Supporting Online Material for

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1 Supporting Online Material for Requirement for Coronin 1 in T Lymphocyte Trafficking and Cellular Homeostasis Niko Föger, Linda Rangell, Dimitry M. Danilenko, Andrew C. Chan* *To whom correspondence should be addressed. acc@gene.com This PDF file includes: Materials and Methods Figs. S1 to S7 Table S1 Published 11 August 26, Science 313, 839 (26) DOI: /science

2 SUPPORTING ONLINE MATERIAL Materials and Methods. Generation of coronin 1 -/- mice The targeting vector to replace exons 2 through 1 of the mouse coronin 1 locus with a neomycin resistance cassette (Fig. S1B) was designed by insertion of a 5.4 kb Spe 1 - Bcg I fragment upstream of exon 2 and a 1.6 kb Hind III fragment downstream of exon 1 into the target vector. The targeting construct was electroporated into 129 RW4 embryonic (ES) stem cells. Targeted ES cells clones were identified by Southern blotting and microinjected into C57BL/6 blastocysts and the resulting male chimeras were crossed with female C57BL/6 mice. Germline transmission was confirmed by polymerase chain reaction and Southern blot analysis of genomic tail DNA. Coronin 1 -/- mice were born at the expected Mendelian ratio, were fertile and did not exhibit any gross developmental abnormalities. The coronin 1 mutant allele was introduced into TCRtransgenic mouse lines by breeding to H-Y TCR + rag2 -/- and DO11.1 TCR + mice. Expression constructs and cell transfection Full-length mouse Coronin 1 (amino acids 1-461) carrying a C-terminal Flag-tag and untagged Coronin 1 were cloned into the bicistronic expression vector IRES2-EGFP (Clontech). The coiled-coil deletion (ΔCC) mutant of Coronin 1 (amino acids 1-425) was generated in the same vector. Coronin 1 S2A and S2D mutants were generated using the QuickChange site directed mutagenesis kit (Stratagene). Constructs were transfected into A2 cells or into in vitro expanded DO11.1 TCR-Tg + CD4 + T cells using the Nucleofector system (Amexa) according to the manufacturer's instructions Flow cytometric analysis Single cell suspensions were stained with monoclonal antibodies (Ab) against CD4, CD8, CD21, CD23, B22, CD44, CD62L, CD69, CXCR4, IgD, H-Y TCR (Pharmingen), IgM (Jackson ImmunoResearch), DO11.1 TCR (Caltag), polyclonal Ab against CCR9 (Abcam) and Annexin V (Pharmingen). Expression of CCR7 was determined by staining with Alexa488-conjugated CCL19-Fc. To assess cellular F-actin and G-actin contents, lymphocytes were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS), permeabilized with.2% Triton X-1 and stained with Alexa488- or Alexa647- conjugated phalloidin (Molecular Probes) or Alexa488-labelled DNase I (Molecular Probes), respectively. Disruption of the mitochondrial transmembrane potential was detected using the MitoCapture TM Mitochondrial Apoptosis Detection Kit (BioVision). Activation of caspase-9 was assessed using the CaspGLOW TM Fluorescein active caspase-9 staining Kit (BioVision). Flow cytometric analysis was performed on a FACSCalibur (Becton Dickinson). Immunohistochemistry and Immunofluorescence microscopy Sections from formalin fixed paraffin embedded tissues were processed for staining with anti-cd3 (DakoCytomation) and anti-b22 (Pharmingen). Sections were incubated with peroxidase- and alkaline phosphatase-conjugated secondary reagents (Vector Laboratories) and developed with NovaRed and Blue Substrate Kit (Vector Laboratories), respectively. For cell polarization, cells were cultured at 37º C for 3 min

