) (6). Consistent with impaired LFA-1 clustering, cells expressing Rab13 DN demonstrated reduced adhesion to ICAM-1 in response to CXCL12 (Fig. 2D). DENND1C KD cells also showed defective LFA-1 clustering in response to CXCL12, which was followed by a reduction in LFA-1– and ICAM-1–dependent adhesion (Fig. 2E). In addition, we examined whether LFA-1 clusters were formed by vesicular transport in experiments with pHluorin, which has the unique property of being fluorescent at a neutral pH and not fluorescent in regions with an acidic pH (pH <6.0), such as the lumen of vesicles. The pH-sensitive fluorescent pHluorin and mRFP were tagged to the N-terminal extracellular domain and C terminus of αL, respectively, which enabled us to distinguish LFA-1 localized on the plasma membrane from that localized in intracellular compartments. Analysis of a representative sample of CXCL12-stimulated cells expressing pHluorin-αL-mRFP (fig. S2B) showed that there are two regions containing LFA-1–mRFP, one of which lacked GFP at time zero. Subsequently, these two regions coalesced into a larger GFP-positive region in the protruding membrane that later formed an LFA-1–containing cluster. Together, these data suggest that Rab13-GTP–dependent vesicle transport is involved in LFA-1 clustering and adhesion.

Next, we examined the effects of Rab13 on the LFA-1–dependent migration of cells on ICAM-1. Expression of Rab13 DA in BAF/LFA-1 cells substantially enhanced CXCL12-stimulated motility on ICAM-1 compared to that in control cells (Fig. 3A). In contrast, expression of Rab13 DN inhibited CXCL12-induced migration (Fig. 3B). We also tested Rab8, a Rab protein closely related to Rab13, and Rab1, which showed an active form–dependent interaction with Mst1 in the yeast two-hybrid screen. Expression of a DN form of Rab8 or Rab1 did not substantially affect CXCL12-induced migration (Fig. 3B). Rab13 accumulated at the leading edge and colocalized with LFA-1 clusters in polarized cells, whereas GFP and GFP-Rab1 did not translocate to the leading edge in response to CXCL12 (Fig. 3C). These results suggest that Rab13 exerts specific functions in LFA-1– and ICAM-1–mediated migration. In experiments with small interfering RNA (siRNA), we examined the requirement of Rab13 for LFA-1–dependent adhesion and migration. The abundance of Rab13 protein in Rab13 KD cells was reduced to about 20% of that in control cells (fig. S3A). LFA-1 clustering was not induced in Rab13 KD cells in response to CXCL12 (fig. S3B). The ability of BAF/LFA-1 cells to adhere to ICAM-1 in response to CXCL12 was substantially diminished by knockdown of Rab13 (fig. S3B). Furthermore, the displacement and velocities of Rab13 KD cells during migration on ICAM-1 in the presence of CXCL12 were reduced to less than 15% of those of control cells (fig. S3C).

Fig. 3 Rab13 is required for optimal cell migration on ICAM-1 and for formation of the immunological synapse.

(A) BAF/LFA-1 cells transfected with plasmids encoding GFP-Rab13 WT or GFP-Rab13 DA or with empty plasmid (Control) were allowed to migrate on ICAM-1 in the presence or absence of CXCL12, and displacement of the cells was measured (n = 50 cells of each group). *P < 0.05, **P < 0.01. (B) The displacement of BAF/LFA-1 cells transfected with plasmids encoding DN forms of GFP-Rab13, Rab8, or Rab1 or with empty plasmid on ICAM-1 was determined (n = 50 cells of each group). *P < 0.01. Representative tracks of each cell type are shown at the bottom. Each line represents a single cell track. (C) Confocal microscopic analysis of the localization of GFP-Rab13 and GFP-Rab1 in transfected BAF/LFA-1 cells in the presence or absence of CXCL12. (D) T lymphocytes were stimulated with an anti-CD3 antibody (1 μg/ml) for the indicated times before being subjected to pull-down assays to precipitate active Rab13, which was detected by Western blotting. The relative amount of Rab13-GTP normalized to that of total Rab13 is shown at the bottom. Western blots are representative of three independent experiments. The means ± SD of the relative amounts of Rab13-GTP at each of the indicated time points after stimulation were 1, 1.4 ± 0.13, 1.9 ± 0.48, 2.5 ± 0.79, and 1.8 ± 0.41. (E) Top: 3A9 T cells expressing GFP-Rab13 were mixed with antigen-pulsed (HEL) or unpulsed CH27 B cells (APCs) for 1 hour at 37°C. Cell-cell conjugates were immunostained with an anti-mouse LFA-1 antibody. Bottom: Three-dimensional views of the colocalization of Rab13 with LFA-1 at the pSMAC of the immunological synapse. (F) Left: T cells transfected with empty plasmid or with plasmid encoding Rab13 DN were mixed with an equal number of APCs and then analyzed with flow cytometry. The numbers in each plot represent the percentage of cell-cell conjugates. Right: 3A9 T cells were incubated with APCs, and the supernatants were harvested for IL-2 measurement. An optical density at 437 nm of 1 was equal to 0.25 ng of recombinant mouse IL-2 per milliliter of culture medium. *P < 0.005. For all graphs, data are means ± SEM of triplicate experiments. For all images, representative cells obtained from one experiment that is representative of at least three independent experiments are shown. Scale bars, 5 μm.

