F-actin disassembly factor MICAL1 binding to Myosin Va mediates cargo unloading during cytokinesis

Fig. 1. MyoVa and MICAL1 colocalized at the midbody via the direct interaction. (A) Domain architecture of MyoVa. (B) Quantitative scatterplot of the total spectra from the binding candidates of GST-MyoVaGTD versus those of GST (control), based on the mass spectrum results of two GTS pull-down samples. P < 0.05, Fisher’s exact test. (C to E) The interaction between endogenous (C) or overexpressed (D) MyoVa and MICAL1 was detected by co-IP and ITC (E) assays. (F) Schematic diagram represents midbody and intercellular bridge between two separating daughter cells during cytokinesis. Cell nucleus in two daughter cells and microtubules in intercellular bridge are indicated with blue circle and green line, respectively. (G to J) Accumulations of endogenous MICAL1 at the midbody in HeLa cells (G) and endogenous MyoVa at the midbodies in HeLa (H), HT1080 (I), and Chinese hamster ovary (CHO) cells (J) during cytokinesis by immunofluorescence. (K to M) HeLa cells were transfected with mCherry-MICAL1 (K) or green fluorescent protein (GFP)–MyoVa (L) individually and cotransfected with GFP-MyoVa and mCherry-MICAL1 (M). β-Tubulin was stained to indicate the intercellular bridge. In each panel, the…

Fig. 2. The interaction between MICAL1 and MyoVa at the midbody is essential for efficient cytokinesis. (A) The GTD-binding motif (GTBM) in MICAL1 was mapped into a loop region connecting LIM and CC domains. Detailed data were shown in fig. S2. (B and C) HeLa cells were treated with the control siRNA (siControl) (B) or the siRNA targeting the MICAL1 mRNA (siMICAL1) (C) for 72 hours and then stained by the antibody against MyoVa. (D and E) In the MICAL1KD cells, Flag-tagged MICAL1 (D) or MICAL1ΔGTBM (E) was transfected for additional 24 hours to rescue the midbody accumulation of MyoVa. Endogenous MyoVa and overexpressed Flag-MICAL1/MICA1ΔGTBM were stained with the antibodies against MyoVa and Flag, respectively. (F and G) Statistical analysis of the intensity fold change of accumulated MyoVa at the midbody (F) and the intensity of overexpressed Flag-MICAL1/MICAL1ΔGTBM at the midbody (G) in siRNA-treated HeLa cells. The fold change represents the MyoVa intensity change ratio between the midbody and its adjacent region. The fold change value around 1 indicates diffused distribution at the midbody, while the value larger than 1 indicates accumulation happening at the midbody. Intensity was…

Fig. 3. MICAL1 coordinates Rab11a-positive vesicle trafficking to the midbody by interacting with MyoVa. (A) Complex structure of MyoVaGTD (purple) and Rab11a (cyan) [Protein Data Bank (PDB) ID: 5JCZ] (28). Inset: Key residue F48 in Rab11a is inserted into the hydrophobic patch in subdomain II of MyoVaGTD. (B) GST pull-down assay confirmed that Rab11a1–177 directly bound to MyoVaGTD, while the Rab11a1–177-F48Q mutant abolished the binding. The gel was stained by Coomassie blue. (C to E) Midbody localization of Rab11a in the HeLa cells transfected with mCherry-tagged Rab11a (C) or Rab11a-F48Q (D) or Rab11a-Q70L (E). (F) Statistical analysis of the intensity fold change and the intensity of overexpressed Rab11a or Rab11a-F48Q or Rab11a-Q70L at the midbody. The fold change represents the Rab11a intensity ratio between the midbody and its adjacent region. Cell number per condition for analysis was indicated. (G and H) Midbody localization of Rab11a in the MICAL1KO cells transfected with the mCherry-tagged Rab11a (G) or Rab11a-Q70L (H), the constitutively active mutant of Rab11a. (I and J) Rescue assay was performed by overexpression of Flag-tagged MICAL1 (I) or MICAL1ΔGTBM (J) in MICAL1KO…

