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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.004333

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Structure and function analysis of the CMS/CIN85 protein family identifies actin-bundling properties and heterotypic-complex formation

Department of Biochemistry, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA

View larger version (78K): [in this window] [in a new window]   Fig. 1. CMS colocalizes with F-actin to dynamic actin structures such as membrane ruffles and podosomes. NIH 3T3 cells, COS-7 cells and podocytes transfected with CMS, were subjected to immunofluorescence. (A) Endogenous paxillin and transiently expressed FLAG-tagged CMS in NIH 3T3 cells showed a different cellular localization pattern, indicating that the cytoplasmic CMS protein is concentrated at leading edges of migratory cells and not to focal adhesions as seen for paxillin. Bars, 20 µm. (B) COS-7 cells transiently expressing CMS were stimulated with PMA for 10 minutes, stained for CMS and F-actin and analyzed by confocal microscopy. CMS was concentrated and colocalized in prominent membrane ruffles indicated by arrows. Bars, 20 µm. (C) Mouse podocytes stably overexpressing Myc-tagged CMS were differentiated for 7 days and subsequently stained for CMS and F-actin. CMS colocalizes with dynamic actin structures such as lamellipodia and podosomes (see arrows). Bars, 10 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 2. CMS colocalizes with podosomes. (A) NIH 3T3 cells ectopically expressing activated Src (Src*3T3) were transduced by retroviral infection with vector and vector carrying CMS. Cells were stained for CMS and F-actin and analyzed for subcellular localization. Prominent rosette-like structures composed of individual podosomes are formed in Src*3T3 cells (see arrows), and colocalization of CMS with these structures in Src*3T3/CMS cells is apparent (see arrows). Bars, 20 µm. (B+C) Confocal immunofluorescence microscopy analysis of CMS and actin in Src*3T3/CMS cells. (B) Images were taken at ventral and dorsal layers. Foot processes composed of podosomes are marked with arrowheads, lamellipodia are marked with an arrow. Bars, 20 µm. (C) Images of two cells taken at the ventral layer show prominent podosome clusters. Bars, 20 µm. Inset left: high-magnification z-axis images of individual podosomes show localization of CMS to the core; bar, 5 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 3. CMS domains contribute cooperatively to F-actin binding. (A) In order to map the actin-binding site(s) on CMS, a series of CMS deletion mutants fused to GST were purified and used in G- and/or F-actin binding assays. Schematic representation of constructs (black bars represent sequences rich in prolines, white regions represent the linker sequence, gray bars represent the CC domain, black lines mark the locations of the putative actin-binding motifs, ovals represent the GST peptides, numbers mark the first and last aa of the CMS peptides and Coomassie-stained gels of purified GST fusion peptides and G-actin are shown. (B) CMS does not bind to G-actin. G-actin-binding by CMS was assayed in a GST pull-down reaction. The GST peptide, GST-tagged actin-binding domain of talin, and the GST-tagged CMS C-terminus (CMS CT) were incubated with G-actin at different pH conditions, and subjected to pull-down and western blot analysis. (C) Mapping of the F-actin binding domain(s) of CMS. GST-tagged CMS peptides along with GST alone or GST-talin were assayed by F-actin pull-down reactions. Samples were washed and analyzed by western blotting to detect precipitated F-actin (left panel). The graph represents the relative values ± standard deviation (s.d.) of bound F-actin to CMS CT, CT CC, CT 524 and CT PR of three independent experiments (n=3). P values were calculated using Student's t test.

View larger version (33K): [in this window] [in a new window]   Fig. 4. The CMS linker region plays a crucial role in maintaining the actin-binding activity of CMS. (A) The linker sequences of CMS and CIN85 were aligned using the BLAST program. Schematic representation of the C-terminal half of CMS and CIN85 depicting the region of identified similarities (boxed area). Sequence alignment of this region is shown below. Black bars represent the PR regions, gray bars represent the CC domain, solid lines mark the linker sequence. (B) Schematic representation of CMS CT in relation to the C-terminus of CIN85 (CIN85 CT), and a hybrid peptide of the C-termini of CMS and CIN85 (Hybrid), in which the linker sequence is replaced by the corresponding sequence of CIN85 (domains in CMS are depicted as in Fig. 3A; black rectangle in CIN85 CT represent sequences rich in prolines, striped rectangle represents linker sequence). Depicted GST-tagged peptides were purified, analyzed using Coomassie-stained acrylamide gels (left panel), and assayed for F-actin-binding activity in GST pull-down reactions. GST was included as a negative control. The reactions were subjected to western blot analysis to score for bound actin. Summary of relative values ± s.d. of actin-binding represents the average of four independent experiments (n=4). P values were calculated using Student's t-test. (C) The PR region of CIN85 contributes to actin-binding. Indicated GST-tagged peptides or GST alone were assessed for F-actin-binding in pull-down reactions. Reactions were analyzed as described.