Previous studies demonstrated that a complex of RAPL and Mst1 was redistributed to the contact zone between lymphocytes and antigen-presenting cells (APCs), where LFA-1 accumulated in response to antigen. Cross-linking the T cell receptor (TCR) complex with anti-CD3 antibodies resulted in about a threefold increase in the abundance of Rab13-GTP in primary mouse T cells (Fig. 3D). To examine whether TCR-dependent activation of Rab13 was involved in the adhesive interaction between T cells and APCs and in the subsequent production of interleukin-2 (IL-2) by the activated T cells, we performed experiments with 3A9 T cells, which have a hen egg lysozyme (HEL)–specific I-Ak–restricted TCR, and I-Ak–bearing CH27 B cells, which act as APCs (22). 3A9 T cells and CH27 APCs interact with each other in the presence of the HEL antigen and the T cells produce IL-2, and this process is dependent on LFA-1–ICAM-1 interactions (22). We transfected 3A9 T cells with plasmid encoding GFP-tagged wild-type Rab13 and found that Rab13 accumulated at contact sites between the HEL-specific 3A9 T cells and HEL-loaded, but not unloaded, CH27 B cells (Fig. 3E). Rab13 was distributed in a ring-shaped pattern at the contact site and colocalized with LFA-1 at the peripheral supramolecular activation cluster (pSMAC) of the immunological synapse (Fig. 3E). Quantitative analysis revealed that the Rab13 accumulated at contact sites containing LFA-1 clusters in 63% of the cell-cell conjugates (fig. S4). Overexpression of Rab13 DN substantially reduced the numbers of conjugates formed between the transfected 3A9 cells and the APCs, and also resulted in decreased IL-2 production (Fig. 3F). These results suggest that Rab13 is involved in the LFA-1– and ICAM-1–dependent migration and adhesion of lymphocytes and in the formation of the immunological synapse.

Rab13 transports LFA-1 to the leading edge in an αL-dependent manner

We next used live-cell imaging to investigate whether Rab13 delivered LFA-1 to the leading edge in a manner dependent on an interaction between LFA-1 and Mst1. For this purpose, we transfected BAF cells expressing GFP–Rab13 wild-type with plasmids encoding the wild-type αL subunit or an αL mutant (Δ1095) that cannot physically associate with RAPL (5) together with plasmid encoding β2-mRFP, and examined the intracellular redistribution of these molecules in cells treated with CXCL12. Wild-type LFA-1 and Rab13 exhibited a similar distribution at the plasma membrane and perinuclear region in unstimulated cells, and then accumulated at the leading edge in response to CXCL12 (Fig. 4A). The cell surface abundance of LFA-1 containing the αL Δ1095 mutant was similar to that of wild-type LFA-1, and it colocalized with Rab13 in unstimulated cells (Fig. 4A). However, the LFA-1 Δ1095 mutant was not present with Rab13 at the leading edge in response to CXCL12 (Fig. 4, A and B). As expected, the cells expressing the αL Δ1095 mutant did not adhere to ICAM-1 in response to CXCL12 (Fig. 4B). The defective binding of the LFA-1 Δ1095 mutant with the RAPL-Mst1 complex impaired the association of LFA-1 with Rab13 in response to CXCL12 (Fig. 4C), suggesting that active Rab13 associated with the cytoplasmic region of the αL subunit as well as with RAPL-Mst1 (fig. S5A).

Fig. 4 Live-cell imaging of LFA-1 clustering in response to chemokine.

(A) Confocal microscopic analysis of BAF cells expressing GFP-Rab13, αL (WT or Δ1095 mutant), and β2-mRFP were either left unstimulated (None) or stimulated with CXCL12. (B) Left: Frequency of the cells represented in (A) that formed LFA-1 clusters (n = 50 cells of each group). Right: BAF cells expressing WT or Δ1095 mutant forms of αL and β2-mRFP were analyzed for their ability to adhere to ICAM-1 in the presence or absence of CXCL12 (n = 50 cells of each group). Data are means ± SEM of triplicate experiments. *P < 0.001. (C) The physical association of WT or Δ1095 mutant LFA-1, Rab13, RAPL, and Mst1 in transfected BAF/LFA-1 cells in the presence or absence of CXCL12 was analyzed by immunoprecipitation and Western blotting with the indicated antibodies. (D) Confocal microscopic analysis of the translocation of GFP-Rab13 and β2-mRFP with WT αL (left) or the Δ1095 mutant αL (right) in BAF cells incubated on a CXCL12-coated surface for the indicated times. Time 0 represents the first time-lapse image; subsequent images were obtained in the same focal plane. (E) Confocal microscopic analysis of the translocation of GFP-Rab13 WT and β2-mRFP with the Δ1095 αL mutant in BAF cells incubated on a CXCL12-coated surface for the indicated times. (F) Confocal microscopic analysis of the translocation of GFP-Rab13 and Lifeact-mCherry in BAF/LFA-1 cells incubated on a CXCL12-coated surface for the indicated times. Confocal images and Western blots are from single experiments that are representative of at least three independent experiments.

