Articles
Synaptotagmin-mediated vesicle fusion regulates cell migration
© 2010 Nature America, Inc. All rights reserved.
Richard A Colvin1,8, Terry K Means1, Thomas J Diefenbach2, Luis F Moita1,3, Robert P Friday1, Sanja Sever4, Gabriele S V Campanella1, Tabitha Abrazinski1, Lindsay A Manice1, Catarina Moita1,3, Norma W Andrews5, Dianqing Wu6, Nir Hacohen1,7 & Andrew D Luster1 Chemokines and other chemoattractants direct leukocyte migration and are essential for the development and delivery of immune and inflammatory responses. To probe the molecular mechanisms that underlie chemoattractant-guided migration, we did an RNA-mediated interference screen that identified several members of the synaptotagmin family of calcium-sensing vesicle-fusion proteins as mediators of cell migration: SYT7 and SYTL5 were positive regulators of chemotaxis, whereas SYT2 was a negative regulator of chemotaxis. SYT7-deficient leukocytes showed less migration in vitro and in a gout model in vivo. Chemoattractantinduced calcium-dependent lysosomal fusion was impaired in SYT7-deficient neutrophils. In a chemokine gradient, SYT7deficient lymphocytes accumulated lysosomes in their uropods and had impaired uropod release. Our data identify a molecular pathway required for chemotaxis that links chemoattractant-induced calcium flux to exocytosis and uropod release. Chemoattractant-directed cell migration is critical for the generation and delivery of immune and inflammatory responses1. Defining the molecular mechanisms that control directed cell movement is therefore essential for understanding the function of cells of the immune response, the host response to infection and tumors, and immune-mediated auto immune and inflammatory diseases. Chemokines and other classical chemoattractants, such as formyl-Met-Leu-Phe (fMLP), C5a and leukotriene B4, induce directed cell migration through the activation of seven-transmembrane-spanning G protein–coupled receptors2. Chemoattractant receptors form a related subfamily of ~50 G protein– coupled receptors that all couple to the pertussis toxin–sensitive cAMPinhibitory heterotrimeric guanine nucleotide–binding protein Gi. Chemokine receptors and other chemoattractant receptors are found on all leukocyte lineages. Activation of chemoattractant receptors transforms a chemical signal in the form of a gradient into a biophysical program that results in leukocyte shape change and directed cell movement3. A leading edge called the lamellopodia and a trailing edge called the uropod characterize the polarized migrating leukocyte that develops after activation of chemoattractant receptors. These complex changes involve cycles of membrane protrusions and contractions, polymerization and depolymerization of F-actin, and adhesion and de-adhesion. These processes are coordinated through the activation of multiple signaling pathways that are only partially understood4,5. After chemokines and chemoattractants bind to the extracellular domains of their cognate G protein–coupled receptor, the Gα and Gβγ
subunits of Gi are liberated. The membrane-bound Gβγ subunits are thought to directly activate downstream signaling pathways, such as the β2 and β3 isoforms of phospholipase C (PLC)6. PLC then hydrolyzes phosphatidylinositol-(4,5)-bisphosphate to produce inositol1,4,5-triphosphate (Ins(1,4,5)P 3) and diacyglycerol. Ins(1,4,5)P 3 induces the mobilization of intracellular calcium stores, which in turn has effects on calmodulin and myosin light-chain kinase7,8. An increase in intracellular free calcium is a hallmark of chemoattractant stimulation and has been linked to directional sensing, cytoskeleton redistribution, traction force regeneration and the relocation of focal adhesions3,4,9,10. The Gβγ subunits also activate phosphatidylinositol-3-OH kinase, which leads to changes in intracellular phosphatidylinositol-3,4,5triphosphate11,12. Studies have indicated that phosphatidylinositol(3,4,5)-triphosphate is an important mediator in the sensing of chemoattractant gradient, facilitating the recruitment of pleckstin homo logy domain–containing proteins to the cell membrane13. The Rho family of small GTPases, which includes Rac1, Cdc42 and RhoA, as well as their exchange factors (for example, Vav) and kinases (for example, ROCK), has also been shown to have critical roles in linking the activation of chemoattractant receptors to cytoskeletal changes and the formation of lamellipodia and uropods14. Rac1 and Cdc42 localize to the leading edge of the cell and regulate the generation of cell protrusions, whereas RhoA localizes to the trailing edge of the migrating cell, where it regulates nonmuscle myosins and cell contraction and de-adhesion14.
1Center
for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital (MGH) and Harvard Medical School, Boston, Massachusetts, USA. 2Ragon Institute of MGH, Massachusetts Institute of Technology (MIT) and Harvard, Boston, Massachusetts, USA. 3Cell Biology of the Immune System Unit, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal. 4Division of Nephrology, Massachusetts General Hospital, Boston, Massachusetts, USA. 5Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA. 6Yale University School of Medicine, Yale University, New Haven, Connecticut, USA. 7Broad Institute, Cambridge, Massachusetts, USA. 8Present address: Novartis Institute for Biomedical Research, Cambridge, Massachusetts, USA. Correspondence should be addressed to A.D.L. (
[email protected]). Received 2 December 2009; accepted 12 April 2010; published online 16 May 2010; doi:10.1038/ni.1878
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Figure 1 Screen of chemotaxis by shRNA identifies synaptotagmins SYT2, SYTL5 and 12 SYT7. (a) Primary chemotaxis screen showing the chemotactic indices of SupT1 cells 10 120 infected with 1,500 arrayed lentiviruses encoding shRNA targeting 300 genes. Horizontal 8 100 80 6 axis, individual samples; vertical axis, natural logarithm (Ln) of the chemotactic index; WT 60 4 boxes outline results for cells infected with viruses targeting genes selected as putative 40 Syt7 –/– 2 positive (green) or negative (red) regulators of chemotaxis. (b) Transwell chemotaxis of 20 P = 0.009 0 0 SupT1 cells infected with viruses containing shRNA targeting genes encoding eGFP 0 1 3 10 30 100 (sheGFP), SYTL5 (shSYTL5), SYT2 (shSYT2) or SYT7 (shSYT7). (c) Transwell chemotaxis CXCL12 (nM) of THP-1 cells infected with viruses targeting genes encoding eGFP, SYTL5 or SYT2. (d) Quantitative PCR analysis of SYT2 mRNA abundance in SupT1 cells infected with viruses targeting genes encoding eGFP or SYT2, presented relative to the abundance of GAPDH mRNA (encoding glyceraldehyde phosphate dehydrogenase). (e) Immmunoblot analysis of extracts from SupT1 cells infected with lentiviruses targeting genes encoding eGFP or SYT2, probed with antibody to SYT2 (anti-SYT2; top) or anti-β-actin (bottom). (f) Quantification of SYT2 protein. (g) Transwell chemotaxis of wild-type (WT) and Syt7−/− primary splenocytes. P values, two-way analysis of variance (ANOVA). Data are representative of an experiment done once (a) or experiments done three times (b,c,g; mean and s.e.m. of two samples), at least four times in duplicate (d; error bars, s.e.m.) or twice (e,f).
