NuMA interacts with phosphoinositides and links the mitotic spindle with the plasma membrane

The positioning and the elongation of the mitotic spindle must be carefully regulated. In human cells, the evolutionary conserved proteins LGN/Gai1-3 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/Gai1-3 are dispensable for NuMA-dependent 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 PtdInsP2 (PIP2) phosphoinositides in vitro. Furthermore, chemical or enzymatic depletion of PIP/PIP2 prevents NuMA cortical localization during mitosis, and conversely, increasing PIP2 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

The nucleoporin Nup205/NPP-3 is lost near centrosomes at mitotic onset and can modulate the timing of this process in Caenorhabditis elegans embryos

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.Regulation of mitosis in time and space is critical for proper cell division. We conducted an RNA interference–based modifier screen to identify novel regulators of mitosis in Caenorhabditis elegans embryos. Of particular interest, this screen revealed that the Nup205 nucleoporin NPP-3 can negatively modulate the timing of mitotic onset. Furthermore, we discovered that NPP-3 and nucleoporins that are associated with it are lost from the nuclear envelope (NE) in the vicinity of centrosomes at the onset of mitosis. We demonstrate that centrosomes are both necessary and sufficient for NPP-3 local loss, which also requires the activity of the Aurora-A kinase AIR-1. Our findings taken together support a model in which centrosomes and AIR-1 promote timely onset of mitosis by locally removing NPP-3 and associated nucleoporins from the NE.V.H. was supported by a Roche postdoctoral fellowship (Mkl/stm 120-2007) and by an MHV postdoctoral fellowship from the Swiss National Science Foundation (PMPD33-118694). Additional support was provided by the Swiss Cancer League (grant KLS 2160-02-2008 to P.G.). Work in the laboratory of P.A. was funded by the Spanish Ministry of Science and Innovation (BFU2010-15478).Peer reviewe

Cortical dynein is critical for proper spindle positioning in human cells

Correct spindle positioning is fundamental for proper cell division during development and in stem cell lineages. Dynein and an evolutionarily conserved ternary complex (nuclear mitotic apparatus protein [NuMA]–LGN–Gα in human cells and LIN-5–GPR-1/2–Gα in Caenorhabditis elegans) are required for correct spindle positioning, but their relationship remains incompletely understood. By analyzing fixed specimens and conducting live-imaging experiments, we uncovered that appropriate levels of ternary complex components are critical for dynein-dependent spindle positioning in HeLa cells and C. elegans embryos. Moreover, using mutant versions of Gα in both systems, we established that dynein acts at the membrane to direct spindle positioning. Importantly, we identified a region within NuMA that mediates association with dynein. By using this region to target dynein to the plasma membrane, we demonstrated that the mere presence of dynein at that location is sufficient to direct spindle positioning in HeLa cells. Overall, we propose a model in which the ternary complex serves to anchor dynein at the plasma membrane to ensure correct spindle positioning

Phosphorylation of SAS-6 by ZYG-1 is critical for centriole formation in C. elegans embryos

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. 2009 Elsevier Inc. All rights reserve

Quantitative Analysis and Modeling Probe Polarity Establishment in C. elegans Embryos

Cell polarity underlies many aspects of metazoan development and homeostasis, and relies notably on a set of PAR proteins located at the cell cortex. How these proteins interact in space and time remains incompletely understood. We performed a quantitative assessment of polarity establishment in one-cell stage Caenorhabditis elegans embryos by combining time-lapse microscopy and image analysis. We used our extensive data set to challenge and further specify an extant mathematical model. Using likelihood-based calibration, we uncovered that cooperativity is required for both anterior and posterior PAR complexes. Moreover, we analyzed the dependence of polarity establishment on changes in size or temperature. The observed robustness of PAR domain dimensions in embryos of different sizes is in agreement with a model incorporating fixed protein concentrations and variations in embryo surface/volume ratio. In addition, we quantified the dynamics of polarity establishment over most of the viable temperatures range of C. elegans. Modeling of these data suggests that diffusion of PAR proteins is the process most affected by temperature changes, although cortical flows appear unaffected. Overall, our quantitative analytical framework provides insights into the dynamics of polarity establishment in a developing system

Aurora A kinase regulates proper spindle positioning in C-elegans and in human cells

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

TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin

TRIM37 is an E3 ubiquitin ligase mutated in Mulibrey nanism, a disease with impaired organ growth and increased tumor formation. TRIM37 depletion from tissue culture cells results in supernumerary foci bearing the centriolar protein Centrin. Here, we characterize these centriolar protein assemblies (Cenpas) to uncover the mechanism of action of TRIM37. We find that an atypical de novo assembly pathway can generate Cenpas that act as microtubule-organizing centers (MTOCs), including in Mulibrey patient cells. Correlative light electron microscopy reveals that Cenpas are centriole-related or electron-dense structures with stripes. TRIM37 regulates the stability and solubility of Centrobin, which accumulates in elongated entities resembling the striped electron dense structures upon TRIM37 depletion. Furthermore, Cenpas formation upon TRIM37 depletion requires PLK4, as well as two parallel pathways relying respectively on Centrobin and PLK1. Overall, our work uncovers how TRIM37 prevents Cenpas formation, which would otherwise threaten genome integrity.Peer reviewe

Combining gonad nuclei spreading and U-Ex microscopy to analyze worm gonad centrioles.

(A) Widefield imaging of ethanol-fixed worm expressing GFP::SAS-7. One layer of nuclei of the gonad is max intensity Z-projected (in this case, a height of 6.25 μm). White, grey, and red bold dashed lines indicate progression through the gonad from the mitotic zone to early and then late meiotic prophase; other white dashed line outlines the gonad. Grey box is magnified in (B). Yellow dashed regions mark 3 oocytes, purple dashed region the spermatheca. Note that centrioles are eliminated in oocytes, prior to fertilization. (B) (Left) Magnification of grey box region from (A). (Middle) Schematic representation of a single nucleus shown in the left and right panels. (Right) Early prophase region of a spread gonad from a worm expressing GFP::SAS-7. Note that spread nuclei are flattened and thus occupy a larger area compared to not spread nuclei. Note also that at this stage, centrioles do not act as microtubule organizing centers [55,56]. (C) Widefield imaging of centrioles in the early prophase region of the gonad from worms expressing GFP::SAS-7 before (left) and after (right) gel expansion. Grey mesh in the background represents the gel matrix.</p