Role of the Midbody Matrix in Cytokinesis: RNAi and Genetic Rescue Analysis of the Mammalian Motor Protein CHO1

Abstract

CHO1 is a kinesin-like motor protein essential for cytokinesis in mammalian cells. To analyze how CHO1 functions, we established RNAi and genetic rescue assays. CHO1-depleted cells reached a late stage of cytokinesis but fused back to form binucleate cells because of the absence of the midbody matrix in the middle of the intercellular bridge. Expression of exogenous CHO1 restored the formation of the midbody matrix and rescued cytokinesis in siRNA-treated cells. By analyzing phenotypes rescued with different constructs, it was shown that both motor and stalk domains function in midbody formation, whereas the tail is essential for completion of cytokinesis after the midbody matrix has formed. During the terminal stage of cytokinesis, different subregions of the tail play distinctive roles in stabilizing the midbody matrix and maintaining an association between the midbody and cell cortex. These results demonstrate that CHO1 consists of functionally differentiated subregions that act in concert to ensure complete cell separation.

Functional Analysis of Human Microtubule-based Motor Proteins, the Kinesins and Dyneins, in Mitosis/Cytokinesis Using RNA Interference

“…Consistent with our findings, MKLP1 and MKLP2 do not interact with each other to form heterodimers in human cells (Neef et al, 2003). MKLP1 is required for the formation of the midbody matrix and MKLP1 and MKLP2 are essential for the completion of cytokinesis (Neef et al, 2003;Matuliene and Kuriyama, 2004).…”

“…More than 30 -70% of MKLP1 and MKLP2 esiRNAtreated cells became bi/multinucleated (Figures 1 and 2). MKLP1 orthologues also have functional roles in cytokinesis Mishima et al, 2002;Goshima and Vale, 2003;Matuliene and Kuriyama, 2004). MKLP-2 (rabkinesin-6/RAB6-KIFL) was originally identified as a Rab6 GTPasebinding protein that is involved in protein transport at the Golgi apparatus (Echard et al, 1998).…”

“…Several studies show that several numbers of kinesins are crucial for spindle assembly and function, chromosome segregation, mitotic checkpoint control, and cytokinesis (Blangy et al, 1995;Schaar et al, 1997;Mountain et al, 1999;Levesque and Compton, 2001;McEwen et al, 2001;Neef et al, 2003;Andrews et al, 2004;Ganem and Compton, 2004;Gruneberg et al, 2004;Kurasawa et al, 2004;Matuliene and Kuriyama, 2004;Mazumdar et al, 2004;Mishima et al, 2004;. Small-molecule inhibitors of specific kinesins perturb mitotic spindle dynamics and arrest cells in mitosis and cytokinesis (Mayer et al, 1999;Sakowicz et al, 2004).…”

Recruitment of Tom1L1/Srcasm to Endosomes and the Midbody by Tsg101

“…The identity of the Tsg101-positive dot structure on the intercellular bridge as the midbody was confirmed by the complete overlap of the GFP-Tsg101 structure with the structure positive for MKLP1 (mitotic kinesin-like protein 1) (Fig. 3, D-D"' and E-E"'), which is a component of the centralspindlin complex and is known to associate with the midbody matrix during cytokinesis (Matuliene and Kuriyama, 2004;reviewed in Glotzer, 2005). In cells with GFP-Tsg101 at the midbody during cytokinesis, 77% of the cells (33 out of 43 cells counted) had mCherry-Tom1L1 (Fig.…”

Calmodulin regulates the post-anaphase reposition of centrioles during cytokinesis

“…In order to examine whether the intercellular bridge associated CaM is also involved in cytokinesis completion, we treated synchronized PtK2 cells with W7 (15 µM) for 4 h at a late stage in cytokinesis when most cells have begun to form intercellular bridges. When the completion of cytokinesis was prolonged by any perturbations, the two daughter cells would be linked by a longer intercellular bridge [12]. As shown in Fig.…”