3 on collagen-coated coverslips in the presence of 25 ng/ml CCL19 (R&D Systems). Cells were fixed with 4% paraformaldehyde in PBS, permeabilized with.2% Triton X- 1 and stained for coronin 1 and talin (Sigma). F-actin and DNA was visualized by staining with phalloidin (Molecular Probes) and DAPI (Molecular Probes), respectively. Phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P 3 ] was detected by staining with a biotinylated mab specific for PI(3,4,5)P 3 (clone RC6F8, Echelon Biosciences) and Streptavidine-AlexaFluor 647 (Molecular Probes). Samples were analyzed by deconvolution microscopy (Delta Vision). Chemotaxis assays Chemotaxis assays were performed using 5-µm transwell filters (Corning Costar) and chemokines from R&D Systems. After 3 h of incubation, cells from whole thymus organ or single cell suspensions that have migrated to the lower chamber were collected and enumerated by flow cytometry. Data are represented as the percentage of input cells migrated in response to chemokines from triplicate samples. In vivo thymic emigration and adoptive transfer Under anesthesia, thymocytes were labeled by intrathymic injection of 1 µl FITC (Molecular Probes; 2mg/ml) in normal saline per thymic lobe. After 2 h, CD4 + or CD8 + recent thymic emigrants were identified by flow cytometry as FITC + cells in blood, spleen and lymph nodes. For adoptive transfers, purified CD4 + thymocytes from coronin 1 +/+ or coronin 1 -/- mice were differentially labeled with either.5 µm CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) or 1 µm CellTracker Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl) amino)tetramethylrhodamine) (Molecular Probes) for 3 min at 37º C. After washing and further incubation for 3 min at 37º C, coronin 1 +/+ and coronin 1 -/- CD4 + thymocytes were then mixed in equal numbers in PBS and 7x1 6 cells were injected intravenously into recipient C57BL/6 mice. An aliquot of this preinjection thymocyte cocktail was analyzed to confirm the preinjection ratio of coronin 1 +/+ to coronin 1 -/- cells. After 2, 2 and 4 hours, blood, spleen and lymph nodes from recipient mice were analyzed by flow cytometry. Immunoblot analysis Western blotting was performed with whole cell lysates separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes and immunoblotted with antibodies against coronin 1, 2 and 3 (Dr. Matthew Thomas), β-actin and GAPDH (Abcam) and cleaved caspase-3 (Cell Signaling Technology). For the analysis of chemokine induced signaling events, in vitro expanded DO11.1 TCR + T cells were stimulated with 25 ng/ml CXCL12 (SDF1α) for various times. Active Rac1 was pulleddown from cell extracts using GST-Pak1-PDB (Pierce), and detected by immunoblotting with anti-rac1 antibody. Cell extracts were also analyzed for phosphorylation of p44/42 MAPK, Akt and p9 RSK using phospho-specific antibodies provided with the Pathscan Multiplex Western Cocktail I (Cell Signaling Technology). To assess cytochrome c release into the cytoplasm, post-mitochondrial extracts were prepared using a subcellular fractionation kit (Pierce) according to the manufacturers instructions. Cytosolic fractions were analyzed by immunoblotting using antibodies against cytochrome c (Pharmingen), β-actin and coronin 1. Effective separation of cytosolic fractions from mitochondria was

4 tested by immunoblotting for VDAC (Biovision). For co-immunoprecipitation studies, cells were lysed in 15 mm NaCl, 1 mm Tris ph 7.5, 1% Brij-97 supplemented with 4 µm latrunculin A (Calbiochem) and protease inhibitor cocktail (Boehringer Mannheim). Flag-tagged proteins were immunoprecipitated using anti-flag M2-agarose beads (Sigma) and immune complexes were analyzed by immunoblotting with anti-flag (Sigma) and anti-arp2 (Santa Cruz) Abs.