Despite the insights noted above, many gaps remain in the understanding of how chemokines and other chemoattractants control this highly integrated multistep process of cell migration. We therefore undertook an RNA-mediated interference (RNAi)-based screen to identify genes previously unknown to be important for chemotaxis. We chose to initially study the effect of gene knockdown on T cell chemotaxis induced by the chemokine CXCL12. CXCL12 and its receptor, CXCR4 (A000636), are essential molecules that direct the migration of essentially all cells, including lymphocytes and hemato poietic stem cells, and are considered to be the primordial chemokine receptor–ligand pair15,16. RESULTS To identify genes and pathways required for chemoattractant-induced chemotaxis, we did a lentivirus-based RNAi screen. As our model system, we examined CXCL12-induced, CXCR4-mediated chemotaxis of lymphocytes. We generated short hairpin RNA (shRNA) molecules expressed from lentiviral vectors based on human immunodeficiency virus type 1 and tested their ability to knock down gene expression in lymphocytes. For this, we coinfected SupT1 cells (a human T cell lymphoma line) with lentivirus expressing an shRNA targeting enhanced green fluorescent protein (eGFP) plus lentivirus expressing eGFP and found that eGFP expression was much lower in those cells than in cells coinfected with lentivirus expressing ‘scrambled’ shRNA and lentivirus expressing eGFP (Supplementary Fig. 1a). We then assessed 1,500 arrayed shRNA-encoding viruses obtained from The RNAi Consortium shRNA Library 17, which targeted 300 genes predominantly but not exclusively encoding kinases and phosphatases, as well as lentiviruses encoding shRNA targeting eGFP, for their ability to affect chemotaxis (Fig. 1a; targeted genes, Supplementary Table 1). After infecting and selecting cells, we assessed their ability to migrate to CXCL12 in a Transwell chemotaxis chamber, in a primary screen18 (Fig. 1a). By secondary screening we confirmed 11 genes that affected CXCL12induced migration. These included two genes, SPP1 (encoding osteopontin) and ZAP70 (encoding the tyrosine kinase Zap70), that have been linked before to chemotaxis19,20. Infection with two viruses targeting distinct regions of SPP1 resulted in less migration 496
of SupT1 cells (Supplementary Fig. 1b,c). The decrease in SupT1 chemotaxis correlated with the decrease in SPP1 mRNA in infected cells (Supplementary Fig. 1c,d). We also identified nine genes previously unknown to influence chemotaxis; these included genes encoding two members of the synaptotagmin family of calcium-dependent regulators of vesicle fusion: SYT2 (A002560), and a related protein, SYTL5. These data demonstrating that synaptotagmins regulate chemotaxis suggested a requirement for vesicle fusion in cell migration and led us to study the role of synaptotagmins in chemotaxis in more detail. Infection with virus targeting SYTL5 mRNA diminished the ability of SupT1 cells to migrate in response to CXCL12 (Fig. 1b). This effect correlated with the ability of the shRNA-encoding virus to diminish SYTL5 mRNA expression (Supplementary Fig. 2a), which suggested that these results were not due to off-target effects of the shRNA. The knockdown of SYTL5 had no effect on the proliferation of SupT1 cells (Supplementary Fig. 2b), which demonstrated that the shRNA targeting SYTL5 was not toxic to the cells. To determine if SYTL5 has a similar role in other cells, we used the virus that knocked down its expression in SupT1 cells to infect THP-1 cells (a human monocytic leukemia cell line). Infection with lentivirus encoding shRNA targeting SYTL5 diminished the ability of THP-1 cells to migrate in response to CCL2, the ligand for the chemokine receptor CCR2 (Fig. 1c). The decrease in SYTL5 protein abundance in SupT1 and THP-1 cells inhibited chemotaxis to different chemokines, which demonstrated that the requirement for SYTL5 in chemotaxis was not limited to one cell type or to one chemokine–chemokine receptor pair. We also identified another member of the synaptotagmin family, SYT2, in our initial screen. Decreasing the abundance of SYT2 mRNA resulted in enhanced migration of SupT1 cells (Fig. 1b) and THP-1 cells (Fig. 1c) in response to chemokines. The enhanced chemotaxis in SupT1 cells correlated with lower abundance of SYT2 mRNA and SYT2 protein (Fig. 1d–f). These data demonstrate that SYT2 is a negative regulator of chemotaxis, analogous to its known role as a negative regulator of exocytosis in mast cells21. We then analyzed 14 additional genes encoding synaptotagmin molecules and synaptotagmin-like molecules for their role in VOLUME 11 NUMBER 6 JUNE 2010 nature immunology
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To further analyze the migration defect caused by SYT7 deficiency, we obtained bone marrow–derived neutrophils (BMDNs) from Syt7−/−and wild-type mice and analyzed their ability to migrate in response to the peptide chemoattractant fMLP with a Zigmond chemotaxis chamber29 and time-lapse video microscopy (Fig. 3 and Supplementary Movies 1 and 2). Syt7−/− BMDNs migrated less than half the distance that wild-type BMDNs migrated (Fig. 3a,b). The migration velocity of neutrophils obtained from Syt7−/− mice was significantly less than that of wild-type BMDNs (Fig. 3c). The diminished chemotaxis was quantitatively similar to the diminshed membrane repair reported for Syt7−/− cells24. SYT7 deficiency also adversely affected the directionality of cell migration (Fig. 3d,e). Consistent with those findings, polarized Syt7−/− cells had less prominent lamellopodia than those of wild-type cells (Supplementary Movies 1 and 2), and the extent of cell polarization was less during cell migration (Fig. 3f). These data demonstrate that SYT7 is a positive regulator of chemotaxis that affects both velocity and directionality in chemoattractant-induced leukocyte migration. To extend our observations to the in vivo setting, we examined the effect of SYT7 deficiency on the recruitment of neutrophils in a model of gout induced by injection of monosodium urate (MSU) crystals into preformed air pouches on the dorsum of mice30–32. At 8 h after injecting PBS or MSU crystals, we lavaged the air pouches and counted recruited cells and determined the frequency of neutrophils (CD11b+Ly6C+) and monocytes (CD11b+Ly6C−) by flow cytometry. The percentage (Fig. 4a) and absolute number (Fig. 4b) of neutrophils recruited into the air pouch by MSU crystals were much lower in Syt7−/− mice (recruitment index (MSU/PBS), 4.4) than in wild-type mice (recruitment index, 42.8). As expected, the air pouches of wild-type and Syt7−/− mice did not have more monocytes (Fig. 4b). However, at 8 h after MSU crystal injection, we recovered fewer monocytes from air pouches of Syt7−/− mice, which suggested a recruitment defect for monocytes as well. As this model is dependent on the induction of interleukin 1β (IL-1β) and CXCL2 (MIP-2; ligand for the neutrophil chemokine receptor CXCR2)
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© 2010 Nature America, Inc. All rights reserved.
chemotaxis by using shRNA targeting each gene in Transwell chemotaxis assays. Only shRNA targeting SYT7, which encodes the lysosomal membrane protein SYT7 (A002565)22, a positive regulator of lysosome exocytosis23, significantly affected the ability of T cells to migrate in response to CXCL12 (Fig. 1b). The efficiency of the knockdown of SYT7 mRNA correlated with the decrease in the migration of SupT1 cells to CXCL12 (Supplementary Fig. 2c,d). Splenocytes obtained from SYT7-deficient (Syt7−/−) mice24 had less ability to migrate in response to CXCL12 than did splenocytes obtained from wild-type mice (Fig. 1g), which confirmed the effect of the SYT7 knockdown. Our findings that SYT7, SYT2 and SYTL5 regulate chemotaxis suggested that vesicle fusion and, in particular, lysosome exocytosis are involved in directed cell migration. To further extend that hypothesis, we used RNAi silencing to analyze genes encoding members of the Rab family of small GTPases that regulate the movement of lysosomes to the membrane for their role in chemotaxis. We chose Rab27a (A001978) because it regulates the exocytosis of secretory lysosomes in cytotoxic T lymphocytes25 and directly interacts with SYTL5 (ref. 26). We also selected Rab3a because it interacts with synapto tagmin family members and has an important role in regulated exocytosis27,28. In cells in which expression of RAB27A or RAB3A mRNA was targeted by shRNA, chemotaxis to CXCL12 was markedly inhibited (Fig. 2a). The lower chemotaxis in SupT1 cells correlated with the decrease in the abundance of RAB27A mRNA and Rab27a protein (Fig. 2b–d). The lower chemotaxis in SupT1 cells similarly correlated with the decrease in RAB3A mRNA (Supplementary Fig. 2e). These data demonstrated that the small GTPases Rab27a and Rab3a, proteins known to regulate lysosome exocytosis and interact with synaptotagmins, also have a role in lymphocyte chemotaxis.