Time-lapse images of BAF/LFA-1 cells on a CXCL12-coated surface demonstrated that wild-type LFA-1 and Rab13 were cotranslocated to the membrane of the developing leading edge (Fig. 4D, movies S1 to S3, and fig. S5B). Rab13 accumulated at the front membrane, which was demarcated from the cell body membrane by the absence of the LFA-1 Δ1095 mutant (Fig. 4D and movies S4 to S6). These data suggest that Rab13 actively transports LFA-1 to the leading edge of the growing membrane, a process that requires the association of the cytoplasmic tail of the αL subunit with RAPL-Mst1.

Although the LFA-1 Δ1095 mutant was absent from the front membrane, it colocalized with the accumulated Rab13 before it extended from the cell body (Fig. 4E and movies S7 to S10, 4-s time point). When the Rab13-enriched projection protruded from the accumulation sites, the LFA-1 Δ1095 mutant did not transfer to that projection during the 8- to 16-s time frame (Fig. 4E and movies S7 to S10). These data suggest that LFA-1 clusters are formed at the same time as the developing Rab13-enriched protrusion. Because formation of the protrusion depends on microtubules and the actin cytoskeleton, we examined the dynamics of microtubules and actin during LFA-1 cluster formation. To visualize microtubule cables, we transfected cells expressing LFA-1–mRFP with plasmid encoding GFP-tagged EB3 (EB3-GFP), which labels the plus ends of growing microtubules. In response to CXCL12, BAF/LFA-1 cells generated protruding membranes in which LFA-1 was enriched; however, the tips of the microtubules visualized by EB3-GFP did not colocalize with the LFA-1–enriched protrusions (fig. S6A). In contrast, when filamentous actin (F-actin) structures were visualized with Lifeact-mCherry (24) in cells expressing GFP–Rab13 wild-type, Rab13 moved to the front of the protrusion along F-actin, and F-actin structures were undetectable after Rab13 reached the leading edge (Fig. 4F).

Mst1 and Rab13 cooperate in F-actin–dependent vesicular transport

VASP is an ENA/VASP family member, and it has an important role in the assembly of actin filaments. VASP contains a proline-rich sequence, which recruits the G-actin–binding protein profilin. Localization of VASP to the leading edge of a migrating cell leads to the local accumulation of profilin, which in turn supplies actin monomers to growing filament ends (25). Some studies reported that the phosphorylation of VASP at Ser157 is important for the localization of VASP to the cell periphery and the induction of elongated protrusions and formation of the leading edge (26, 27). Because activation of the kinase Hippo (Mst1/2) leads to the phosphorylation of Ena by Warts kinase (Lats1) in Drosophila border cells, we examined the involvement of Mst1-dependent phosphorylation of VASP in chemokine-dependent F-actin assembly. As expected, overexpression of Mst1 increased the phosphorylation of VASP at Ser157 compared to that in untransfected cells (Fig. 5A). CXCL12 stimulated an increase in the phosphorylation of VASP at Ser157, whereas VASP phosphorylation was decreased in Mst1 KD cells (Fig. 5B), suggesting that CXCL12 leads to the phosphorylation of VASP Ser157 in an Mst1-dependent manner. Consistent with previous findings (26, 27), we found that wild-type VASP accumulated at the tip of growing F-actin filaments or at the leading edge of cells stimulated with CXCL12, whereas accumulation of the VASP S157A mutant was substantially reduced (Fig. 5C and fig. S6B). LFA-1 clustering occurred at the protrusion where wild-type VASP accumulated at the tip and along F-actin in CXCL12-stimulated cells (Fig. 5D and fig. S6C). The overexpression of the VASP S157A mutant substantially reduced the formation of LFA-1 clusters and the adhesion of cells to ICAM-1 (Fig. 5D). These data suggest that Mst1 promotes F-actin polymerization through the phosphorylation of VASP, which might be crucial for the Rab13-dependent vesicular transport of LFA-1.

Fig. 5 Roles of Mst1 and Rab13 in the actin-dependent transport of LFA-1.

(A) COS cells transfected with plasmid encoding VASP with or without plasmid encoding V5-Mst1 were analyzed by Western blotting to examine the phosphorylation of VASP at Ser157. (B) Control or Mst1 KD BAF/LFA-1 cells were stimulated with CXCL12 for the indicated times and then were analyzed by Western blotting with the antibodies against the indicated proteins. (C) Confocal microscopic analysis of the translocation of GFP-VASP (WT or S157A) and Lifeact-mCherry in transfected BAF/LFA-1 cells incubated on a CXCL12-coated surface for the indicated times. The graph shows the ratio of the abundance of GFP-VASP WT or GFP-VASP S157A at the tip of F-actin relative to that in the rest of the cell (n = 50 cells of each group). *P < 0.02. (D) Left: Confocal microscopic analysis of BAF/LFA-1 cells expressing GFP-VASP (WT or S157A) and Lifeact-mCherry that were stimulated with CXCL12 for 10 min and then were immunostained with anti–LFA-1 antibody. The z-stack analysis of representative CXCL12-stimulated cells is shown. Center: Frequency of those cells expressing VASP WT or VASP S157A that showed LFA-1 patches (n = 30 cells of each group). Right: BAF/LFA-1 cells expressing VASP WT or VASP S157A were analyzed for their ability to adhere to ICAM-1 in the presence of CXCL12 (n = 30 cells of each group). *P < 0.03. (E) Top: Halo-tagged myosin Va expressed in COS cells was pulled down with GDP-loaded or GTPγS-loaded Rab13-GST. Bottom: Lysates of COS cells transfected with plasmids encoding myosin Va and GFP-Rab13 (WT or DA) were immunoprecipitated and analyzed by Western blotting with the indicated antibodies. (F) Top left: Western blotting analysis of the myosin Va head domain in BAF/LFA-1 cells. Bottom left: Frequency of LFA-1 clustering in the indicated cells in the absence or presence of CXCL12 (n = 50 cells of each group). Top right: Confocal microscopic analysis of the indicated cells incubated in the absence or presence of CXCL12 and immunostained with anti–LFA-1 antibody. Bottom right: The ability of the indicated cells to adhere to ICAM-1 in the presence of CXCL12 was analyzed (n = 50 cells of each group). *P < 0.01. For all images, one or two cells obtained from single experiments that are representative of at least three independent experiments are shown. Scale bars, 5 μm. Data in bar graphs are means ± SEM of triplicate experiments.

