In mitotic ARHGEF10-knockdown cells, we observed multiple spindle poles, each of which contains two centrioles and the PCM. One possible mechanism for the formation of such supernumerary centrosomes is aborted cell division [1
]. Aneuploid cells generated through aberrant mitotic exit contain more than two centrosomes, which may form multipolar spindles in next M phase. Initially, we thought that aberrant division of cells lacking ARHGEF10 may frequently occur because RhoA is known to be involved in many facets of mitotic and cytokinetic control. However, the occurrence of multinucleated cells was not affected by ARHGEF10 knockdown in contrast to treatment with a ROCK inhibitor, which significantly enhanced the formation of multinucleated cells (Figure ). Furthermore, normal ingression of the cleavage furrow was, in fact, detected in ARHGEF10-knockdown cells as well (Figure ). Therefore, it is not feasible that ARHGEF10 is responsible for RhoA activation in mitosis and cytokinesis. Instead, ARHGEF10 may regulate RhoA during interphase.
Deregulated centrosome duplication also leads to the formation of multiple centrosomes and multipolar spindles [1
]. Thus, the multi-centrosome phenotype observed in ARHGEF10-knockdown cells may be ascribed to disorder in centrosome duplication. Localization of ARHGEF10 in centrosomes strongly supports this notion. Centriole duplication is initiated by the activation of cyclin-dependent kinase 2 around the G1/S transition, concurrent with DNA replication, and short daughter centrioles elongate during S and G2 phases [17
]. Following completion of centriole elongation at the G2/M transition, centrosome maturation and separation occur, with a pair of centrioles in each centrosome. Centrosome duplication must occur precisely once every cell cycle, being inextricably coupled with other cell cycle-dependent events. To ensure this, cells are afforded mechanisms (1) to limit centriole duplication to once in every cell division cycle and (2) to limit the number of progenitor centrioles to one per pre-existingcentriole.
Plenty of molecules that positively regulate centrosome duplication, including Polo-like kinase-4 [16
], cyclin-dependent kinase 2 [17
] and SPD-2 [20
], have been reported. The phenotype termed centriole overduplication (production of multiple daughter centrioles arranged around each parental centriole) was observed when a positive regulator of centriole duplication, for instance Polo-like kinase-4, is overexpressed [16
] due to a disordered mechanism to limit the number of progenitor centrioles. The supernumerary centrosome formation described in Figure is distinct from this phenotype, and therefore, the ARHGEF10-RhoA pathway may not be responsible for the copy number control of procentrioles duplicated from a single parental centriole.
One major mechanism to limit centriole duplication to once in every cell division cycle involves a protein-digesting enzyme termed separase, which is also responsible for sister chromatid separation [21
]. In the normal cell division cycle, centrioles are prevented from duplicating again during late S and G2 phases, being tightly connected or engaged. At the end of mitosis, the engaged centriole pair becomes dissociated, losing their strict orthogonal orientation. This process, called disengagement, is triggered by the action of separase, and is prerequisite for a new round of centriole duplication [21
]. Thus, intrinsic block to centriole reduplication throughout the cell cycle and timely activation of separase represent a mechanism to ensure that a new round of duplication can occur only after passage through M phase. Although speculative, disruption of the ARHGEF10-RhoA-dependent signaling pathway by knocking down its component may permit an excess cycle of centriole duplication if sustained engagement of centrioles or suppression of separase activity during late S and G2 phases requires this pathway. The observation that only two among three centrosomes in ARHGEF10-knockdown cells exhibit diminished ARHGEF10 expression (Figure ) may be consistent with the idea that only a centrosome that lacks ARHGEF10 undergoes an excess duplication cycle before mitosis.
Multiple mitotic spindles were also observed in cells deficient in the Polo-like kinase-1 target Kizuna [23
]. Unlike Kizuna-depleted multipolar cells, however, we did not detect fragmentation of the PCM in ARHGEF10-knockdown cells, suggesting that the ARHGEF10-RhoA pathway is not implicated in stabilization of the PCM in early mitotic phase.
In this paper, we further identified the motor protein KIF3B as a binding partner of ARHGEF10 (Figure ). KIF3B has been implicated in a variety of physiological responses, including determination of left-right asymmetry [24
], axonal transport [25
], assembly and maintenance of the excitation-contraction-coupling membranes in skeletal muscle [26
], cytokinesis [27
] and glucose transport in adipocytes [28
]. We observed multiple mitotic spindle poles in KIF3B-knockdown cells (Figure ), resembling phenotypes observed following expression of a dominant-negative mutant of KIF3B [29
]. Considering that ARHGEF10 associates with KIF3B in the centrosome, KIF3B may regulate spindle pole formation through the ARHGEF10-RhoA pathway. KIF3B knockdown may also cause supernumerary centrosomes through cytokinesis defects independently of ARHGEF10 because a dominant-negative mutant of KIF3B caused chromosomal aneuploidy [29
]. The mechanisms may be more complicated because it is reported that silencing of another motor protein KIFC5A causes centrosome amplification primarily through reduplication and partly as a result of defects incytokinesis [30