Figure 4: GTP-bound Rab35 directly interacts with MICAL1 and contributes to its localization at the intercellular bridge.

(a) Domains of MICAL1. CH, calponin homology; LIM, Lin1, Isl-1 and Mec3; CC, predicted coiled-coil. (b) S. cerevisiae reporter strain expressing indicated GAD- and LEX fusion proteins, and grown on selective medium with or without Histidine. (c) Recombinant GST-tagged wild-type Rab35 proteins loaded with either GDP or GTPγS were incubated with recombinant His-tagged MICAL1^879–1067 or MICAL1^879-1026. Western blot anti-6xHis and Ponceau S red staining are presented. Input: 1%. (d) Flag-tagged proteins from HEK 293 T cells transfected with the indicated plasmids were immunoprecipitated and revealed with anti-Flag antibodies (Input: 4%). Endogenous co-immunoprecipitated MICAL1 is revealed with anti-MICAL1 antibodies. (e) A TALEN-edited HeLa cell line expressing GFP-Rab35^endogenous was transfected with plasmids encoding mCherry-MICAL1. Fluorescent and phase contrast pictures are displayed. Scale bars, 10 μm. (f) Percentage of cells with GFP-MICAL1 at the intercellular bridge in control- or Rab35^S22N-expressing cells (N=3). ***P<0.001 (χ²-tests). n=256–170 cells per condition. (g) Same as in b for the indicated GAD- and LEX fusion proteins. (h) Cells transfected with GFP-MICAL1^1–1026 truncated mutant (green) were stained with β−tubulin (red) and CHMP4B (blue). Scale bar, 10 μm. (i) Distribution of the abscission times and mean abscission times in control- and MICAL1-depleted cells transfected with the indicated plasmids (N=3). No statistical differences between red and black curves; P<0.032 between blue and green and P=0.000 between red or black and other curves (KS test). NS, not significant; **P<0.01 (t-test). n=154–299 cells per condition. Error bars represent s.d. Red arrow: midbody and orange arrow: future abscission site.