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Figure 2 Knockdown of RAB27A and RAB3A P < 0.05 inhibits T cell chemotaxis. (a) Transwell 2,500 120 3.0 chemotaxis of SupT1 cells infected with 2,000 100 2.5 viruses targeting genes encoding eGFP, Rab27a sheGFP 80 1,500 2.0 (control) shRAB3a 60 or Rab3a. (b) Quantitative PCR analysis of 1.5 P = 0.007 Anti-Rab27a 1,000 40 1.0 RAB27A mRNA abundance in SupT1 cells 500 20 shRAB27a 0.5 Anti-actin infected with viruses targeting genes encoding P = 0.01 0 0 0 eGFP or Rab27a, presented relative to GAPDH 0 1 10 100 CXCL12 (nM) mRNA abundance. (c) Immunoblot analysis of extracts of SupT1 cells infected with lentiviruses targeting genes encoding eGFP or Rab27a, probed with anti-Rab27a (top) or anti-β-actin (bottom). (d) Quantification of Rab27a protein, presented as the ratio of the Rab27a to β-actin in lysates of cells infected with lentivirus targeting RAB27A, normalized to the ratio in lysates of cells infected with lentivirus targeting the gene encoding eGFP. P values, two-way ANOVA (a) or Student’s t-test (b). Data are representative of at least three similar experiments (a; mean and s.e.m. of two samples) or experiments done at least four times (b; error bar, s.e.m.) or twice (c,d).
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Figure 3 Migration of wild-type and SYT7-deficient neutrophils ex vivo. (a,b) Tracks of neutrophil migration, derived from stacks of images taken every 15 s for 30 min during migration in a Zigmond chamber in a gradient of fMLP. Data are representative of four similar experiments. (c) Migration velocity of wild-type and Syt7−/− BMDNs. Each symbol represents an individual cell; small horizontal lines indicate the mean. Data are compiled from four separate experiments. (d,e) Migration vectors, presented as Rose plots of data obtained in a,b. P = 0.00002 (d) or 0.7 (e; Rayleigh test for both). Data are representative of four similar experiments. (f) Frequency of polarized cells 25 min after exposure to an fMLP gradient (n = 100 cells examined at 25 min and categorized as polarized or unpolarized). P values, Student’s t-test. Data are compiled from four similar experiments (error bars, s.e.m.).
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Articles Figure 4 Migration of wild-type and SYT75 P = 0.0001 WT Syt7–/– deficient neutrophils in vivo in a model of WT 2.5 WT 45.50 81.94 51.70 Syt7–/– 16.40 4 gout. (a) Flow cytometry of cells recruited P = 0.0003 Syt7–/– 2.0 into the air pouch of a wild-type mouse and 3 1.5 a Syt7−/− mouse injected with MSU crystals; 2 1.0 P = 0.001 cells were stained with anti-Ly6G and anti1 0.5 CD11b. Numbers in quadrants indicate percent 1.65 0.02 2.79 0.02 0 0 cells in each. (b) Total cells, neutrophils PBS MSU PBS MSU PBS MSU PBS MSU PBS MSU Ly6G + + Total PMN Mono (PMN; CD11b Ly6C ) and monocytes (Mono; IL-1β MIP-2 + – CD11b Ly6C ) recruited into the air pouches of wild-type mice (n = 11) and Syt7−/− mice (n = 11) injected with MSU crystals or wild-type mice (n = 6) and Syt7−/− mice (n =6) injected with PBS (control). P values, Student’s t-test. (c) Enzyme-linked immunosorbent assay of IL-1β and CXCL2 (MIP-2) in supernatants of wild-type and Syt7−/− thioglycolate-recruited peritoneal macrophages primed with lipopolysaccharide, then stimulated with PBS or MSU crystals. No significant difference, wild-type versus Syt7−/− (Student’s t-test). Data are representative of three (a,b) or two (c) experiments (error bars, s.e.m.). CD11b
in resident macrophages, we determined the ability of Syt7−/− macrophages to secrete IL-1β and CXCL2 after being activated with MSU crystals. Peritoneal macrophages derived from wild-type and Syt7−/− mice showed no difference in their ability to secrete IL-1β or CXCL2 (MIP-2) after being stimulated with MSU crystals (Fig. 4c) or heat-killed Legionella pneumophilia (data not shown), which demonstrated that the secretion of IL-1β and chemokines was not impaired in SYT7-deficient cells (Fig. 4c). Together these data demonstrate that SYT7 is required for chemokine-dependent recruitment of neutrophils in vivo. To begin to explore the mechanism by which synaptotagmin and Rab proteins influence CXCL12-mediated chemotaxis, we determined if knockdown of the genes encoding these molecules affected the surface expression of CXCR4. We infected cell lines with virus expressing shRNA targeting SYTL5, SYT2, SYT7, RAB27A or RAB3A and assessed CXCR4 surface expression and CXCR4 internalization after stimulation with CXCL12 (Fig. 5a). There was no significant difference between any of the knockdown cell lines or between
Syt7−/− splenocytes and cells with shRNA-mediated knockdown of eGFP or wild-type cells in their surface expression of CXCR4 (Fig. 5a,b). Similarly, CXCL12-induced CXCR4 internalization was not significantly altered in any of the knockdown cell lines or in Syt7−/− cells versus cells with shRNA-mediated knockdown of eGFP or wild-type cells (Fig. 5a,b). Actin polymerization is also required for effective directional cell migration33. We therefore determined if silencing of SYT7, SYTL5, SYT2, RAB27A or RAB3A affected F-actin polymerization (Fig. 5c). Knockdown of SYT7, SYT2, RAB27A or RAB3A mediated by shRNA had a minimal effect on chemoattractant-induced F-actin poly merization, as measured by flow cytometry (Fig. 5c). Splenocytes obtained from Syt7−/− mice had F-actin polymerization equivalent to that of cells obtained from wild-type mice after stimulation by CXCL12, which confirmed our data obtained with shRNA-mediated knockdown of SYT7 (Fig. 5d). In contrast, shRNA-mediated knockdown of SYTL5 resulted in less F-actin polymerized after CXCL12 stimulation (Fig. 5c). Knockdown of SYT7, SYTL5, SYT2 or RAB27A
Figure 5 CXCR4 cell surface expression and sheGFP shSYTL5 chemokine-induced F-actin polymerization and 1.6 1.6 WT shSYT2 Syt7 –/– calcium release. (a) Cell surface expression of shSYT7 1.2 1.2 shRab3a CXCR4 on shRNA-expressing SupT1 cells (key) shRab27a stimulated with CXCL12 (concentration, horizontal 0.8 0.8 axis). Differences not significant (Student’s 0.4 0.4 t-test). (b) Cell surface expression of CXCR4 on −/− wild-type and Syt7 splenocytes stimulated with 0 0 0 1 10 0 1 10 CXCL12 (concentration, horizontal axis), analyzed CXCL12 (nM) CXCL12 (nM) by flow cytometry. Differences not significant (Student’s t-test). (c) F-actin polymerization in 250 WT sheGFP 250 sheGFP shRNA-expressing SupT1 cells (key) stimulated shSYTL5 Syt7 –/– 200 shSYT2 shRab27a 200 for various time (horizontal axis) with CXCL12, shSYT7 shSYT7 * shRab3a analyzed by flow cytometry. *P < 0.02, versus 150 150 * shRab27a shSYT2 cells expressing shRNA targeting the gene 100 100 shSYTL5 encoding eGFP at the same time point (Student’s * 50 t-test). (d) Flow cytometry of F-actin content in 50 sheGFP wild-type splenocytes (n = 3 mice) and Syt7−/− 0 0 + BAPTA 0 1 5 0 1 5 0 10 20 30 40 50 60 splenocytes (n = 3 mice) after stimulation with Time (min) Time (min) Time (s) CXCL12 for various times (horizontal axis). (e) Calcium flux in SupT1 cells infected with WT 1.2 WT WT 1.0 0.8 Syt7 –/– Syt7 –/– shRNA-expressing lentivirus (right margin), Syt7 –/– 0.8 0.6 0.8 loaded with the fluorescent calcium indicator 0.6 0.4 0.4 Fluo-8 and activated with 10 nM CXCL12, 0.4 0.2 0.2 presented as emission at 520 nM after activation 0 0 0 at 480 nM; curves were displaced on the vertical 0 1 3 6 12 0 1 3 6 12 0 1 3 6 12 Time (min) Time (min) Time (min) axis for clarity. Baselines of original curves were similar (data not shown). (f) GTP-bound RhoA, Cdc42, and Rac in cytoplasmic extracts of wild-type and Syt7−/− splenocytes stimulated for various times (horizontal axis) with 10 nM CXCL12. No significant difference, wild type versus Syt7−/− (Student’s t-test). Data represent one of three similar experiments (a,b; average and s.e.m. of two samples), at least three experiments (c; average and s.e.m. of two or three replicates of one to four per gene), an experiment done three times (d; error bars, s.e.m.) or experiments done in triplicate and repeated three times (e), or are representative of three experiments (e) or an experiment done in triplicate two times (f; error bars, s.e.m.).