Myosin Va was reported to mediate Rab GTPase–dependent vesicular transport (2830). Because the transcripts of the myosin Va gene undergo tissue-specific alternative splicing, we cloned a complementary DNA (cDNA) encoding myosin Va from lymphocytes, expressed it in COS cells, and examined the association between myosin Va and Rab13. We found that myosin Va specifically bound to Rab13-GTP (Fig. 5E). In unstimulated cells, myosin Va was present in the cytoplasm, where it partially colocalized with wild-type Rab13 near the Golgi (fig. S7A). Upon stimulation of the BAF/LFA-1 cells with CXCL12, myosin Va moved and transiently colocalized with wild-type Rab13 and LFA-1 at the leading edge (fig. S7, A and B). Overexpression of the head domain of myosin Va inhibited the clustering of LFA-1 in CXCL12-stimulated cells (Fig. 5F) and inhibited the LFA-1– and ICAM-1–dependent migration and adhesion of BAF/LFA-1 cells (Fig. 5F and fig. S7C).

Rab13 is indispensable for lymphocyte trafficking

Finally, we examined the activation and function of Rab13 in primary mouse T cells. We found that Rab13 was activated in T cells upon exposure to the CC chemokine CCL21 (Fig. 6A). To generate Rab13−/− mice, we used gene targeting to generate mice carrying floxed Rab13 alleles (Rab13f/f) in which exon 1 containing the initiation codon was flanked with loxP sites. We then crossed this mice with CAG-Cre transgenic mice to delete exon 1 ubiquitously. Southern and Western blotting analysis confirmed that Rab13 protein was not detectable because of the deletion of exon 1 in Rab13−/− T cells (fig. S8A). Rab13−/− mice were born with the expected Mendelian frequencies and grew normally. There was no difference in the ratio of naïve T cells between Rab13f/f and Rab13−/− mice at 6 to 8 weeks old (fig. S8B).

Fig. 6 Impaired redistribution of Mst1 and LFA-1 to the front membrane in Rab13-deficient T cells in response to chemokine.

(A) Primary mouse T cells were stimulated with 100 nM CCL21 for the indicated times and then were subjected to pull-down assays with GST–MICAL-L2-C. The Western blot shown is representative of three independent experiments. (B) Confocal microscopic analysis of the localization of Mst in T cells incubated in the absence or presence of CCL21 for 10 min. Z-stack analysis of the stimulated cells (yellow asterisks in the upper figure) is shown at the bottom. Line profiles of Mst along the arrows (X-Y) of z-stack confocal images are shown. The bar graph shows the percentages of polarized cells that exhibited an increase in Mst staining intensity at the front membrane in the absence or presence of CCL21 (n = 50 cells or each group). *P < 0.02. (C) The cell surface expression of LFA-1 in T cells and B cells from Rab13f/f and Rab13−/− mice was determined by flow cytometry. (D) Confocal microscopic analysis of T cells from Rab13f/f and Rab13−/− mice that were stimulated with CCL21 and then immunostained with anti–LFA-1 and anti-CD44 antibodies. The graph shows the percentages of polarized cells in the absence or presence of CCL21 (n = 50 cells of each group). *P < 0.02. For each image, three cells obtained from an experiment that is representative of at least three independent experiments are shown. Scale bars, 5 μm. For each bar graph, data are means ± SEM of triplicate experiments.

We examined whether a deficiency in Rab13 in primary lymphocytes affected the subcellular localization of Mst1 in response to chemokine. As previously described (6), Mst1 translocated to the front membrane of Rab13f/f T cells upon stimulation with CCL21, whereas Mst1 did not translocate to the leading edge, but remained diffusely distributed in the cytoplasm of Rab13−/− T cells (Fig. 6B). The abundance of CCR7, the receptor for CCL21, in T cells from Rab13−/− mice was similar to that in T cells from Rab13f/f mice (fig. S8B). These results suggest that Rab13 is required for the chemokine-dependent redistribution of Mst1 in primary T cells. Next, we examined the effect of Rab13 deficiency on LFA-1 clustering. The abundance of LFA-1 in T and B cells from Rab13−/− mice was similar to that in T and B cells from Rab13f/f mice (Fig. 6C). About 30% of CCL21-stimulated T cells from wild-type mice showed polarized morphologies with a leading edge and uropod, to which LFA-1 and CD44 were clustered, respectively (Fig. 6D). However, in most CCL21-treated Rab13−/− T cells, the redistribution of LFA-1 was impaired (Fig. 6D).

