JCS007641 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig.S1. Sequence similarity between coronin 1A and coronin 1B. Alignment between human coronin 1A and 1B was performed using ClustalX 1.83, combined with the secondary structural information from the pdb file (2AQ5) of murine coronin 1A, and are presented by ESPRIPT.

• Supplemental Figure S2 -

  Fig. S2. Coronin 1B protein purification. (A) Diagram of coronin 1B protein expression vector. (B) Visualization of coronin 1B purification steps by Coomassie Blue staining of SDS-PAGE. Lane 1, whole-cell lysate clarified by centrifugation; lane 2, flow-through from the Talon column; lane 3, Talon beads; lane 4, StrepTactin beads.

• Supplemental Figure S3 -

  Fig. S3. Pelleting of actin filaments polymerized from ATP- and ADP-G-actin. Filaments were polymerized from the indicated concentrations of ATP- and ADP-G-actin and pelleted as described in Material and Methods.

• Supplemental Figure S4 -

  Fig. S4. The R30D mutation does not disrupt coronin 1B protein structure. (A) Limited protease K digestion of coronin 1B. 6 μg of wild-type or the R30D mutant protein was mixed with 20 ng protease K and incubated at 55°C. Reactions were stopped at fixed time points by mixing with 2×SDS sample buffer and immediate boiling. Samples were separated by PAGE and visualized by silver staining. (B) Circular dichroism profiles of both wild-type coronin 1B and the R30D mutant protein.

• Supplemental Figure S5 -

  Fig. S5. Lentiviral-based knockdown/rescue system for coronin 1B. (A) Target sequence of an shRNA designed to specifically knock down mouse or rat, but not human coronin 1B. (B) Diagram of the modified lentiviral vector combining shRNA expression from the Pol III U6 promoter with GFP-tagged coronin 1B expression from the MSCV 5′ LTR promoter, which is used as the rescue construct and replaces endogenous coronin 1B with a GFP tagged version. (C) Rat2 fibroblasts were infected with the knockdown/rescue lentivirus (green), which replaces the endogenous coronin 1B with the GFP-tagged coiled-coil deletion mutant (lacking the epitope for the coronin 1B antibody). Cells were immunostained for endogenous coronin 1B (red) to check the efficiency of knockdown. (D) Rat2 fibroblasts were infected with the knockdown/rescue lentivirus which co-expresses the coronin 1B shRNA and the GFP-tagged human coronin 1B. GFP-positive cells were isolated by fluorescent-activated cell sorting. Cell lysate from these cells or uninfected control cells were compared by immunoblotting for coronin 1B expression. Our coronin 1B antibody (4245.Exp) has fivefold higher affinity for human coronin 1B than the endogenous rat protein (data not shown), indicating that re-expression levels are close to physiological levels.

• Supplemental Figure S6 -

  Fig. S6. Coronin 1B cannot oligomerize post lysis. (A) Diagram of post-lysis oligomerization test. In order to confirm that post-lysis oligomerization of coronin 1B does not occur, we separately transfected HEK293 cells with either Myc- or GFP-tagged coronin 1B, prepared lysates and mixed them together. We then tested for cross-immunoprecipitation (IP) with antibodies against the different tags. (B) Results of post-lysis oligomerization test described above. No cross-IP is observed with post lysis mixing. Arrow head, GFP-tagged coronin 1B; arrow, Myc-tagged version; line, endogenous protein; asterisk, mouse IgG bands.

• Supplemental Figure S7 -

  Fig. S7. The binding of both coronin 1A and coronin 1B to F-actin is antagonized by cofilin. (A) Representative Coomassie-Blue-stained gel showing the F-actin binding ability of coronin 1A/1B in the presence of 2 μM. (B) Representative Coomassie-Blue-stained gel showing the F-actin binding ability of coronin 1A/1B in the presence of 2 μM cofilin and 5 μM phalloidin.

• Supplemental Figure S8 -

  Fig. S8. Coronin 1B recovery kinetics in FRAP. (A) In FRAP analysis, circled regions in DIC image and fluorescence images were subjected to bleaching: Spot1, lamellipodia; Spot2, cytoplasm. (B) One-phase exponential decay Y=Span*exp(−K*X)+Plateau was used to fit the fluorescence data from Fig. 7E. (C) Experiments listed in Fig. 7G were subjected to curve fitting as described in B. Recovery half-life (0.69/K) was calculated from the kinetics and presented as the mean ± s.e.m. (**P<0.01.)

• Supplemental Figure S9 -

  Fig. S9. Coronin 1B ΔCC mutant binds poorly to F-actin, loses leading edge enrichment, and fails to rescue coronin-1B-depletion-induced slow motility. (A) S2 cells were transfected with coronin 1B ΔCC mutant tagged with V5-6xHis and immunostained for F-actin (probed by phalloidin) and the ΔCC mutant (probed using anti-V5 antibody). (B) Representative Coomassie-Blue-stained gel showing the F-actin binding ability of the coronin 1B ΔCC mutant (1.1 μM total actin). (C) Rat2 cells were infected with retrovirus expressing GFP-tagged coronin 1B ΔCC mutant and immunostained for endogenous coronin 1B and cortactin. (D) Rat2 cells were infected with retrovirus expressing GFP-tagged wild-type or ΔCC mutant coronin 1B. Cells were lysed and exogenous expressed protein was immunoprecipitated using anti-GFP antibodies. Immunoprecipitates were subjected to immunoblotting for endogenous coronin 1B and Arp2/3 complex (probed using p34Arc subunit). (E) Rat2 fibroblasts were infected with lentivirus expressing coronin 1B shRNA (KD1B, without or with co-expression of GFP-tagged wild-type or ΔCC mutant coronin 1B) or the control shRNA (NS), and the cells were subjected to single-cell tracking. Data are presented as mean cell speed, error bars indicate 95% confidence intervals.