Fig. 4. MICAL1 controls the unloading of Rab11a-positive vesicles at the midbody. (A and B) Normal (A) and MICAL1KO cells (B) expressing mCherry-Rab11a-Q70L were observed under a time-scaled SIM. The boundaries of two daughter cells during cytokinesis were indicated with dashed lines, and the middle regions of midbody were circled. Scale bars, 1 μm. The midbody regions were enlarged in the time-series snapshots to show the movements of Rab11a-positive vesicles indicated by red arrowheads. The corresponding overlapped figures in the right panel were calculated under time-lapse color mode by ImageJ. (C and D) FRAP analysis of Rab11a-positive vesicles at the midbody in normal (C) and MICAL1KO cells (D) expressing GFP-MyoVa and mCherry-Rab11a-Q70L. The bleaching region was indicated by a dashed circle, and β-tubulin was stained with SiR-tubulin to indicate the intercellular bridges. Scale bars, 1 μm. (E) Recovery efficiency of GFP-MyoVa and mCherry-Rab11a-Q70L at the midbody. Cell number per condition for analysis was indicated. (F) HeLa cells expressing PAGFP-tagged Rab11a-Q70L were photoactivated at the midbody region or the cytosol region. The two regions were indicated by dashed circles.…

Fig. 5. MICAL1 competes with Spires in binding with MyoVa. (A) Cartoon representation of the overall structure of MyoVaGTD (purple) in complex with MICAL1GTBM (blue). The GTBM region of MICAL1 binds to the subdomain I of MyoVaGTD. (B) Structure-based sequence alignment of the GTBM regions from MICAL1, Spir1/2, and MLPH. The residues important for the interaction with MyoVaGTD were highlighted in red and indicated by stars. (C) Structural comparison of the MyoVaGTD-bound GTBMs in MICAL1 (blue), Spir2 (cyan, PDB ID: 5JCY), and MLPH (orange, PDB ID: 4KP3). The surface of MyoVaGTD was shown in light purple. The main interface residues of the three GTBM peptides were shown as sticks, corresponding to the highlighted residues in (B). (D) ITC-derived binding affinity between MyoVaGTD or MyoVaGTD-I1559E and the three GTBM peptides. #, previously reported binding affinity (46). (E) GST pull-down assay was performed to confirm the competition between Spir2 and MICAL1 in the binding to MyoVa. The gel was stained by Coomassie blue. (F) Confocal imaging of HeLa cells cotransfected with GFP-MyoVa and Flag-MICAL1. (G and H) Confocal imaging of HeLa cells triple-transfected with GFP-MyoVa, Flag-MICAL1,…

Fig. 6. Enzymatic activity of MICAL1 is required for Rab11a-positive vesicle unloading at the midbody. (A to D) Overexpression of the catalytically inactive MICAL1-3G3W mutant in MICAL1KO cells failed to rescue the midbody accumulation of either endogenous MyoVa (A) or overexpressed Rab11a-Q70L (C). Statistical analysis of the intensity fold change of endogenous MyoVa (B) and Rab11a-Q70L (D) and the corresponding intensity of overexpressed MICAL1 at the midbody in MICAL1KO cells, respectively. The fold change represents the MyoVa or Rab11a intensity ratio between the midbody and its adjacent region. Cell number per condition for analysis was indicated. (E) FRAP analysis of Rab11a-positive vesicles at the midbody in the MICAL1KO cells coexpressing mCherry-Rab11a-Q70L with GFP-tagged MICAL1, MICAL1-3G3W, or MICAL1ΔGTBM. The bleaching region was indicated by a dashed circle, and β-tubulin was stained with SiR-tubulin to indicate the intercellular bridge. Scale bars, 1 μm. (F) Recovery efficiency of Rab11a-postive vesicles at the midbody in the MICAL1KO cells cotransfected with mCherry-Rab11a-Q70L and GFP-tagged MICAL1, MICAL1-3G3W, or GFP-MICAL1ΔGTBM. Cell number per condition for analysis…