Dynein is anchored at the plasma membrane by a ternary complex comprising NuMA–LGN–Gα and thus ensures correct spindle positioning
Spindle positioning and spindle elongation are critical for proper cell division. In human cells, an evolutionary conserved ternary complex (NuMA/LGN/Gai) anchors dynein at the cortex during metaphase, thus ensuring correct spindle positioning. Whether this complex contributes to anaphase spindle elongation is not known. More generally, the mechanisms coupling mitotic progression with spindle behaviour remain elusive. Here, we uncover that levels of cortical dynein markedly increase during anaphase in a NuMA-dependent manner. We demonstrate that during metaphase, CDK1-mediated phosphorylation at T2055 negatively regulates NuMA cortical localization and that this phosphorylation is counteracted by PPP2CA phosphatase activity. We establish that this tug of war is essential for proper levels of cortical dynein and thus spindle positioning during metaphase. Moreover, we find that upon CDK1 inactivation in anaphase, the rise in dephosphorylated NuMA at the cell cortex leads to cortical dynein enrichment, and thus to robust spindle elongation. Our findings uncover a mechanism whereby the status of NuMA phosphorylation coordinates mitotic progression with proper spindle function.
How living systems break symmetry in an organized manner is a fundamental question in biology. In wild-type Caenorhabditis elegans zygotes, symmetry breaking during anterior-posterior axis specification is guided by centrosomes, resulting in anterior-directed cortical flows and a single posterior PAR-2 domain. We uncover that C. elegans zygotes depleted of the Aurora A kinase AIR-1 or lacking centrosomes entirely usually establish two posterior PAR-2 domains, one at each pole. We demonstrate that AIR-1 prevents symmetry breaking early in the cell cycle, whereas centrosomal AIR-1 instructs polarity initiation thereafter. Using triangular microfabricated chambers, we establish that bipolarity of air-1(RNAi) embryos occurs effectively in a cell-shape and curvature-dependent manner. Furthermore, we develop an integrated physical description of symmetry breaking, wherein local PAR-2-dependent weakening of the actin cortex, together with mutual inhibition of anterior and posterior PAR proteins, provides a mechanism for spontaneous symmetry breaking without centrosomes.
The positioning and the elongation of the mitotic spindle must be carefully regulated. In human cells, the evolutionary conserved proteins LGN/Ga i1-3 anchor the coiled-coil protein NuMA and dynein to the cell cortex during metaphase, thus ensuring proper spindle positioning. The mechanisms governing cortical localization of NuMA and dynein during anaphase remain more elusive. Here, we report that LGN/Ga i1-3 are dispensable for NuMAdependent cortical dynein enrichment during anaphase. We further establish that NuMA is excluded from the equatorial region of the cell cortex in a manner that depends on the centralspindlin components CYK4 and MKLP1. Importantly, we reveal that NuMA can directly associate with PtdInsP (PIP) and PtdInsP 2 (PIP 2 ) phosphoinositides in vitro. Furthermore, chemical or enzymatic depletion of PIP/PIP 2 prevents NuMA cortical localization during mitosis, and conversely, increasing PIP 2 levels augments mitotic cortical NuMA. Overall, our study uncovers a novel function for plasma membrane phospholipids in governing cortical NuMA distribution and thus the proper execution of mitosis.
Centrioles are evolutionarily conserved macromolecular structures that are fundamental to form cilia, flagella, and centrosomes. Centrioles are 9-fold symmetrical microtubule-based cylindrical barrels comprising three regions that can be clearly distinguished in the Chlamydomonas reinhardtii organelle: an ∼100-nm-long proximal region harboring a cartwheel; an ∼250-nm-long central core region containing a Y-shaped linker; and an ∼150-nm-long distal region ending at the transitional plate. Despite the discovery of many centriolar components, no protein has been localized specifically to the central core region in Chlamydomonas thus far. Here, combining relative quantitative mass spectrometry and super-resolution microscopy on purified Chlamydomonas centrioles, we identified POB15 and POC16 as two proteins of the central core region, the distribution of which correlates with that of tubulin glutamylation. We demonstrated that POB15 is an inner barrel protein within this region. Moreover, we developed an assay to uncover temporal relationships between centriolar proteins during organelle assembly and thus established that POB15 is recruited after the cartwheel protein CrSAS-6 and before tubulin glutamylation takes place. Furthermore, we discovered that two poc16 mutants exhibit flagellar defects, indicating that POC16 is important for flagellum biogenesis. In addition, we discovered that WDR90, the human homolog of POC16, localizes to a region of human centrioles that we propose is analogous to the central core of Chlamydomonas centrioles. Moreover, we demonstrate that WDR90 is required for ciliogenesis, echoing the findings in Chlamydomonas. Overall, our work provides novel insights into the identity and function of centriolar central core components.
Accurate spindle positioning is essential for error-free cell division. The one-cell Caenorhabditis elegans embryo has proven instrumental for dissecting mechanisms governing spindle positioning. Despite important progress, how the cortical forces that act on astral microtubules to properly position the spindle are modulated is incompletely understood. Here, we report that the PP6 phosphatase PPH-6 and its associated subunit SAPS-1, which positively regulate pulling forces acting on spindle poles, associate with the Aurora A kinase AIR-1 in C. elegans embryos. We show that acute inactivation of AIR-1 during mitosis results in excess pulling forces on astral microtubules. Furthermore, we uncover that AIR-1 acts downstream of PPH-6-SAPS-1 in modulating spindle positioning, and that PPH-6-SAPS-1 negatively regulates AIR-1 localization at the cell cortex. Moreover, we show that Aurora A and the PP6 phosphatase subunit PPP6C are also necessary for spindle positioning in human cells. There, Aurora A is needed for the cortical localization of NuMA and dynein during mitosis. Overall, our work demonstrates that Aurora A kinases and PP6 phosphatases have an ancient function in modulating spindle positioning, thus contributing to faithful cell division.
Despite being essential for proper cell division, the mechanisms governing centrosome duplication are incompletely understood and represent an important open question in cell biology. Formation of a new centriole next to each existing one is critical for centrosome duplication. In Caenorhabditis elegans embryos, the proteins SPD-2, ZYG-1, SAS-6, SAS-5, and SAS-4 are essential for centriole formation, but the mechanisms underlying their requirement remain unclear. Here, we demonstrate that the kinase ZYG-1 phosphorylates the coiled-coil protein SAS-6 at serine 123 in vitro. Importantly, we show that this phosphorylation event is crucial for centriole formation in vivo. Furthermore, we establish that such phosphorylation ensures the maintenance of SAS-6 at the emerging centriole. Overall, our findings establish that phosphorylation of the evolutionarily conserved protein SAS-6 is critical for centriole formation and thus for faithful cell division.
Microtubule asters must be positioned precisely within cells. How forces generated by molecular motors such as dynein are integrated in space and time to enable such positioning remains unclear. In particular, whereas aster movements depend on the drag caused by cytoplasm viscosity, in vivo drag measurements are lacking, precluding a thorough understanding of the mechanisms governing aster positioning. Here, we investigate this fundamental question during the migration of asters and pronuclei in C. elegans zygotes, a process essential for the mixing of parental genomes. Detailed quantification of these movements using the female pronucleus as an in vivo probe establish that the drag coefficient of the male-asters complex is approximately five times that of the female pronucleus. Further analysis of embryos lacking cortical dynein, the connection between asters and male pronucleus, or the male pronucleus altogether, uncovers the balance of dynein-driven forces that accurately position microtubule asters in C. elegans zygotes.
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