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Figure 6 Effect of calcium on chemotaxis, F-actin polymerization, and chemokine-induced expression of LAMP-1. (a) Transwell chemotaxis of SupT1 cells toward CXCL12 with and without (0) treatment with 10 μM BAPTA. P value, two-way ANOVA. Data are representative of three similar experiments (mean and s.e.m. of three samples). (b) F-actin polymerization with and without treatment with 10 μM BAPTA. Results not significant (Student’s t-test). Data are from one of three similar experiments (average and s.e.m. of two samples). (c) Flow cytometry analysis of the cell surface expression of LAMP-1 on wild-type BMDNs the presence (right) or absence (left) of C5a stimulation (10 nM), with or without BAPTA treatment (left margin). Numbers in outlined areas indicate percent LAMP-1+ cells. Data are representative of three experiments done in triplicate. (d) Flow cytometry analysis of the cell surface expression of LAMP-1 on wild-type and Syt7−/− BMDNs at baseline (without chemoattractant) and after C5a stimulation (+ C5a). Data are representative of three experiments done in triplicate. (e) Laser-scanning confocal microscopy of the localization of LAMP-2 and cathepsin-L (CatL) in the podocyte cell membrane before (0 CK; top row) and after (bottom row) stimulation with 10 nM CXCL11. Left, staining with anti-LAMP-2; middle, staining with anti-cathepsin-L; right, merged images of LAMP-2 (red), cathepsin-L (green) and overlay (yellow). Original magnification, ×40. Results are representative of three experiments.
also had no significant effect on calcium release after chemokine stimulation (Fig. 5e), which demonstrated that the effects of synapto tagmins on chemotaxis are downstream of calcium release. Because activation of the small GTPases RhoA, Cdc42 and Rac is also critical for cell migration14, we studied the effect of SYT7 on chemokine-induced activation of these GTPases. We obtained splenocytes from wild-type and Syt7−/− mice and analyzed activated RhoA, Cdc42 and Rac in the cells at rest and at 1, 3, 6 and 12 min after stimulation with CXCL12 (Fig. 5f). CXCL2 induced equivalent activation profiles for these GTPases in cells obtained Syt7−/− mice and wild-type mice (Fig. 5f), which demonstrated that SYT7 is not directly involved in GTPase activation after chemokine stimulation. Synaptotagmin proteins contain calcium-sensing domains that mediate their role in calcium-dependent vesicle fusion22. As a transient increase in intracellular free calcium is a hallmark of chemoattractant receptor activation, it is possible that synaptotagmins have a similar role in mediating calcium-dependent vesicle fusion after chemo attractant stimulation, which may also be required for chemotaxis. To begin to test this, we first studied the effect of the intracellular calcium chelator BAPTA on chemokine-induced calcium flux and chemotaxis. As expected, BAPTA blocked calcium influx after chemo kine stimulation (Fig. 5e). BAPTA also completely inhibited the migration of SupT1 cells and Jurkat cells (a human T cell leukemia line) to CXCL12 (Fig. 6a and Supplementary Fig. 3a). In contrast, BAPTA had no effect on chemokine-induced polymerization of F-actin (Fig. 6b and Supplementary Fig. 3b), which suggested that an increase in intracellular free calcium is required for chemotaxis but is not required for F-actin polymerization. SYT7 (ref. 22) and Rab27a34 are known positive regulators of lysosome exocytosis, whereas SYT2 (ref. 21) is a known negative regulator of lysosome exocytosis. We therefore investigated whether stimulation with chemoattractants induced calcium-dependent lysosome fusion and exocytosis. To determine if stimulation with chemoattractants results in the fusion of lysosomes with the cell membrane, we analyzed expression of the lysosomal marker LAMP-1 on the surface of BMDNs after stimulation with the chemoattractant C5a (Fig. 6c). Cell-surface expression of LAMP-1 was markedly enhanced after C5a stimulation (Fig. 6c). Consistent with those findings, extracellular nature immunology VOLUME 11 NUMBER 6 JUNE 2010
β-hexosaminidase activity increased after the addition of fMLP to HL-60 human promyelocytic leukemia cells (Supplementary Fig. 4), which demonstrated that chemoattractants induce lysosome–cell membrane fusion that results in the release of lysosomal contents into the extracellular milieu. Similar to its effect on chemotaxis, BAPTA resulted in much lower chemoattractant-induced expression of LAMP-1 on the cell surface (Fig. 6c). These data demonstrate that chemoattractant stimulation results in calcium-dependent fusion of LAMP-1+ vesicles with the cell membrane. We next tested the effect of SYT7 deficiency on chemoattractantinduced cell surface expression of LAMP-1. Neutrophils derived from wild-type and Syt7−/− mice had similar basal surface expression of LAMP-1 (Fig. 6d). However, the induction of cell surface expression of LAMP-1 after C5a stimulation was markedly attenuated in Syt7−/− neutro phils compared with that in wild-type neutrophils (Fig. 6d). To localize chemokine-induced lysosome fusion, we imaged podocytes, which are 40 times larger than leukocytes. Podocytes express the chemokine receptor CXCR3 (ref. 35) and have been shown to migrate in response to chemokines36. We stimulated podocytes with the CXCR3 ligand CXCL11 and found that the lysosomal markers cathepsin-L and LAMP-2 localized to the plasma membrane (Fig. 6e), which demonstrated again that chemokine stimulation results in the fusion of lysosomes with the cell membrane. Together these data suggest that synaptotagmins function downstream of chemoattractant-induced calcium release and upstream of the fusion of LAMP-1+ vesicles with the plasma membrane. To visualize the role of SYT7 in vesicle localization during chemotaxis, we used laser-scanning confocal microscopy to analyze activated wild-type and Syt7−/− CD4+ T cells migrating in a CXCL10 gradient (Fig. 7a). We used the red fluorescent dye LysoTracker Red to visualize intracellular vesicles with a low pH, including lysosomes. Migrating Syt7−/− T cells had much more accumulation of LysoTracker Red in the distal ends of their uropods (Fig. 7a and Supplementary Movies 3–6). To quantify the accumulation of LysoTracker Red, we sampled fluorescence intensity of cell bodies and uropods of polarized cells and then normalized those results to cell area. The cell bodies of polarized wild-type and Syt7−/− lymphocytes had a similar area, whereas the uropods of Syt7−/− lymphocytes were marginally smaller than those of wild-type lymphocytes (Fig. 7b). Syt7−/− T cells showed 499
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twofold greater LysoTracker Red fluorescence across the cell body and over fourfold greater normalized intensity in the uropod than that of wild-type cells (Fig. 7c). The accumulation of LysoTracker Red–stained vesicles in Syt7−/− lymphocytes is consistent with less fusion of these vesicles with the cell membrane than that of migrating wild-type lymphocytes. In addition, the ratio of uropod to cellbody fluorescence intensity was twice as high in polarized Syt7−/− cells as in polarized wild-type cells (Fig. 7d), which demonstrated that LysoTracker Red–stained vesicles were distributed differently in Syt7−/− cells exposed to a chemokine gradient. Coincident with the accumulation of LysoTracker Red described above, uropods in Syt7−/− T cells seemed to have difficulty releasing from the substrate, which resulted in cells that seemed to be stuck in place by a tethered tail (Fig. 7e and Supplementary Movies 3–6). Over the 30 min of observation, 6.14% of polarized wild-type T cells (7 of 114) demonstrated an adherent phenotype, whereas 81.48% of polarized Syt7−/− T cells (88 of 108) demonstrated an adherent phenotype (Fig. 7f). Furthermore, comparison of the average time of adherence of wild-type and Syt7−/− T cells with the adherent phenotype at their uropods showed that Syt7−/− T cells had an average adherence time of 29.43 ± 0.36 min, whereas for the wild-type cells it was 20.46 ± 3.76 min (Fig. 7g). Thus, the few wild-type T cells with the adhesion phenotype had a shorter duration period of adhesion, whereas the majority of Syt7−/− T cells remained adherent at their uropods for the duration of the 30-minute observation period. The phenotype we observed for the impaired migration of Syt7−/− neutrophils and T cells suggests a previously unknown role for synaptogamins and calcium-dependent vesicle fusion in the de-adhesion process required for cell migration. 500
Figure 7 Localization of LysoTracker Red–stained vesicles during cell migration. (a) Confocal microscopy of LysoTracker Red localization during the migration of activated CD4+ lymphocytes from wild-type mice and Syt7−/− mice in a gradient (wedge) of CXCL10 (Dunn chemotaxis chamber). Original magnification, ×20. Results are representative of four experiments. (b) Cell body and uropod surface area of wild-type and SYT7-deficient activated CD4+ T cells migrating in a CXCL10 gradient. *P = 0.01 (Student’s t-test). Data are representative of four experiments (n = 20 cells per group; error bars, s.e.m.). (c) LysoTracker Red localization in wild-type and Syt7−/− activated CD4+ T cells migrating in a CXCL10 gradient. *P = 0.000001 (Student’s t-test). Data are representative of four experiments (n = 20 cells per group; error bars, s.e.m.). (d) LysoTracker Red fluorescence intensity in polarized wildtype and Syt7−/− cells (uropod/cell body). *P = 0.00025 (Student’s t-test). Data are representative of four experiments (error bars, s.e.m.). (e) Laser-scanning confocal microscopy of polarized, activated wild-type and Syt7−/− CD4+ lymphocytes (time series). Outlines (far left) indicate initial locations of polarized cells followed over time in the subsequent images. Original magnification, ×20. Results are representative of four experiments. (f) Adherence of polarized wild-type cells (n = 114) and Syt7−/− cells (n = 108) over a 30-minute period. *P = 8.3 × 10−30 (χ2 test). Data are representative of four experiments (error bars, s.e.m.). (g) Duration of the adhesion of polarized adherent wild-type cells (n = 6) and Syt7−/− cells (n = 88). *P = 0.00000045 (Student’s t-test). Data are representative of four experiments (error bars, s.e.m.).