The ability of T cells to adhere to ICAM-1 in response to CCL21 was substantially reduced by Rab13 deficiency (Fig. 7A). The displacement and velocity of Rab13−/− T cells during migration on ICAM-1 in the presence of CCL21 were decreased to 43 and 58% of those in control T cells, respectively (Fig. 7B and movies S11 to S14). These data suggest that Rab13 is involved in the clustering of LFA-1 and the efficient adhesion and migration of primary T cells in response to chemokine. Because impaired LFA-1 clustering leads to a diminished lymphocyte homing capability (8, 9), we next examined the effects of Rab13 deficiency on lymphocyte homing into the spleen and lymph nodes. For this purpose, we differentially labeled Rab13f/f and Rab13−/− T cells and injected them into normal mice. After 1 hour, the numbers of cells from both populations were almost equally present in the blood, whereas the extent of trafficking of Rab13−/− T cells into the spleen and peripheral lymph nodes was reduced to about 50 and 30%, respectively, of that of control cells (Fig. 7C). We also examined the transfer of injected cells to the liver and lung, as previously reported (31), but there was no increase in the number of Rab13−/− T cells (fig. S8C), suggesting that exclusion from lymphoid tissues might affect cell survival. Because LFA-1 is not involved in lymphocyte homing to the spleen (32), we analyzed the chemotaxis of Rab13-deficient T cells to CCL21 in transwell migration assays. The numbers of Rab13−/− T cells that were attracted by CCL21 and migrated through the uncoated transwell filter were substantially reduced compared with those of Rab13f/f cells (fig. S8D). On the other hand, the numbers of Rab13f/f T cells that transmigrated through ICAM-1–coated transwell filters were diminished compared to the numbers of cells that migrated through uncoated filter, which was due to adhesion to ICAM-1, whereas there was no corresponding decrease in the numbers of migrating Rab13−/− cells (fig. S8D). These data suggest that Rab13 deficiency affects not only the LFA-1–dependent adhesive activity of cells but also their chemotaxis.

Fig. 7 Loss of Rab13 impairs the trafficking of lymphocytes in vivo.

(A) After incubation with or without CCL21 for 10 min, the shear stress–resistant adhesion of T cells from Rab13f/f and Rab13−/− mice to ICAM-1 was measured (n = 50 cells of each group). *P < 0.02. (B) Top: The displacement and velocity of T cells from the indicated mice were measured on ICAM-1 in the presence or absence of CCL21 (n = 50 cells of each group). *P < 0.001. Bottom: Representative tracks of single cells in the presence of CCL21. (C) Left: Analysis of the homing of adoptively transferred T cells from Rab13f/f and Rab13−/− mice to the blood, spleens, and peripheral lymph nodes (LNs) of recipient mice by flow cytometry, as described in Materials and Methods. Numbers besides the boxed areas indicate the ratio of T cells from Rab13−/− mice to that from Rab13f/f mice. Right: Ratios of T cells from Rab13−/− mice to T cells from Rab13f/f mice (n = 4 experiments). *P < 0.001. (D) Left: Pictures and frozen sections of spleens and inguinal lymph nodes from Rab13f/f and Rab13−/− mice. The frozen sections were immunostained with anti-CD3 and anti-B220 antibodies to visualize T cells and B cells, respectively (×100). Data shown are representative of five independent experiments. Right: Numbers of T cells and B cells from the spleens, lymph nodes, and blood of Rab13f/f and Rab13−/− mice (n = 10). *P < 0.001. For each bar graph, data are means ± SEM of triplicate experiments.

Because of the defective homing ability of Rab13-deficient cells, Rab13−/− mice had smaller lymphoid tissues, such as spleens and lymph nodes, than did wild-type mice (Fig. 7D). Immunohistological analysis showed decreases in the sizes and cellular densities of B cell follicles and T cell areas of the secondary lymphoid tissues of Rab13−/− mice, although the architecture of the lymphoid tissues, including the segregation of T cells and B cells, appeared to be normal (Fig. 7D). The numbers of T cells and B cells in the peripheral lymph nodes and spleen of Rab13−/− mice were reduced compared to those of wild-type mice, although there was no substantial difference in their numbers in the blood (Fig. 7D). These data suggest that Rab13 plays indispensable roles in lymphocyte trafficking.

DISCUSSION

Here, we first demonstrated a substantial link between Mst1 and Rab13. The Rab13-dependent clustering of LFA-1 generates adhesion sites on ICAM-1 at the front membrane, which leads to high-speed migration by lymphocytes. Rab13-deficient mice demonstrated hypoplastic lymphoid tissues caused by the impaired trafficking capacity of lymphocytes; however, the digestive and sex organs in these mice were rather enlarged, as was described for mice deficient in Mst1 and Mst2 (7), confirming a previously uncharacterized connection between Rab13 and Mst1 in vivo.

