Ndel1, a protein located at the subdistal appendage of mother centriole, functions as an upstream regulator of the trichoplein–Aurora A pathway that suppresses ciliogenesis in proliferating cells.

The primary cilium is a non-motile and microtubule-enriched protrusion ensheathed by plasma membrane. Primary cilia function as mechano/chemosensors and signaling hubs and their disorders predispose to a wide spectrum of human diseases. Most types of cells assemble their primary cilia in response to cellular quiescence, whereas they start to retract the primary cilia upon cell-cycle reentry. The retardation of ciliary resorption process has been shown to delay cell-cycle progression to the S or M phase after cell-cycle reentry. Apart from this conventional concept of ciliary disassembly linked to cell-cycle reentry, recent studies have led to a novel concept, suggesting that cells can suppress primary cilia assembly during cell proliferation. Accumulating evidence has also demonstrated the importance of Aurora-A (a protein originally identified as one of mitotic kinases) not only in ciliary resorption after cell-cycle reentry but also in the suppression of ciliogenesis in proliferating cells, whereas Aurora-A activators are clearly distinct in both phenomena. Here, we summarize the current knowledge of how cycling cells suppress ciliogenesis and compare it with mechanisms underlying ciliary resorption after cell-cycle reentry. We also discuss a reciprocal relationship between primary cilia and cell proliferation.

Rho/Ras family small GTPases are known to regulate numerous cellular processes, including cytoskeletal reorganization, cell proliferation, and cell differentiation. These processes are also controlled by Ca 2+ , and consequently, cross talk between these signals is considered likely. However, systematic quantitative evaluation has not yet been reported. To fill this gap, we constructed optogenetic tools to control the activity of small GTPases (RhoA, Rac1, Cdc42, Ras, Rap, and Ral) using an improved light-inducible dimer system (iLID). We characterized these optogenetic tools with genetically encoded red fluorescence intensity-based small GTPase biosensors and confirmed these optogenetic tools’ specificities. Using these optogenetic tools, we investigated calcium mobilization immediately after small GTPase activation. Unexpectedly, we found that a transient intracellular calcium elevation was specifically induced by RhoA activation in RPE1 and HeLa cells. RhoA activation also induced transient intracellular calcium elevation in MDCK and HEK293T cells, suggesting that generally RhoA induces calcium signaling. Interestingly, the molecular mechanisms linking RhoA activation to calcium increases were shown to be different among the different cell types: In RPE1 and HeLa cells, RhoA activated phospholipase C epsilon (PLCε) at the plasma membrane, which in turn induced Ca 2+ release from the endoplasmic reticulum (ER). The RhoA–PLCε axis induced calcium-dependent nuclear factor of activated T cells nuclear translocation, suggesting that it does activate intracellular calcium signaling. Conversely, in MDCK and HEK293T cells, RhoA–ROCK–myosin II axis induced the calcium transients. These data suggest universal coordination of RhoA and calcium signaling in cellular processes, such as cellular contraction and gene expression.

We previously reported that vimentin, GFAP, and desmin (type III intermediate filament [IF] proteins) are mitotically phosphorylated by CDK1, Aurora-B, and Rho-kinase. This phosphorylation is critical for efficient separation of these IFs and completion of cytokinesis. Keratin 5 (K5) and K14 form a heterodimer, which constitutes IF network in basal layer cells of stratified squamous epithelia. Here, we report that the solubility of K5/K14 increased in mitosis. The in vitro assays revealed that three mitotic kinases phosphorylate K5 more than K14. We then identified Thr23/Thr144, Ser30, and Thr159 on murine K5 as major phosphorylation sites for CDK1, Aurora-B, and Rho-kinase, respectively. Using site- and phosphorylation-state-specific antibodies, we demonstrated that K5-Thr23 was phosphorylated in entire cytoplasm from prometaphase to metaphase, whereas K5-Ser30 phosphorylation occurred specifically at the cleavage furrow from anaphase to telophase. Efficient K5/K14-IF separation was impaired by K5 mutations at the sites phosphorylated by these mitotic kinases. K5-Thr23 phosphorylation was widely detected in dividing K5-positive cells of murine individuals. These results suggested that mitotic reorganization of K5/K14-IF network is governed largely through K5 phosphorylation by CDK1, Aurora-B, and Rho-kinase.