DISCUSSION In an RNAi-based genetic screen to identify genes encoding molecules that function in leukocyte chemotaxis, we have identified genes encoding molecules known to regulate vesicle trafficking and fusion. We found that three genes in the synaptotagmin family encoding calcium-sensing membrane proteins regulated chemoattractantinduced directed cell migration. We identified SYT7 and the related protein SYTL5 as positive regulators of chemotaxis, whereas we found SYT2 to be a negative regulator of chemotaxis. Those findings are consistent with the opposing roles of these proteins in regulating calciumdependent lysosomal exocytosis21,22. We also identified Rab27a and Rab3a, small GTPases that regulate lysosome transport and interact with the synaptotagmin proteins identified37, as positive regulators of chemotaxis. We used SYT7-deficient mice to further characterize the role of SYT7 in cell migration. Neutrophils derived from those mice migrated more slowly and with less directionality than did neutrophils derived from wild-type mice. Furthermore, SYT7-deficient mice showed a neutrophil recruitment defect in an in vivo model of MSU crystal–induced gout, even though SYT7-deficient and wildtype macrophages secreted equivalent amounts of IL-1β and CXCL12 (MIP-2) in response to MSU crystals. Exploring the mechanism by which synaptotagmins influence chemotaxis, we found that SYT7 as well as calcium flux were required for chemoattractant-induced cell surface expression of LAMP-1. By confocal video imaging of lymphocytes migrating in a chemokine gradient, we observed that LysoTracker Red–stained vesicles accumulated in the uropods of the SYT7-deficient cells, which also suggested that SYT7 mediates chemokine-induced fusion of lysosomes with the cell membrane in migrating cells. Furthermore, we found that SYT7 was required for effective de-adhesion and uropod detachment. Collectively, our data identify a molecular pathway required for chemotaxis mediated by synaptotagmins and Rab proteins that involves chemoattractantinduced vesicle exocytosis and uropod release. The idea that membrane flow and vesicle trafficking have a role in cell migration has been considered for many years. In 1962 it was suggested that there is a membrane cycle during cell migration; experiments showed that 100-nM beads move around the cell surface during cell migration38. Later it was shown that membrane flows toward VOLUME 11 NUMBER 6 JUNE 2010 nature immunology
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Articles the rear of the migrating cell and caps at the front of the cell39–41. In 1983 further evidence for vesicle trafficking during cell migration was presented showing that the surface of the HeLa human cervical cancer cell becomes rough during cell movement, thus suggesting membrane recycling42. Subsequently, it has been shown that lipids move to the front, whereas proteins move to the rear, of migrating cells43. Consistent with that, in 1994 it was shown that recycling receptors in endocytic vesicles are routed to the plasma membrane of the leading lamella of migrating chick fibroblasts44. These descriptive studies all suggested that vesicle trafficking occurs during cell migration, but they did not explore the molecular mechanisms that mediate this process or demonstrate the functional importance of vesicle trafficking in cell migration. We have now done both by defining a role for several proteins (SYT7, SYTL5, SYT2, Rab27a and Rab3a) in a pathway that controls vesicle trafficking in chemotaxis and, using STY7deficient mice, we have demonstrated that this process is important for chemotaxis in vitro and for cell migration in vivo. Exploring the mechanism by which synaptotagmin and Rab proteins influence chemotaxis, we found that chemokine receptor abundance and chemokine-induced receptor internalization were not affected by knockdown of SYT7, SYTL5, SYT2, RAB27A or RAB3A. Our findings differ from those of a study that found that another synaptotagmin, SYT3, regulates recycling and surface expression of CXCR4 in the TAM2D2 mouse T cell line45. This suggests that the role of SYT3 may be different from that of the synaptotagmins identified in our study. In addition, we also found that chemokineinduced polymerization of F-actin and calcium flux were not affected by knockdown of SYT7, SYT2, RAB27A or RAB3A. Knockdown of SYTL5, however, resulted in less F-actin polymerization after chemo kine stimulation, which suggests that this synaptotagmin-like protein may have an effect on cells distinct from that of the other synaptotagmins studied. Together these findings suggested that synaptotagmins participate in chemotaxis downstream of the expression of chemo attractant receptors and initial signal transduction. Given the known function of synaptotagmins in mediating calcium-induced fusion of vesicles with the cell membrane, we suspected that synaptotagmins may have a similar role in chemoattractant-induced chemotaxis. Ins(1,4,5)P3, generated via chemoattractant receptor activation of PLC, is thought to induce the increase in intracellular free calcium by activating Ins(1,4,5)P3 receptors on the endoplasmic reticulum, which then open up calcium channels, resulting in the release of free calcium into the cytoplasm. In migrating cells, calcium is thought to contribute to many processes, including directional sensing, c ytoskeleton redistribution (through effects on myosin light-chain kinase or calmodulin kinase, among others), traction force regeneration and the relocation of focal adhesions3,4,9,10. Additionally, it has been suggested that calcium is important for uropod release in migrating neutrophils46. However, an absolute requirement for calcium flux in cell migration has been controversial. PLC-β2 and PLC-β3 are the two main PLC isoforms found in leukocytes, and neutrophils obtained from mice doubly deficient in PLC-β2 and PLC-β3 have a blunted calcium flux and enhanced migration compared with that of wild-type neutrophils, which suggests that calcium may not be required for chemotaxis47. In contrast, lymphocytes obtained from mice doubly deficient in PLC-β2 and PLC-β3 have no calcium flux and much less chemotaxis48, which suggests that PLC-β2 and PLC-β3 are required for calcium flux and chemotaxis in lymphocytes but not in neutrophils, in which other PLC isoforms may also contribute to localized calcium influx and chemotaxis. Similar to results obtained in our experiments with BAPTA, lymphocyte chemotaxis has been shown to be inhibited by calcium chelation48. Notably, nature immunology VOLUME 11 NUMBER 6 JUNE 2010