Loss of Mst1 impaired the activation of Rab13 and the extension of projections from the cell body in lymphocytes. The kinase activity of Mst1 is required for LFA-1 clustering at the leading edge of cells (6). Mst1 not only associates with the cytoplasmic tail of the αL subunit and mediates Rab13-dependent LFA-1 transport but also activates Rab13 and promotes the development of F-actin filaments in response to chemokine by controlling the phosphorylation of DENND1C and VASP (fig. S9). A study reported that NDR1/2 (nuclear Dbf2-related 1/2), downstream effectors of Mst1, phosphorylates Rabin8, a GEF for Rab8, and activates Rab8, which is critical for ciliogenesis (33) and dendritic spine development (34). DENND1C does not have any motifs that are targeted by NDR family kinases; thus, it might be a direct substrate of Mst1. Thus, our findings suggest that Rab13 is a key signaling effector of Mst1 that facilitates the migration and adhesion of lymphocytes; however, we cannot exclude the possibility that other Rab family GTPases could also be involved in this function.

Although internalization and recycling play an important role in cell migration through integrins (18, 19, 3537), our previous reports demonstrated that the Rap1-RAPL-Mst1 signaling pathway does not affect Rab5- and Rab11-dependent endocytosis or LFA-1 recycling (6, 38). LFA-1 clustering at the leading edge depends on the cytoplasmic tail of the αL subunit, but not on β2, and LFA-1 clusters do not colocalize with Rab4, Rab5, or Rab11 (6, 38). The internalization and recycling of LFA-1 on vesicles is thought to be promoted by the GTPase Rap2 (39). Rap2 colocalizes with the TfR, which is consistent with the other reports linking intracellular integrins to recycling vesicles that express the TfR (39). However, RAPL and Mst1 colocalize with vesicular compartments distinct from those containing TfR (38). Thus, it is likely that LFA-1–transporting vesicles containing Rap2 are distinct from those of the Rap1-RAPL-Mst1 pathway that require Rab13. Once the cells adhered to ICAM-1 through LFA-1 clusters, they often moved, keeping a similar adhesion point. We speculate that Rab13-dependent LFA-1 clusters that form at the front membrane might be essential for the generation of a new attachment point, which supports skipping movements and a rapid change in directionality, and that Rap2-dependent LFA-1 endocytosis and recycling is necessary for static movement after attachment.

We showed that Rab13 colocalized with an LFA-1–containing ring at the immunological synapse, and that inhibiting Rab13 impaired the formation of the immunological synapse between T cells and APCs, indicating that Rab13 likely plays an indispensable role in distributing LFA-1 at these contact sites. T cells recognize as few as 10 activating peptide-bound major histocompatibility complexes (pMHCs) among about 1 million self-pMHC molecules on the surface of APCs (40). The recognition of activating pMHC complexes results in acute T cell arrest and stable signal integration through the formation of three SMACs: a central SMAC that contains clustered antigen receptors and kinases; a peripheral SMAC enriched in LFA-1; and a distal SMAC that is marked by a critical tyrosine phosphatase (41). Our results suggest that Rab13-dependent vesicular transport is involved in the formation of LFA-1 rings and T cell activation when T cells interact with APCs.

Naïve lymphocytes recirculate continually between peripheral lymphoid tissues through the blood and lymphatic systems for the purpose of immunosurveillance. Recirculating naïve lymphocytes enter across the high endothelial venule (HEV) into lymphoid tissues by means of specialized interactions with capillaries. Chemokine-dependent activation of LFA-1 in lymphocytes is crucial for firm adhesion of the cells to ICAM-1 on the HEV and subsequent transmigration into the tissues. We found that impaired LFA-1 activation in Rab13-deficient lymphocytes was one of the main causes of the reduced lymph node sizes observed in Rab13−/− mice, but we could not exclude the possibility that these findings may be in part a result of defects in stromal cells.

Lymphocytes are the most dynamic cells in the body in terms of their motility, and they can migrate rapidly within secondary lymphoid organs in search of cognate antigens that are presented by dendritic cells, and in the extravascular inflamed interstitial tissues. The cooperative association of Mst1 with Rab13 coupled the clustering of LFA-1 with the development of cell protrusions that might facilitate the generation of attachment sites in the direction of cell migration. This adhesion machinery acts as a driving force to enable the high-velocity movement of lymphocytes and promote lymphocyte trafficking in vivo. Elucidating the roles of the active movement of lymphocytes in vivo with Rab13-deficient mice has shed light on the adhesion-dependent mechanisms that regulate immune surveillance and immune responses.

MATERIALS AND METHODS

Cell culture and mice

BAF pro-B cell lines, the 3A9 T cell hybridoma, and CH27 B lymphoma cells were maintained as previously described (22). C57BL/6 mice were obtained from Shimizu Laboratory Supplies and were used as wild-type mice. CAG-Cre mice were provided by J.-i. Miyazaki (Osaka University, Osaka, Japan). Floxed Rab13 mice and CAG-Cre mice were bred, and Rab13-deficient mice without CAG-Cre were maintained under specific pathogen–free conditions. Homozygous mice were obtained by interbreeding the heterozygous mice. For all experiments, 7- to 8-week-old littermates were used. All experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of Kitasato University (Kanagawa, Japan). T cells were isolated from the lymph nodes or spleens of C57BL/6 mice with MidiMACS (Mitenyi Biotec).