View larger version (34K): [in this window] [in a new window]   Fig. 5. CMS binding to F-actin in whole-cell extracts mirrors the binding measured with purified components. Cell lysates from 293T cells expressing the FLAG-tagged CMS constructs depicted in the scheme were incubated with exogenous F-actin and tested for their ability to co-sediment (lanes 3 to 12). F-actin was sedimented by centrifugation at 100,000 g. The soluble (S) and actin-bound fractions (P) were resolved by SDS-PAGE and analyzed by western blot for the presence of F-actin and CMS. Lanes 1 and 2 represent negative controls without F-actin. The CC domain and PR region deletion mutants of CMS show a diminished F-actin binding activity, similarly to the results of GST pull-downs assays (Fig. 3). F-actin-binding was scored: +++, strong; ++, medium; +, low; –, no binding.

View larger version (34K): [in this window] [in a new window]   Fig. 6. Heterotypic oligomerization of CMS with CIN85 mediated by the CC domain. (A) Lysates from 293T cells transiently expressing wild-type Flag-tagged CMS, deletion constructs (CMS CC, CMS 628 CMS 605; positions of the introduced stop codons are marked with triangles in panel E), and vector control were incubated with 10 µg purified GST-CIN85 CT. Similarly, control and CMS expressing lysates were incubated with GST and GST-CMS CT (negative and positive controls, respectively). Precipitates were analyzed by western blotting with FLAG antibody (left panel), equal expression of the CMS constructs is represented in the right panels. (B) Expression of endogenous CIN85 in mouse podocytes. PCR was performed on cDNA generated from randomly growing immortalized mouse podocytes. Amplification of the 5' region of CIN85 was performed with mouse-specific primer m1 (positioned in exon 1) and mmn (positioned in exon 9) lane 2. Amplification of the 3' region of CIN85, including the CC domain, was performed with primer m6 (positioned in exon 19) and mc (positioned in exon 24) lane 3. PCR primers m1, mmn and mc are as described (Buchman et al., 2002). Amplified and sequenced cDNAs (a, b, c) are indicated with arrows; a, 5' region of CIN85; b, full-length 3' region of CIN85; c, 3' region of CIN85 exon 21; *, additional amplified cDNA of unknown nature; lane 1, negative control. (C) An additive effect on actin-binding was observed when CMS and CIN85 were combined in one reaction (protein ratio 1:1). Pull-down assays performed with the indicated peptides were analyzed as described above. (D) Binding of endogenous CMS to CIN85 CT PR. Whole-cell extracts (1.5-2 mg) of podocytes and MCF-7 cells were incubated with GST or GST-CIN85 CT PR and analyzed by western blotting for association with CMS (pellet). Equal amounts of supernatant (supernat.) were loaded and analyzed for CMS and actin. (E) CMS/CIN85 family sequence alignment (different species) of the distal C-terminus containing the CC domain performed with the Clustal W program. Conserved amino acids are color-coded (red, small and hydrophobic; blue, acidic; magenta, basic; green, hydroxyl, amine or basic-Q residue). Consensus symbols are depicted (*, identical residues;:, conserved substitutions;., semi-conserved substitutions). Coiled-coil probabilities are given for the CMS/CIN85 family members [Paircoil program, Berger et al. (Berger et al., 1995)].

View larger version (60K): [in this window] [in a new window]   Fig. 7. CMS and CIN 85 crosslink individual F-actin filaments into bundles. (A) Biochemical actin bundling assay. Freshly polymerized non-muscle actin was incubated for 30 minutes with -actinin, GST and CMS CT (left panel), or with GST, CMS CT, CT PR, CT CC and CIN85 CT. The F-actin bundles were separated from soluble F-actin filaments by centrifugation, and the pellets were subjected to western blot analysis. CMS CT and CIN85 CT caused actin pelleting, with a detectable depletion of the actin amount in the CMS CT supernatant. (B) TEM of negatively stained preparations of actin filaments and F-actin crosslinked by CMS CT and -actinin. F-actin bundles formed when freshly polymerized, non-muscle F-actin was incubated with CMS CT, CIN85CT or -actinin (positive control). The CC domain is crucial for the formation of actin filament crosslinks, because in the presence of CMS CC no bundles were formed similarly to GST (negative control). Bars, 100 nm. (C) These findings allow us to propose a model for the interaction of CMS with F-actin. The CC domain plays a dual role: being important in actin binding and filament crosslinking.

View larger version (32K): [in this window] [in a new window]   Fig. 8. The CC domain and PR region are important for cell migration. (A) Podocytes were transfected with two different concentrations of CMS siRNA or control siRNA in triplicate. After 48 hours cells were subjected to serum-stimulated Boyden chamber migration assay. Cell lysates were analyzed by western blotting for CMS and actin. (B) Podocytes expressing mutated forms of CMS display reduced migration. Cells stably expressing CMS, CMS CC, CMS PR or vector control were subjected to a serum-stimulated migration assay for 4 hours. Data represent the mean ± s.d. of triplicates from three independent experiments (n=3; right panel; *P<0.01; **P=0.056). (C) Transformed NIH 3T3 cells display reduced migration. Src*3T3 cells expressing the indicated peptides were subjected to a serum-stimulated migration assay for 2 hours. Data represent the mean ± s.d. of triplicates from three independent experiments (n=3; *P<0.0001; **P<0.002). P values were calculated using Student's t test. Protein expression was determined by western blot analysis.

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