we found that calcium chelation also inhibited the chemotaxis of Jurkat T cells, even though a detectable calcium flux is not typically evident in these cells after chemokine stimulation. It is likely that calcium is required at specific intracellular microdomains during chemotaxis49 that may not always be appreciated in a wholecell calcium-efflux assay. As mentioned above, the identification of synaptotagmin family members in our RNAi screen suggested that chemokine-induced release of calcium may induce the fusion of vesicles with the cell membrane and this may in turn be required for effective chemotaxis. Synaptotagmin family members, including SYT7, SYT2 and SYTL5, contain calcium-sensing domains that are critical for the regulation of calcium-induced vesicle fusion23. We therefore sought to determine if chemoattractant-induced calcium flux has a role in synaptotagmin-mediated vesicle fusion during chemotaxis. In support of our hypothesis, we found that chemoattractants induced cell surface expression of LAMP-1, which suggested that chemoattractant receptor signaling can induce the fusion of lysosomes with the cell surface. We also found that this process required both calcium and SYT7, which suggests that calcium-induced vesicle fusion contributes to effective chemotaxis. This result is consistent with the observation that VAMP-7, a SYT7-regulated SNARE protein required for calciumdependent fusion of lysosomes with the cell membrane, has a role in epithelial cell migration50. We also used live-cell video microscopy of LysoTracker Red–labeled wild-type and SYT7-deficient lymphocytes migrating in a chemo kine gradient to visualize the movement of lysosomes in migrating lymphocytes. Although we were unable to visualize the movement of individual vesicles with this technique, we found that LysoTracker Red–stained vesicles accumulated to a greater extent in the uropods of SYT7-deficient cells than in those of wild-type cells. This phenotype was consistent with the failure of lysosome–cell membrane fusion in SYT7-deficient cells. We also unexpectedly found that SYT7-deficient cells had difficulty releasing uropods and had prolonged attachment to the substrate. This phenotype was reminiscent of that of neutrophils treated with a calcium chelator46. Uropod release and de-adhesion are active regulated processes required for effective cell migration. De-adhesion is a calcium-dependent process that requires activity of the RhoA kinase ROCK and nonmuscle myosin heavy chain IIA in the uropod46,51–53. Our data suggest that SYT7-dependent fusion of intracellular vesicles with the cell membrane may be required for the delivery of cargo important for the detachment of uropods from the substrate. The delivered cargo may be proteins known to accumulate in the uropods of migrating cells or phospholipids required for the cell membrane dynamics associated with uropod release. The identification of these factors awaits further investigation. Of potential clinical relevance to our study, Chediak-Higashi syndrome is a congenital immunodeficiency that results from a mutation in LYST (encoding the lysosomal trafficking regulator LYST)54. Neutrophils obtained from patients with this syndrome have impaired function, including defective chemotaxis. Our study raises the possibility that the impaired neutrophil chemotaxis seen in Chediak-Higashi syndrome may be a direct consequence of a defect in lysosomal transport. Our data showing that SYT7-deficient cells had impaired chemotaxis ex vivo and cell migration in vivo demonstrate that chemokine-induced vesicle transport and fusion pathways are important in leukocyte migration. Methods Methods and any associated references are available in the online version of the paper at http://www.nature.com/natureimmunology/. 501
Articles Accession codes. UCSD-Nature Signaling Gateway (http://www. signaling-gateway.org): A000636, A002560, A002565 and A001978. Note: Supplementary information is available on the Nature Immunology website.
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Acknowledgments We thank the Immune Circuits and RNAi Platform groups of the Broad Institute and W. Xu for discussions and the Imaging Core of the Ragon Institute of MGH, MIT and Harvard. Supported by the US National Institutes of Health (R01 DK074449 to A.D.L. and R01 GM064625 to N.W.A.), the Defense Advanced Research Projects Agency (W81XWH-04-C-0139 to N.H.), Fundação LusoAmericana para o Desenvolvimento (L.F.M.) and Fundação para a Ciência e a Tecnologia (L.F.M.). AUTHOR CONTRIBUTIONS R.A.C. designed the experiments, did the screening, collected and analyzed data and wrote the manuscript; T.K.M. did air-pouch experiments, macrophage stimulation and GTPase assays and helped edit the manuscript; T.J.D. did confocal microscopy and analyzed lymphocytes; L.F.M. and C.M. helped with initial screens; R.P.F. assisted with air-pouch experiments; S.S. did confocal microscopy of podocytes; G.S.V.C. helped with experiments; T.A. and L.A.M. collected data; N.W.A. provided mouse strains; D.W. assisted with video migration assays of neutrophils; N.H. helped design experiments, analyzed data and helped edit the manuscript; A.D.L. helped design experiments, analyzed data and edited the manuscript; and all authors discussed results and commented on the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Published online at http://www.nature.com/natureimmunology/. Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/. 1. Viola, A. & Luster, A.D. Chemokines and their receptors: drug targets in immunity and inflammation. Annu. Rev. Pharmacol. Toxicol. 48, 171–197 (2008). 2. Murphy, P.M. The molecular biology of leukocyte chemoattractant receptors. Annu. Rev. Immunol. 12, 593–633 (1994). 3. Ridley, A.J. et al. Cell migration: integrating signals from front to back. Science 302, 1704–1709 (2003). 4. Van Haastert, P.J. & Devreotes, P.N. Chemotaxis: signalling the way forward. Nat. Rev. Mol. Cell Biol. 5, 626–634 (2004). 5. Kehrl, J.H., Hwang, I.Y. & Park, C. Chemoattract receptor signaling and its role in lymphocyte motility and trafficking. Curr. Top. Microbiol. Immunol. 334, 107–127 (2009). 6. Wu, D., LaRosa, G.J. & Simon, M.I. G protein-coupled signal transduction pathways for interleukin-8. Science 261, 101–103 (1993). 7. Malchow, D., Bohme, R. & Rahmsdorf, H.J. Regulation of phosphorylation of myosin heavy chain during the chemotactic response of Dictyostelium cells. Eur. J. Biochem. 117, 213–218 (1981). 8. Liu, G. & Newell, P.C. Regulation of myosin regulatory light chain phosphorylation via cyclic GMP during chemotaxis of Dictyostelium. J. Cell Sci. 107, 1737–1743 (1994). 9. Pettit, E.J. & Fay, F.S. Cytosolic free calcium and the cytoskeleton in the control of leukocyte chemotaxis. Physiol. Rev. 78, 949–967 (1998). 10. Brundage, R.A., Fogarty, K.E., Tuft, R.A. & Fay, F.S. Calcium gradients underlying polarization and chemotaxis of eosinophils. Science 254, 703–706 (1991). 11. Stoyanov, B. et al. Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase. Science 269, 690–693 (1995). 12. Stephens, L. et al. A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein βγ subunits. Cell 77, 83–93 (1994). 13. Hannigan, M.O., Huang, C.K. & Wu, D.Q. Roles of PI3K in neutrophil function. Curr. Top. Microbiol. Immunol. 282, 165–175 (2004). 14. Thelen, M. & Stein, J.V. How chemokines invite leukocytes to dance. Nat. Immunol. 9, 953–959 (2008). 15. Zou, Y.R., Kottmann, A.H., Kuroda, M., Taniuchi, I. & Littman, D.R. Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393, 595–599 (1998). 16. Ma, Q. et al. Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc. Natl. Acad. Sci. USA 95, 9448–9453 (1998). 17. Root, D.D., Hacohen, N., Hahn, W.C., Lander, E.S. & Sabatini, D.M. Genome-scale loss-of-function screening with a lentiviral RNAi library. Nat. Methods 3, 715–719 (2006). 18. Colvin, R.A., Campanella, G.S., Sun, J. & Luster, A.D. Intracellular domains of CXCR3 that mediate CXCL9, CXCL10, and CXCL11 function. J. Biol. Chem. 279, 30219–30227 (2004). 19. Koh, A. et al. Role of osteopontin in neutrophil function. Immunology 122, 466–475 (2007).