Antibodies and reagents

Fluorescent isothiocyanate (FITC)–conjugated anti-mouse LFA-1 antibody (M17/4; BD PharMingen), anti-human LFA-1 antibody (TS2/4, TS1/18; American Type Culture Collection), anti-mouse CD3 antibody (145-2C11; BD PharMingen), and Alexa Fluor 633–conjugated goat anti-rat or anti-mouse immunoglobulin G (IgG) (Invitrogen) were used for immunostaining. Antibodies specific for Mst1/2, Rab13 (Millipore), T7 (Novagen), Myc (9E10; American Type Culture Collection), V5 (Novagen), GFP (Clontech), β-actin (Sigma), VASP, VASP pSer157, myosin Va, EEA1, ERK (Cell Signaling Technology), DENND1C, CD71 (TfR), GM130, integrin-β2 (Santa Cruz Biotechnology), and human LFA-1 (BD Transduction Laboratories) and horseradish peroxidase (HRP)–conjugated goat anti-rat, anti-rabbit, or anti-mouse IgG (Cell Signaling Technology) were used for immunoprecipitations and Western blotting. FITC-conjugated anti-mouse CD3 and phycoerythrin-conjugated anti-mouse B220 were used for flow cytometric analysis and immunostaining of mouse lymphoid tissues. The Golgi was stained with BODIPY TR ceramide. Phos-tag-biotin and streptavidin-HRP (Wako Pure Chemical Industries) were used to detect phosphorylated proteins.

DNA constructs and transfections

cDNAs encoding Rab13, Rab8, and Rab1 were subcloned into pEGF-C1 (Clontech) as previously described (42). The DA (Q67L) and DN (T22N) mutants were also subcloned similarly. A cDNA construct encoding full-length MST1 and the N-terminal V5-epitope tag was subcloned into pcDNA3 and pcDNA4 (Life Technologies). The Mst1ΔC (1 to 330) and Mst1ΔN (331 to 487) mutants were also subcloned similarly. A cDNA sequence encoding the coiled-coil domain of MICAL-L2 was subcloned into the pGEX vector (GE Healthcare Bio-Sciences) and expressed in Escherichia coli BL21 as a GST fusion protein. A cDNA sequence encoding myosin Va was cloned into pcDNA3. A cDNA sequence encoding human VASP was purchased from the Kazusa DNA Research Institute. Venus, mRFP1, and mCherry were fused to the C termini of Mst1, the αL subunit, the β2 subunit, VASP, myosin Va, and Lifeact with a linker (GGGGS)4. cDNA encoding DENND1C was cloned into pEF-T7. The pH-sensitive fluorescent pHluorin and mRFP were fused to the N-terminal extracellular domain and C terminus of the αL subunit, respectively. Lifeact-mCherry was provided by A. Ida (Kyoto University). cDNAs encoding the wild-type or mutant (1095) αL and β2 subunits of LFA-1 were previously described (3). All constructs were verified by sequencing.

RNA-mediated interference and gene expression by lentiviral transduction

siRNA-mediated knockdown was used to reduce the abundances of mouse Mst1, Rab13, and DENND1C. A 19-nucleotide, sense siRNA sequence (5′ to 3′) or a scrambled control siRNA sequence was introduced into BAF/LFA-1 cells with a lentiviral vector that also did or did not encode GFP (a gift from H. Miyoshi, RIKEN, Wako, Japan), which contained the siRNA construct under the control of the H1 promoter cassette. The production and concentration of lentivirus particles were assessed as previously described (6). The transduction efficiencies were greater than 90%.

Immunoprecipitation and Western blotting analysis

Cells were transfected with the indicated expression plasmids with polyethylenimine and were lysed in buffer [1% (v/v) NP-40, 150 mM NaCl, 25 mM tris-HCl (pH 7.4), 10% (v/v) glycerol, 2 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, and 0.1 mM aprotinin]. Immunoprecipitations and Western blotting were performed as previously described (5).

Flow cytometric analysis

Flow cytometric analysis was performed as described previously (3). Cells (1 × 106) were incubated in staining buffer containing antibodies (5 μg/ml) on ice for 30 min. The cells were washed twice with the staining buffer, further incubated with FITC or allophycocyanin-conjugated goat anti-mouse IgG F(ab′)2 fragments (1 μg/ml, eBioscience), and subjected to flow cytometric analysis with a Gallios flow cytometer (Beckman Coulter).

Immunofluorescence staining

Cryostat sections of frozen tissues (10-μm thickness) were fixed with acetone, air-dried, and stained with the indicated antibodies. Chemokine-stimulated T lymphocytes were stained with the indicated antibodies as described previously (9). Stained samples were observed with a confocal laser microscope (LSM510 META, Carl Zeiss). Cells with segregated LFA-1 and CD44 accompanied with elongated cell shapes were considered to be polarized cells. Cells showing increases of more than fivefold in the staining intensity of a molecule on the front membrane compared to the peak value of intensity on the sides and rear of the cell were considered to have patches of the molecule in the front of the cells.

Pull-down assays

Rab13-GTP protein was pulled down with GST fused to the coiled-coil region of MICAL-L2 (amino acid residues 806 to 1009) (GST–MICAL-L2-C) as previously described (21). Briefly, 107 cells were lysed in ice-cold lysis buffer [1% Triton X-100, 50 mM tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, and 0.5 mM aprotinin] and were incubated with GST–MICAL-L2-C fusion proteins coupled to glutathione agarose beads for 1 hour at 4°C. The beads were washed three times with lysis buffer and then subjected to Western blotting analysis.

