A central question in cell proliferation is what controls cell cycle transitions. Although classical experiments indicate that accumulation of mitotic cyclins drives the G2/M transition in embryonic cells, the trigger for mitotic entry in somatic cells remains unknown.

Seki and Rutz describe an optimized Cas9/RNP transfection approach to enable highly efficient CRISPR-mediated gene knockout in primary mouse and human T cells without T cell receptor stimulation that results in near complete loss of target gene expression at the population level.

We report here an efficient functional genomic analysis by combining information on the gene expression profiling, cellular localization, and loss-of-function studies. Through this analysis, we identified Cep55 as a regulator required for the completion of cytokinesis. We found that Cep55 localizes to the mitotic spindle during prometaphase and metaphase and to the spindle midzone and the midbody during anaphase and cytokinesis. At the terminal stage of cytokinesis, Cep55 is required for the midbody structure and for the completion of cytokinesis. In Cep55-knockdown cells, the Flemming body is absent, and the structural and regulatory components of the midbody are either absent or mislocalized. Cep55 also facilitates the membrane fusion at the terminal stage of cytokinesis by controlling the localization of endobrevin, a v-SNARE required for cell abscission. Biochemically, Cep55 is a microtubule-associated protein that efficiently bundles microtubules. Cep55 directly binds to MKLP1 in vitro and associates with the MKLP1-MgcRacGAP centralspindlin complex in vivo. Cep55 is under the control of centralspindlin, as knockdown of centralspindlin abolished the localization of Cep55 to the spindle midzone. Our study defines a cellular mechanism that links centralspindlin to Cep55, which, in turn, controls the midbody structure and membrane fusion at the terminal stage of cytokinesis.

Ca2+ signals control cell migration by regulating forward movement and cell adhesion. However, it is not well understood how Ca2+-regulatory proteins and second messengers are spatially organized in migrating cells. Here we show that receptor tyrosine kinase and phospholipase C signaling are restricted to the front of migrating endothelial leader cells, triggering local Ca2+ pulses, local depletion of Ca2+ in the endoplasmic reticulum, and local activation of STIM1, supporting pulsatile front retraction and adhesion. At the same time, the mediator of store-operated Ca2+ influx STIM1 is transported by microtubule plus ends to the front. Furthermore, higher Ca2+ pump rates in the front relative to the back of the plasma membrane enable effective local Ca2+ signaling by locally decreasing basal Ca2+. Finally, polarized phospholipase C signaling generates a diacylglycerol gradient towards the front that promotes persistent forward migration. Thus, cells employ an integrated Ca2+ control system with polarized Ca2+ signaling proteins and second messengers to synergistically promote directed cell migration.

Through a convergence of functional genomic and proteomic studies, we identify Bora as a previously unknown cell cycle protein that interacts with the Plk1 kinase and the SCF–β-TrCP ubiquitin ligase. We show that the Bora protein peaks in G2 and is degraded by proteasomes in mitosis. Proteolysis of Bora requires the Plk1 kinase activity and is mediated by SCF–β-TrCP. Plk1 phosphorylates a conserved DSGxxT degron in Bora and promotes its interaction with β-TrCP. Mutations in this degron stabilize Bora. Expression of a nondegradable Bora variant prolongs the metaphase and delays anaphase onset, indicating a physiological requirement of Bora degradation. Interestingly, the activity of Bora is also required for normal mitotic progression, as knockdown of Bora activates the spindle checkpoint and delays sister chromatid segregation. Mechanistically, Bora regulates spindle stability and microtubule polymerization and promotes tension across sister kinetochores during mitosis. We conclude that tight regulation of the Bora protein by its synthesis and degradation is critical for cell cycle progression.