5 A C ovary cerebrum heart kidney liver lung muscle spleen -/- +/+ coronin 1 coronin 2 coronin 3 β-actin GAPDH 5 probe 3 probe B wildtype allele 11.3 kb kb wildtype allele 8.2 kb mutant allele kb kb kb mutant allele 3.6 kb D thymus 11.3 kb ATG 1 11 spleen spleen 8.2 kb B S SS B S TK S neo 6.5 kb 3.6 kb B S SS B S B S neo 1 11 mutant locus bone marrow marrow B 1. kb Fig. S1 wildtype locus targeting vector coronin Coronin 1 coronin Coronin 2 coronin Coronin 3 β-actin beta-actin 5 probe 3 probe Figure S1. (A) Tissue expression of coronins 1, 2 and 3. Whole lysates of the indicated tissues were separated by SDS-PAGE and analyzed by immunoblotting for coronin1, 2 and 3. Protein loading was assessed by immunoblotting for β-actin and GAPDH. (B) Gene targeting strategy. Endogenous (wild-type) coronin 1 locus (top), targeting vector (middle) and targeted (mutant) coronin 1 locus. Replacement of exons 2 through 1 with a neomycin resistance cassette by homologous recombination converts the 11.3 kb wild-type Bgl II fragment into a 6.5 kb fragment as detected with the 5' probe. The 8.2 kb wild-type Sal I fragment is converted into a 3.6 kb fragment in the mutant locus as detected with the 3' probe. The 5' and 3' coronin 1 flanking probes used for southern blotting are indicated. B, Bgl II; S, Sal I. (C) Southern blot analysis of genomic DNA from coronin 1 +/+ and coronin 1 -/- mice. Genomic DNA was digested with Bgl II (left panel) or Sal I (right panel) and hybridized with the 5' and 3' DNA probes indicated in B, respectively. (D) Expression of coronin 1 (top), 2 and 3 (middle) in thymocytes, splenocytes and bone marrow cells from coronin 1 +/+ and coronin 1 -/- mice was examined by western blot analysis. Protein loading was assessed by analysis of ß-actin expression (bottom).

6 E 33 6 F Thymus Fig. S1 Blood Spleen FL3-H 19 5 FL3-H FL3-H FL3-H CD4 G B22 CD8 3 2 Bone Marrow Lymph Node CD R5 R2 R3 R4 IgM R3 R2 R4 R5 FL3-H CD8 FL3-H Figure S1 (E-F) Representative flow cytometric profiles of CD4 and CD8 positive lymphocytes in blood, spleen and lymph nodes (E) and thymus (F) from coronin 1 +/+ and coronin 1 -/- mice. (G) Normal B cell development in coronin 1 -/- mice. Bone marrow derived cells from coronin 1 +/+ and coronin 1 -/- mice were stained for indicated cell surface markers and analyzed by flow cytometry.

7 H Spleen (B22 + ) J Thymus (CD4 + ) Fig. S1 IgD CD69 IgM CD62L R5 R4 7 8 R5 R4 CD I counts CD23 CD4 - CD8 - CD4 + CD8 + CD4 + CD8 + +/+ -/- CD69 Figure S1 (H) Normal B cell development in coronin 1 -/- mice. Splenic B cells from coronin 1 +/+ and coronin 1 -/- mice were stained for indicated cell surface markers and analyzed by flow cytometry. (I) Flow cytometric analysis of CD69 expression on thymic subsets from coronin 1 +/+ (black lines) and coronin 1 -/- (red lines) mice. (J) Flow cytometric analysis of CD4 + thymocytes from coronin 1 +/+ and coronin 1 -/- mice stained for CD4, CD8, CD62L and CD69.