Analysis of lymphocyte migration on ICAM-1

Migration of lymphocytes on ICAM-1 was performed as previously described with a Delta T Open Dish System (Bioptecs Inc.) with immobilized recombinant human or mouse ICAM-1–Fc (0.5 μg/ml) with or without chemokine (9, 12). A total of 2 × 105 cells were loaded onto the ICAM-1–coated dish. Phase-contrast images were obtained with an Olympus Plan Fluorito 10×/0.3 numerical aperture objective every 30 s for 30 min at 37°C with a heated stage for ΔT dishes. The frame-by-frame displacements and lymphocyte velocities were calculated by automatically tracking individual cells with MetaMorph software (Molecular Devices). In each field, 50 randomly selected cells were manually tracked to measure the median velocity and displacement from the starting point.

Confocal microscopy and time-lapse imaging

Immunostaining and live-cell imaging were performed as previously described (6). Nonadherent cells incubated with or without chemokine were fixed in suspension and immobilized on poly-l-lysine–coated slides before staining. Confocal images were obtained with a 63× objective lens. 4′,6-Diamidino-2-phenylindole (Life Technologies) was used to visualize the nuclei. Time-lapse confocal images were also obtained in multitrack mode. Line profiles of the confocal images were obtained with the software Image-Pro Plus (Media Cybernetics).

Detachment assay

Adhesion assays with ICAM-1–coated plates were performed as previously described with a temperature-controlled parallel flow chamber (FCS2, Bioptechs Inc.) with immobilized recombinant ICAM-1–Fc (0.5 μg/ml) (9). The cells were incubated with 100 nM CXCL12 (R&D Systems) for 10 min and then shear stress was applied for 1 min at 2 dynes/cm2.

T cell stimulation by APCs and IL-2 measurement using conjugation assays

3A9 T cells (3 × 104) were cultured with CH27 B cells (3 × 104) as APCs with or without HEL (100 μg/ml, Sigma). The amount of IL-2 in the culture supernatants was measured with the IL-2–dependent cell line CTLL-2, as described previously (22). APCs were incubated for 16 hours with or without HEL (100 μg/ml) and then were labeled with CellMask Deep Red plasma membrane stain (Life Technologies). T cells were labeled with 0.1 μM 5,6-carboxyfluorescein diacetate (CFSE; Life Technologies). T cells were incubated with an equal number of APCs (1 × 105 cells) for 30 min at 37°C and then were analyzed with a FACSCalibur flow cytometer (Becton Dickinson).

Homing assay

Lymphocytes were adoptively transferred as described previously (8). Purified T cells from Rab13f/f or Rab13−/− mice were labeled with 1 μM CFSE and 10 μM 5-(and-6)(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR; Life Technologies). Equal numbers of labeled control (Rab13f/f) and Rab13-deficient cells (total: 1 × 106) were injected intravenously into normal C57BL/6 mice. After 1 hour, inguinal and axillary lymph node cells, splenocytes, and peripheral blood mononuclear cells were analyzed by flow cytometry. Reversal of the fluorescent dyes gave the same results.

Transwell migration assays

Purified T cells were added to transwell inserts with 5-μm-diameter pores (Kurabo Industries) at 2 × 106/ml in 500 μl of RPMI medium containing 1% fetal bovine serum (FBS). Into the lower chambers was added 500 μl of RPMI, 1% FBS containing CCL21. After incubation for 1 hour, the numbers of cells that migrated to the lower wells were counted. In some experiments, the inserts were coated with recombinant ICAM-1–Fc beforehand.

Statistical analysis

Student’s two-tailed t test was used to compare experimental groups, and P < 0.05 was considered to be statistically significant.

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/7/336/ra72/DC1

Fig. S1. Effects of Rab13 on the kinase activity of Mst1 and the localization of Rab13 and LFA-1.

Fig. S2. Involvement of vesicular transport in the clustering of LFA-1.

Fig. S3. Analysis of the motility of Rab13-knockdown cells.

Fig. S4. Quantitation of the accumulation of Rab13 at contact sites between T cells and APCs.

Fig. S5. LFA-1 associates with Mst1 and is cotranslocated to the leading edge with Rab13.

Fig. S6. Involvement of VASP in the clustering of LFA-1 in response to CXCL12.

Fig. S7. Colocalization of Rab13 and myosin Va.

Fig. S8. Generation of Rab13-deficient mice and analysis of the cell surface markers and chemotaxis of Rab13-deficient T cells.

Fig. S9. Model for the Rab13- and Mst1-dependent activation of LFA-1 in lymphocytes.

Table S1. Summary of the binding of Rab family GTPases to Mst1.

Movies S1 to S14.

REFERENCES AND NOTES

Acknowledgments: We thank M. Kim, Y. Ouchi, D. Ishizuka, and H. Suda for technical assistance. Funding: This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan. Author contributions: K.K. designed and performed the experiments and wrote the paper; M.O., A.N., K.E., and S.I. performed the experiments; and T.K. and M.F. contributed to the analysis of the Rab GTPases and commented on the experiments and paper. Competing interests: The authors declare that they have no competing interests.
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