Abstract
During cytokinesis, the antiparallel array of microtubules forming the central spindle organizes the midbody, a structure that anchors the ingressed cleavage furrow and guides the assembly of abscission machinery. Here, we identified a role for the flavoprotein monooxygenase MICAL3, an actin disassembly factor, in organizing midbody-associated protein complexes. By combining cell biological assays with cross-linking mass spectrometry, we show that MICAL3 is recruited to the central spindle and the midbody through a direct interaction with the centralspindlin component MKLP1. Knock-out of MICAL3 leads to an increased frequency of cytokinetic failure and a delayed abscission. In a mechanism independent of its enzymatic activity, MICAL3 targets the adaptor protein ELKS and Rab8A-positive vesicles to the midbody, and the depletion of ELKS and Rab8A also leads to cytokinesis defects. We propose that MICAL3 acts as a midbody-associated scaffold for vesicle targeting, which promotes maturation of the intercellular bridge and abscission.
Highlights
Cytokinesis is the last step of cell division, during which the cytoplasm is partitioned between the two daughter cells
We showed that mitotic kinesin-like protein 1 (MKLP1) recruits MICAL3 to the central spindle, whereas MICAL3 participates in targeting to the midbody its binding partners ELKS and Rab8A, and the loss of both ELKS and Rab8A caused cytokinesis defects
Previous work has shown that centralspindlin represents a major binding hub for different factors involved in cytokinesis [4], and our current work added MICAL3 to the list of MKLP1 interactors
Summary
MICAL3 Interacts with MKLP1—To get insight into the function of MICAL3, we performed a mass spectrometrybased screen for its binding partners. No rescue was observed with GFP alone, we could completely rescue the binucleated phenotype by either the fulllength MICAL3 or its enzymatically dead mutant, but not with the MICAL3 deletion mutant lacking the MKLP1-binding C terminus (Fig. 5F) These data indicate that MICAL3 contributes to the stabilization and/or maturation of the intercellular bridge in a way that does not require its monooxygenase activity. We have examined whether the localization of CRIK or KIF14 was perturbed in MICAL3 knock-out cells and found this not to be the case (Fig. 8F) These data do not rule out that ELKS, localized to the midbody through its interaction with MICAL3, somehow affects the function of CRIK-containing protein complexes, they indicate that MICAL3 does not participate in the targeting of this signaling protein complex to the intercellular bridge, in line with the data showing that CRIK directly interacts with MKLP1 [28]. We conclude that the complex of MICAL3 and ELKS helps to target Rab8A to the intercellular bridge, and that all three proteins are required to the proper maturation of the bridge, which prevents the bridge recession and promotes timely abscission
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