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ONLINE METHODS
Cells. SupT1 cells (a gift from the NIH AIDS Research and Reference Reagent Program), Jurkat cells and THP-1 cells (American Type Culture Collection) were grown in complete RPMI medium containing 10% (vol/vol) FBS, nonessential amino acids and l-glutamine. HEK293T cells (human embryonic kidney cells; American Type Culture Collection) were grown in complete DMEM containing 10% (vol/vol) FBS, nonessential amino acids and l-glutamine. Podocytes were cultured in RPMI medium containing 10% (vol/vol) FBS as reported55.
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Purification of BMDNs. BMDNs were purified as reported56. Marrow cavities of the tibias and femurs of 8-week-old mice were flushed with complete DMEM containing 10% (vol/vol) FCS. After hypotonic lysis of red blood cells, mature neutrophils were isolated by centrifugation for 30 min at 28 °C and 500g over discontinuous Percoll gradients consisting of 55% (vol/vol), 65% (vol/vol) and 75% (vol/vol) Percoll in PBS. Mature neutrophils were recovered at the interface of the 65% and 75% fractions. Neutrophil purity varied between 70% and 90%. CD4+ T lymphocyte purification and activation. CD4+ T lymphocytes were purified from spleens obtained from wild-type and Syt7−/− mice by magnetic bead separation according to the manufacturer’s instructions (DynalInvitrogen). Cells (5 × 105) were plated in 1 ml complete RPMI medium containing 10% (vol/vol) FCS and anti-CD28 (1:1,000 dilution; 37.51; 553294; BD Pharmingen) on plates coated with anti-CD3 (2 μg/ml; 145-2C11; 553057; BD Pharmingen). On day 3, cells were provided complete RPMI medium containing IL-2 (5 ng/ml). Cells were analyzed on days 6–10. Splenocyte purification. Spleens were isolated from wild-type or Syt7−/− mice. Individual cell suspensions were obtained by disruption of spleens in RPMI medium, followed by passage through a 70-μm cell strainer. Cells were spun for 7 min at 470g and the resulting cell suspensions were subjected to hypotonic lysis of red blood cells. Cells were washed twice, then resuspended in complete RPMI medium with 10% (vol/vol) FCS and assayed the next day. Syt7−/− mice. Syt7−/− mice on the C57BL/6 background were generated as reported24. C57BL/6 mice were obtained from the National Center for Biotechnology Information. All mice were bred and used at Massachusetts General Hospital under protocols approved by the Subcommittees on Research and Animal Care. Lentivirus production. Plasmids encoding lentiviruses expressing shRNA molecules were obtained from The RNAi Consortium shRNA Library57. Plasmids were purified with a QIAprep Miniprep kit (Qiagen). Plasmids were then transfected into HEK293T cells by a three-plasmid system for the production of lentivirus57,58. Lentivirus infection and selection. Cells were placed in 96-well tissue culture dishes (2 × 104 cells per well) and were infected with 5 μl unconcentrated shRNA lentiviral supernatant and polybrene (7.5 μg/ml). Cells were spun for 30 min at 830g and were incubated for 4 d. Infected cells were selected in complete RPMI medium containing 10% (vol/vol) FBS and puromycin (3 μg/ml) and were tested 2 weeks after infection. Transwell chemotaxis assay. Chemotaxis was assayed in 96-well chemotaxis chambers with polycarbonate membranes (pore size, 5 μm; Neuroprobe) as reported18. CyQUANT dye mix (Molecular Probes–Invitrogen) was used for quantification of migrating cells as reported 18. After 2 h of incubation, fluorescence was measured in a CytoFluor fluorescent plate reader (Applied Biosystems). Primary cell chemotaxis was measured by flow cytometry. Migrating cells were placed into a flow cytometry tube containing 20,000 1-μm latex beads (Invitrogen). Events were collected until 4,000 beads were accumulated and the number of cells per 4,000 beads was obtained. The statistical significance of chemotaxis data was determined by ANOVA. For the primary screen, the number of cells infected with each arrayed virus was determined with Alamar Blue according to the manufacturer’s directions (US Biological). CXCL12 was placed in the bottom well of a Neuroprobe
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chemotaxis chamber (10 nM per well). Cells were spun (100 μl, containing on average 4 × 104 cells), then were resuspended in 50 μl chemotaxis buffer and placed on top of the filter in the chemotaxis chamber. After 3 h of incubation, the number of cells migrating to the lower chamber was determined with CyQUANT. The chemotactic index was determined by division of the number of migrating cells (as determined by CyQUANT fluorescence) by the number of input cells (as determined by Alamar Blue fluorescence) and the results were normalized to 1 on the basis of the migration of cells expressing eGFPspecific shRNA. The mean and s.d. of chemotactic indices for all infected cell lines on each plate was determined. Genes were selected for further study if two or more shRNAs targeting the gene changed the chemotactic index of the infected cells by approximately 1 or more s.d. in the same direction. For the secondary screen, after selection in puromycin, cells were counted in a hemocytometer and their ability to migrate to CXCL12 in a Neuroprobe chemotaxis chamber was tested. Cells (2.5 × 104) were placed on top of the filter on the chamber. RPMI medium with 1% (wt/vol) BSA was placed in the bottom well of the chamber with 0, 1, 10 or 100 nM CXCL12. Migration of cells to the lower chamber was quantified with CyQUANT and compared with that of cells infected with viruses expressing eGFP-specific shRNA. Gene identifiers and shRNA target sequences are in Supplementary Table 2. Quantitative PCR. Total RNA obtained with the RNeasy kit (Qiagen) was treated with DNase I (Invitrogen). Total RNA (10–50 ng) was used as a template for the reverse-transcription reaction; 50 μl cDNA was synthesized with oligo(dT)15, random hexamers, and MultiScribe reverse transcriptase (Applied Biosystems). All reactions used the same conditions and the same reverse-transcription master mix to minimize differences in reverse-transcription efficiency. Quantitative PCR used 5 μl cDNA, 12.5 μl 2× SYBR Green master mix (Stratagene) and 250 nmol sense and antisense primers. Oligonucleotide primer sequences designed on the PrimerBank website59 and obtained from Integrated DNA Technologies were as follows: SYTL5, 5′-ATGATCCTGGGCGTCCTAAAG-3′ and 5′-TCCCACTTCTACGTTTTGCTTC-3′; SYT2, 5′ -TCCCTGGACTAT GATTTTCAGGC-3′ and 5′-GTCTTCCGATGGACTTTGGTC-3′; SYT7, 5′-ACTGGTGTCAGCGCAAACT-3′ and 5′-GCAGGCAACTTGATAGCC TTCT-3′; Rab3a, 5′-AAGACCATCTATCGCAACGACA-3′ and 5′-CCATAGC GCCCCGGTAGTA-3′; and Rab27a, 5′-GAAACTGGATAAGCCAGCTA CAG-3′ and 5′-ATATTTCTCTGCGAGTGCTATGG-3′. Emitted fluorescence for each reaction was measured three times during the annealing-extension phase, and amplification plots were analyzed with MX4000 software, version 3.0 (Stratagene). The quantity of gene expression was generated by comparison of the fluorescence generated by each sample with standard curves of known quantities, and the calculated number of copies was divided by the number of copies of the housekeeping genes GAPDH and B2M (encoding β2-microglobulin). Immunoblot analysis. Cellular extracts were prepared with Nonidet P-40 buffer (150 mM NaCl, 1% (vol/vol) Nonidet P-40 and 50 mM Tris, pH 8) and Complete Protease Inhibitors (Roche). After separation by PAGE, proteins were transferred to nitrocellulose membranes. Anti-SYT2 (ab68854; Abcam) and anti-Rab27a (ab55567; Abcam) were added at a dilution of 1:1,000 in a solution of 1% (wt/vol) dehydrated milk in Tris-buffered saline with 0.05% (vol/vol) Tween-20. Analysis of β-actin served as a loading control; anti-βactin (AC-15; ab6276; Abcam) was added to the blots at a dilution of 1:10,000 in a solution of 1% (wt/vol) dehydrated milk in Tris-buffered saline with 0.05% (vol/vol) Tween-20. Blots were developed with ECL Plus reagents (GE Healthcare). Video migration assays. BMDNs were seeded on coverslips (4 × 104 cells each) and then were placed on a Zigmond chemotaxis chamber (Neuroprobe). Either fMLP or CXCL1 (KC; data not shown), at a concentration of 100 nM in 1% (wt/vol) BSA, was placed in the right well of the Zigmond chamber and RPMI medium with 1% (wt/vol) BSA was added to the left well. After a 5-minute equilibration period, brightfield images were obtained every 15 s for 30 min with a SPOT charge-coupled device camera controlled by SPOT software. Stacks of images were analyzed by ImageJ software (National Institutes of Health) with the chemotaxis plug-in for analysis of the track, velocity and direction of each moving cell (Ibidi).
