Rab11 endosomes coordinate centrosome number and movement following mitotic exit

The last stage of cell division involves two daughter cells remaining interconnected by a cytokinetic bridge that is cleaved in a process called abscission. During pre-abscission, we identified that the centrosome moves in a Rab11-dependent manner towards the cytokinetic bridge in human cells grown in culture and in an in vivo vertebrate model, Danio rerio (zebrafish). Rab11-endosomes are dynamically organized in a Rab11-GTP dependent manner at the centrosome during pre-abscission and this organization is required for the centrosome protein, pericentrin, to be enriched at the centrosome. Using zebrafish embryos, we found that reduction in pericentrin expression or optogenetically disrupting Rab11-endosome function inhibited centrosome movement towards the cytokinetic bridge and abscission resulting in daughter cells prone to being binucleated and/or having supernumerary centrosomes. These studies suggest that Rab11-endosomes contribute to centrosome function during pre-abscission by regulating pericentrin organization resulting in appropriate centrosome movement towards the cytokinetic bridge and subsequent abscission.

APC/CFZR-1 regulates centrosomal ZYG-1 to limit centrosome number

Aberrant centrosome numbers are associated with human cancers. The levels of centrosome regulators positively correlate with centrosome number. Thus, tight control of centrosome protein levels is critical. In Caenorhabditis elegans, the anaphase-promoting complex/cyclosome and co-activator FZR-1 (APC/CFZR-1) ubiquitin ligase negatively regulates centrosome assembly through SAS-5 degradation. In this study, we report the C. elegans ZYG-1 (Plk4 in human) as a potential substrate of APC/CFZR-1. Inhibiting APC/CFZR-1 or mutating a ZYG-1 destruction (D)-box leads to elevated ZYG-1 levels at centrosomes, restoring bipolar spindles and embryonic viability to zyg-1 mutants, suggesting that APC/CFZR-1 influences centrosomal ZYG-1 via D-box motif. We also show the Slimb/βTrCP-binding (SB) motif is critical for ZYG-1 degradation, substantiating a conserved mechanism by which ZYG-1/Plk4 stability is regulated by SCFSlimb/βTrCP-dependent proteolysis via the conserved SB motif in C. elegans. Furthermore, we show that co-mutating ZYG-1 SB and D-box motifs stabilizes ZYG-1 in an additive manner, suggesting that APC/CFZR-1 and SCFSlimb/βTrCP ubiquitin ligases function cooperatively for timely ZYG-1 destruction in C. elegans embryos where ZYG-1 activity remains at threshold level to ensure normal centrosome number.

Cep215 is essential for morphological differentiation of astrocytes

Cep215 (also known as Cdk5rap2) is a centrosome protein which is involved in microtubule organization. Cep215 is also placed at specific subcellular locations and organizes microtubules outside the centrosome. Here, we report that Cep215 is involved in morphological differentiation of astrocytes. Cep215 was specifically localized at the glial processes as well as centrosomes in developing astrocytes. Morphological differentiation of astrocytes was suppressed in the Cep215-deleted P19 cells and in the Cep215-depleted embryonic hippocampal culture. We confirm that the microtubule organizing function of Cep215 is critical for the glial process formation. However, Cep215 is not involved in the regulation of cell proliferation nor cell specification. Based on the results, we propose that Cep215 organizes microtubules for glial process formation during astrocyte differentiation.

APC/CFZR-1 Controls ZYG-1 Levels to Regulate Centrosome Assembly

Aberrant centrosome numbers are associated with human cancers. The levels of centrosome regulators positively correlate with centrosome number. Thus, tight control of centrosome protein levels is critical. In Caenorhabditis elegans, the anaphase-promoting complex/cyclosome and co-activator FZR-1 (APC/CFZR-1) ubiquitin ligase negatively regulates centrosome assembly through SAS-5 degradation. In this study, we identify the C. elegans ZYG-1 (Plk4 in human) as a new substrate of APC/CFZR-1. Inhibiting APC/CFZR-1 or mutating a ZYG-1 destruction (D)-box leads to elevated ZYG-1 levels at centrosomes, restoring bipolar spindles and embryonic viability to zyg-1 mutants, suggesting that APC/CFZR-1 targets ZYG-1 for proteasomal degradation via D-box motif. We also show the Slimb/βTrCP-binding (SB) motif is critical for ZYG-1 degradation, substantiating a conserved mechanism by which ZYG-1/Plk4 stability is regulated by SCFSlimb/βTrCP-dependent proteolysis via the conserved SB motif in C. elegans. Furthermore, inhibiting both APC/CFZR-1 and SCFSlimb/βTrCP, by co-mutating ZYG-1 SB and D-box motifs, stabilizes ZYG-1 in an additive manner, conveying that APC/CFZR-1 and SCFSlimb/βTrCP ubiquitin ligases function cooperatively for timely ZYG-1 destruction in C. elegans embryos where ZYG-1 activity remains at threshold level to ensure normal centrosome number.

Gravin regulates centrosome function through PLK1

We propose to understand how the mitotic kinase PLK1 drives chromosome segregation errors, with a specific focus on Gravin, a PLK1 scaffold. In both three-dimensional primary prostate cancer cell cultures that are prone to Gravin depletion and Gravin short hairpin RNA (shRNA)–treated cells, an increase in cells containing micronuclei was noted in comparison with controls. To examine whether the loss of Gravin affected PLK1 distribution and activity, we utilized photokinetics and a PLK1 activity biosensor. Gravin depletion resulted in an increased PLK1 mobile fraction, causing the redistribution of active PLK1, which leads to increased defocusing and phosphorylation of the mitotic centrosome protein CEP215 at serine-613. Gravin depletion further led to defects in microtubule renucleation from mitotic centrosomes, decreased kinetochore-fiber integrity, increased incidence of chromosome misalignment, and subsequent formation of micronuclei following mitosis completion. Murine Gravin rescued chromosome misalignment and micronuclei formation, but a mutant Gravin that cannot bind PLK1 did not. These findings suggest that disruption of a Gravin–PLK1 interface leads to inappropriate PLK1 activity contributing to chromosome segregation errors, formation of micronuclei, and subsequent DNA damage.