8 % FITC positive cell number (x1 3 ) A cell number C CCR7 CXCR4 CCR9 thymus blood spleen cells (x1 3 /ml) cell number (x1 3 ) lymph node D cell number (x1 4 ) /+ -/- thymic organ CD4 + DP CD8 + SP CD4 + B migration (% of input) migration (% of input) CCL19 CXCL12 control CD4 + naive CD4 + effector/ memory Fig. S2 4 4 (ng/ml) Fig. S2. (A) Flow cytometric analysis of chemokine receptor expression levels on CD4 + thymocytes from coronin 1 +/+ (black lines) and coronin 1 -/- (red lines) mice. Thymocytes were stained with CCL19-Ig to detect CCR7 expression (left), or with antibodies against CXCR4 (middle) and CCR9 (right) in combination with anti-cd4 and anti-cd8 antibodies (control staining is shown in grey). (B) Transwell migration of coronin 1 +/+ (open bars) and coronin 1 -/- (closed bars) splennic CD4 + naive (CD44 low CD62L high ) (top) and effector/memory (CD44 high CD62L low ) (bottom) T cells in response to CCL19 (left), CXCL12 (middle) and medium control (right). Data represent the percent of input cells migrated in response to the indicated chemokine with error bars from duplicate samples. (C) In vivo analysis of thymic egress. Recent thymic emigrants were detected by flow cytometry 2 h after intrathymic FITC injection. The data represent the number of FITC + CD4 + lymphocytes detected in blood, spleen and lymph nodes of coronin 1 +/+ (open bars, n=4) and coronin 1 -/- (closed bars, n=3) mice. Percent FITC + thymocytes were monitored as a control for efficient intrathymic FITC labeling. (D) In vitro emigration of thymocytes in whole thymus organ transwell chemotaxis assays. Data represent numbers of CD4 + CD8 + and CD4 + thymocytes that emigrated from coronin 1 +/+ (open bars, n=3) and coronin 1 -/- (closed bars, n=3) thymi in the presence of CCL19.

9 A % of Max 1 8 counts /+ =194 % of Max 1 8 counts /- =121 B Fig. S sec (SDF1α) P-p9RSK P-Akt P-p44/42 MAPK C +/+ -/ sec 15 sec 3 sec CD4 phalloidin (F-actin) non stimulated 5 µm 5 µm PIP3 pseudocolor CD4 25 CCL19 stimulated 5 µm 5 µm PIP3 pseudocolor high eif4e Fig. S3 (A) F-actin polymerization in naive CD4 + T cells in response to CCL19. Coronin 1 +/+ and coronin 1 -/- T cells were stimulated for the indicated times with CCL19 (25 ng/ml). F- actin content in naive CD4 + CD44 low T cells was analyzed by flow cytometry. One representative experiment of four is shown. (B) In vitro expanded T cells from coronin 1 +/+ and coronin 1 -/- DO11.1 TCR + mice were stimulated with CXCL12 (SDF1α) for the indicated times. Whole cellular lysates were immunoblotted with a cocktail of phosphospecific antibodies against p9rsk, Akt and p44/42 MAPK. Equal protein loading was assessed by blotting for eif4e. low (C) Localization of phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P 3 ] in chemokine (CCL19) stimulated T cells. CD4 + CD62L high naive T cells from coronin 1 +/+ and coronin 1 -/- mice, either unstimulated or stimulated for 3 min with CCL19 were fixed and surface stained for CD4 (Pharmingen). After permeabilization, cells were further stained with a biotinylated mab specific for PI(3,4,5)P 3 (clone RC6F8, Echelon Biosciences) and Streptavidin-AlexaFluor 647 (Molecular Probes). Samples were analyzed by deconvolution microscopy (Delta Vision).