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In vivo recruitment assay. The subcutaneous air-pouch model of MSU crystal–induced neutrophilic inflammation was used as described 32. Subcutaneous air pouches were generated by injection of 3 ml air into the subcutaneous tissue on the back of anesthetized female mice. Air pouches were reinflated 3 d later. The next day, MSU crystals (3 mg) were injected into the pouches; 8 h later, pouches were washed with 3 ml cold PBS. Cell counts were obtained with a hemocytometer. The frequency of neutrophils was determined by staining of the cells with anti-Ly6G (RB6-8C5; 12-5931; BD Pharmingen) and anti-CD11b (M1/70; 17-0112; eBiosciences), followed by flow cytometry to determine the percentage of double-positive cells. The total number of neutrophils recruited into each air pouch was obtained by multiplication of the total number of cells by the percentage of neutrophils. Statistical significance was determined with Student’s t-test. Cytokine and chemokine secretion assay. Macrophages were obtained from wild-type C57BL/6 or Syt7−/− mice by intraperitoneal injection of thioglycolate and were seeded into 24-well tissue culture plates (1 × 106 cells per well) in 1 ml RPMI medium with 10% (vol/vol) FBS added. Cells were mock stimulated or primed for 18 h with lipopolysaccharide (50 ng/ml) and then were stimulated for 8 h with MSU crystals (250 μg/ml) or heat-killed Legionella pneumophilia. Cytokine concentrations (IL-1β and CXCL2 (MIP-2)) were determined by sandwich enzyme-linked immunosorbent assay (Peprotech (IL-1β) or R&D Systems (CXCL2 (MIP-2)). Samples were tested in duplicate. Lysosome staining. CD4+ lymphocytes activated with anti-CD3 (145-2C11; 553057; BD Pharmingen) and anti-CD28 (37.51; BD Pharmingen) were incubated for 20 min with LysoTracker Red according to the manufacturer’s directions (Molecular Probes–Invitrogen). Cells were allowed to adhere to fibronectin-coated coverslips (4 × 104 cells each) and were loaded onto a Dunn chemotaxis chamber containing 100 nM CXCL10 in the outer ring. After 20 min of incubation at 37 °C, time-lapse images were obtained every 10 s for 30 min with a Zeiss LSM confocal microscope (Carl Zeiss). Polarization of cells was assessed by identification of asymmetrical uropods and lamellipods. Cell and uropod surface area and fluorescence intensity of wild-type and Syt7−/− cells were measured with Zen software (Carl Zeiss). Statistical significance was determined with Student’s t-test. The frequency of polarized cells that adhered was determined by analysis of the polarized cells for the adherent phenotype. The null hypothesis was rejected with the χ2 test. Similarly, the time each adherent cell remained attached to the substrate was determined during the time-series images. The statistical significance of adherence time was determined with Student’s t-test. LAMP-1 surface expression. Differentiated BMDNs (1 × 105) obtained from wild-type C57BL/6 or Syt7−/− mice were resuspended in RPMI medium and phycoerythrin- or fluorescein isothiocyanate–conjugated anti-LAMP (1:100 dilution; H4A3; 555801 or 555800; BD Pharmingen). Chemokines were added to the cells and cells were incubated for 3 h at 37 °C. For analysis of the effect of calcium on chemokine-induced cell surface expression of LAMP-1, cells were treated for 30 min at 37 °C with 10 μM BAPTA (1,2bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid) before addition of the chemokine. Cells were washed, were fixed with 1% (vol/vol) paraformaldehyde and then were analyzed on a FACSCalibur flow cytometer. Release of b-hexosaminidase. Cells (1 × 104) were resuspended in RPMI medium with 1% (wt/vol) BSA added. Cells were then incubated for 30 min with no stimulant, fMLP or phorbol 12-myristate 13-acetate plus ionomycin and were spun at 82g for 5 min. Cell supernatants (20 μl) were added for 1 h to substrate buffer (20 μl; 1 mM p-nitrophenyl N-acetyl-β-d-glucosamine
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in 0.05 M citrate buffer, pH 4.5). Stop buffer (33 mM glycine, 83 mM sodium carbonate and 67 mM sodium chloride, pH 10.7) was added and absorbance was measured at 415 nM on a SpectraMax Plus plate reader (Molecular Devices). Staining of LAMP-2 and cathepsin-L. Serum-starved podocytes incubated at 37 °C were stimulated for 10 min with 0 nM or 100 nM CXCL11. Cells were fixed and made permeable and were stained with anti-LAMP-2 (ABL-93; Developmental Studies Hybridoma Bank at the University of Iowa) and anti–cathepsin-L (generated for S.S. by Pocono Rabbit Farm and Laboratory). Images were obtained on a Zeiss LSM confocal microscope (Carl Zeiss). CXCR4 internalization. Internalization was assayed as reported18. After being incubated with CXCL12, cells were washed with ice-cold flow cytometry buffer (PBS containing 1% (wt/vol) BSA), then were analyzed for surface expression of CXCR4 with phycoerythrin-conjugated anti-CXCR4 (12GS; 555974; BD Biosciences). F-actin polymerization. SupT1 cells or primary splenocytes (1 × 105) in RPMI medium with 1% BSA were incubated for various times at 37 °C with CXCL12 (10 nM). After incubation, cells were fixed with 2% (vol//vol) paraformaldehyde, were made permeable with Fix & Perm (Caltag-Invitrogen) and were stained with phalloidin–Alexa Fluor 647 or phalloidin–Alexa Fluor 488 (Molecular Probes–Invitrogen). F-actin polymerization was subsequently measured by flow cytometry in a FACSCalibur and data were analyzed with FlowJo (TreeStar). F-actin polymerization was determined by calculation of the mean fluorescence index of the cells at each time point after stimulation with chemokine. Calcium flux. Cells were loaded with 2.5 mM Fluo-8 according to the manufacturer’s directions (ABD Biosystems). Cells were placed in flat-welled 96-well tissue culture plates (1 × 106 cells per well) coated with poly-l-lysine in PBS without calcium and magnesium, with 1% (wt/vol) BSA added. After acclimation to a temperature of 37 °C, baseline measurements of fluorescence with an excitation of 490 nM and emission of 525 nM were obtained on a Thermo plate reader (Thermo Fisher Scientific). CXCL12 (50 nM) was added to each well and fluorescence measurements were obtained for an additional 2 min. GTPase activation. Splenocytes isolated from wild-type and Syt7−/− mice were diluted to a density of 1 × 106 cells per ml and were seeded into six-well plates. Splenocytes (2 × 106) were stimulated for 0, 1, 3, 6 and 12 min with 10 nM mouse CXCL12. Cell lysates were used in subsequent enzyme-linked immunosorbent assays for measurement of activation of the GTPases Rac1, RhoA, and Cdc42 according to the manufacturer’s protocol (G-LISA; Cytoskeleton). Absorbance at 490 nm was measured on a SpectraMax (Molecular Devices) and data were analyzed with SoftMax Pro.
55. Mundel, P. et al. Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp. Cell Res. 236, 248–258 (1997). 56. Kim, N.D., Chou, R.C., Seung, E., Tager, A.M. & Luster, A.D. A unique requirement for the leukotriene B4 receptor BLT1 for neutrophil recruitment in inflammatory arthritis. J. Exp. Med. 203, 829–835 (2006). 57. Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298 (2006). 58. Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996). 59. Wang, X. & Seed, B.A. PCR primer bank for quantitative gene expression analysis. Nucleic Acids Res. 31, e154 (2003).
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