10 A C no antigen with antigen (OVA-petide) (OVA-peptide) Fig. S4 no antigen Coronin 1 phalloidin DAPI 5 µm 5 µm 5 µm 5 µm counts B cpm (x1 3 ) µm OVA CFSE OVA peptide (µm) Coronin 1 phalloidin (F-actin) pseudocolor T APC APC T Fig. S4 (A) Purified CD4 + DO11.1 TCR + coronin 1 +/+ (black lines) or coronin 1 -/- (red lines) T cells were labeled with 1 µm CFSE (carboxyfluorescein diacetate succimidyl ester), washed and stimulated with or without.1 µm OVA peptide ( ) in the presence of irradiated syngeneic spleen cells as antigen-presenting cells. After 3 days, cell cycling, as measured by dilution of the CFSE dye, was monitored by flow cytometric analysis. (B) Purified CD4 + DO11.1 TCR + coronin 1 +/+ (open bars) and coronin 1 -/- (closed bars) T cells (1x1 5 ) were stimulated with the indicated concentrations of OVA peptide ( ) in the presence of irradiated syngeneic spleen cells (5x1 5 ) as antigen-presenting cells in triplicate cultures. After 48 h, cultures were pulsed with 1 µci [ 3 H]thymidine and its incorporation was measured 16 h later in a β-scintillation counter. (C) F-actin accumulation at the T cell/antigen presenting cell contact site. CD4 + DO11.1 TCR + T cells from coronin 1 +/+ and coronin 1 -/- mice were mixed with OVA-peptide ( ) pulsed A2 cells, conjugated by short centrifugation and incubated at 37º C for 2 min. Cells were fixed, permeabilized and stained for coronin 1 and F-actin (phalloidin) and DNA with 4',6'-diamidino-2-phenylindole hydrochloride (DAPI). Individual and overlay fluorescence is shown by deconvolution analysis. Numbers of cell conjugates with F-actin accumulation at the T cell/apc interface out of total cell pairs counted were: coronin 1 +/+ : 38 of 79; coronin 1 -/- : 4 of 85). APC T APC T high low

11 Fig. S5 A Cytochrome c β-actin Coronin-1 VDAC 4 4 hours Whole-cell lysate B % annexin V effector/memory T cells ice 37º C B cells ice 37ºC Fig. S5. (A) Cytochrome c release into the cytosol in in vitro cultured T cells from coronin 1 +/+ and coronin 1 -/- mice. Purified CD4 + CD62L high naïve T cells were cultured for the indicated times at 37º C in normal medium, and post-mitochondrial extracts were prepared. Cytosolic fractions were subjected to immunoblotting with anti-cytochrome c, anti-β-actin and anti-coronin 1 mab's. Effective separation of cytosolic fractions from mitochondria was controlled for by immunoblotting for VDAC. (B) Spontaneous cell death in cultures of purified memory/effector (CD4 + CD62L low ) and B cells from coronin 1 +/+ (open bars) and coronin 1 -/- (closed bars) mice. Cells were cultured at 37 C in normal medium (RPMI/1% FCS) or kept on ice for 18h. Dead cells were determined by Annexin V staining of duplicate cultures. Spontaneous cell death in cultures of purified memory/effector (CD4 + CD62L low ) and B cells from coronin 1 +/+ (open bars) and coronin 1 -/- (closed bars) mice. Cells were cultured at 37 C in normal medium (RPMI/1% FCS) or kept on ice for 18h. Dead cells were determined by Annexin-V staining of duplicate cultures.

12 % of Max /+ control GFP-neg GFP-med GFP-high Fig. S6 2 counts % of Max FL4-H /- control % of Max /- Coronin1 FL FL4-H phalloidin (F-actin) FL4-H Fig S6. Transfection of coronin 1 -/- T cells with a coronin 1 expression construct reconstitutes basal F-actin levels. The cellular F-actin content of in vitro expanded coronin 1 -/- DO11.1 TCR + T cells transfected with a wild type (WT) coronin 1-IRES-GFP expression vector (bottom) was assessed by phalloidin staining. Flow cytometric histograms are gated on GFP-negative (neg), GFP-intermediate (med) and GFP-high expressing cell subsets. F- actin levels of coronin 1 +/+ (top) and coronin 1 -/- (middle) T cells transfected with GFP-only expressing vector are shown as control

13 Fig. S7 Coronin1 - /- : GFP-control 3 11 Coronin1 FL (1-461) Coronin1 FL (1-461) FLAG Coronin1 S2A (1-461) FLAG Coronin1 S2D (1-461) FLAG Coronin1 ΔCC (1-425) FLAG 3 58 Coronin1 +/+ : GFP-control Coronin1 Fig. S7. Expression levels of coronin constructs in in vitro expanded coronin 1 -/- DO11.1 TCR + T cells transiently transfected with the indicated coronin 1 IRES-GFP expression constructs. Flow cytometric histograms show intracellular staining for coronin 1 and FLAG-epitope based on the GFPpositive cell gating that has also been used to determine intracellular F-actin contents (see Fig. 4G). Staining of control transfected coronin 1 +/+ and coronin 1 -/- T cells is shown for comparison. The relative mean fluorescence intensity is given for each histogram. FLAG

14 Thymus coronin 1 +/+ coronin 1 -/- p-value cell no. (x1 6 ) 138. ± ± CD4 - CD8 - (%) 2.2 ± ± CD4 + CD8 + (%) 83.2 ± ± CD4 + CD8 - (%) 11.8 ± ± CD4 - CD8 + (%) 3.3 ± ±.5.3 CD4 - CD8 - cell no. (x1 6 ) 3. ± ± CD4 + CD8 + cell no. (x1 6 ) ± ± CD4 + CD8 - cell no. (x1 6 ) 16.6 ± ± CD4 + CD8 - CD69low cell no. (x1 6 ) 13.2 ± ± CD4 + CD8 - CD69high cell no. (x1 6 ) 3.4 ± ±.52.2 CD4 - CD8 + cell no. (x1 6 ) 4.5 ± ± CD4 - CD8 + CD69low cell no. (x1 6 ) 2.9 ± ± CD4 - CD8 + CD69high cell no. (x1 6 ) 1.6 ±.17.5 ± E-4 PBMC Spleen red blood cells (x1 6 ) 9.7 ± ± platelets (x1 3 ) 82. ± ± white blood cells (x1 3 ) 9.2 ± ± E-4 lymphocytes (x1 3 ) 8.1 ± ± E-4 neutrophils (x1 3 ).71 ± ± monocytes (x1 3 ).13 ±.5.5 ±.1.18 eosinophils (x1 3 ).23 ±.2.9 ±.3.2 basophils (x1 3 ).14 ±..4 ±..7 cell no. (x1 6 ) 72.6 ± ± CD4 + cell no. (x1 6 ) 12.8 ± ±.83.1 CD4 + CD25 + cell no. (x1 6 ) 1.61 ± ± CD4 + CD44low cell no. (x1 6 ) 1.5 ± ± E-4 CD4 + CD44high cell no. (x1 6 ) 2.3 ± ± CD8 + cell no. (x1 6 ) 5.1 ± ± CD8 + CD44low cell no. (x1 6 ) 3.8 ±.7 1. ±.26.3 CD8 + CD44high cell no. (x1 6 ) 1.3 ± ± B22 + cell no. (x1 6 ) 47.6 ± ± Table S1. Blood cell counts (n=6) and lymphoid subsets in thymi (n=6), spleens (n=8) and lymph nodes (n=8) are compared between coronin 1 +/+ and coronin 1 -/- mice. Mice were 5-7 weeks of age and lymphocytes were stained for the indicated cell surface markers and analyzed by flow cytometry. Statistical analysis was performed by two-tailed Student's t test. Lymph nodes inguinal cell no. (x1 6 ) 5.5 ± ± E-5 CD4 + cell no. (x1 6 ) 2.7 ±.21.3 ± E-8 CD4 + CD44low cell no. (x1 6 ) 2.57 ±.2.29 ± E-8 CD4 + CD44high cell no. (x1 6 ).15 ±.2.4 ±.1.1 CD8 + cell no. (x1 6 ).9 ±.15.1 ± E-4 CD8 + CD44low cell no. (x1 6 ).76 ±.12.7 ± E-5 CD8 + CD44high cell no. (x1 6 ).15 ±.3.5 ±.1.2 B22 + cell no. (x1 6 ) 1.6 ± ±.3.33 Table S1