A CTDNEP1-lipin 1-mTOR regulatory network restricts ER membrane biogenesis to enable chromosome motions necessary for mitotic fidelity

SummaryThe endoplasmic reticulum (ER) dramatically restructures in open mitosis to become excluded from the mitotic spindle; however, the significance of ER reorganization to mitotic progression is not known. Here, we demonstrate that limiting ER membrane biogenesis enables mitotic chromosome movements necessary for chromosome biorientation and prevention of micronuclei formation. Aberrantly expanded ER membranes increase the effective viscosity of the mitotic cytoplasm to physically restrict chromosome dynamics – slowed chromosome motions impede correction of mitotic errors induced by transient spindle disassembly, leading to severe micronucleation. We define the mechanistic link between regulation of ER membrane biogenesis and mitotic fidelity by demonstrating that a CTDNEP1-lipin 1-mTOR regulatory network limits ER lipid synthesis to prevent chromosome missegregation. Together, this work shows that ER membranes reorganize in mitosis to enable chromosome movements necessary for mitotic error correction and reveal dysregulated lipid metabolism as a potential source of aneuploidy in cancer cells.

Reproductive Isolation from Incompatible Hybrid Mitotic Networks (HyMEN)

At the end of mitosis the Mitotic Exit Network (MEN) pathway triggers complex tasks which mainly include the spindle disassembly and the nuclear envelopes assembly. In the course of telophase, which often lasts less than an hour and corresponds to only about 2% of the entire cell cycle’s duration, spatial and temporal cues are integrated to ensure that cytokinesis occurs after the genome has partitioned between mother and daughter cells. From the end of anaphase through telophase, sequential components of a Ras-like GTPase signaling pathway are controlled by a set of different spatial and temporal signals. Successful propagation of these signals through multi-step transduction requires a remarkable sequential coordination. By considering that cells lacking proper MEN function fail to exit from mitosis, I argue that in a hybrid genome impaired coordination between two diverged MENs is prone to result in critical mitotic defects, from late anaphase through telophase. The so-called HyMEN model of hybrid incompatibility depicted here can be regarded as an extension of the Bateson-Dobzhansky-Muller model of speciation, centered on the MEN.

Shugoshin promotes efficient activation of spindle assembly checkpoint and timely spindle disassembly

Shugoshin proteins are evolutionary conserved across eukaryotes with some species-specific cellular functions ensuring the fidelity of chromosome segregation. Shugoshin being present at various subcellular locales, acts as an adaptor to mediate various protein-protein interactions in a spatio-temporal manner. Here, we characterize shugoshin (Sgo1) in the human fungal pathogen, Candida albicans. Interestingly, we discover a novel in vivo localization of Sgo1 along the length of the mitotic spindle. Further, Sgo1 performs a hitherto unknown function of facilitating timely disassembly of spindle in this organism. We observe that Sgo1 retains its centromeric localization and performs its conserved functions that include regulating the centromeric condensin localization, chromosome passenger complex (CPC) maintenance and sister chromatid biorientation. We identify novel roles of Sgo1 as a spindle assembly checkpoint (SAC) component with functions in maintaining the SAC proteins, Mad2 and Bub1, at the kinetochores, in response to faulty kinetochore-microtubule attachments. These findings provide an excellent evidence of the functional rewiring of shugoshin in maintaining genomic stability.

A Noncanonical Hippo Pathway Regulates Spindle Disassembly and Cytokinesis During Meiosis in Saccharomyces cerevisiae

Meiosis in the budding yeast Saccharomyces cerevisiae is used to create haploid yeast spores from a diploid mother cell. During meiosis II, cytokinesis occurs by closure of the prospore membrane, a membrane that initiates at the spindle pole body and grows to surround each of the haploid meiotic products. Timely prospore membrane closure requires SPS1, which encodes an STE20 family GCKIII kinase. To identify genes that may activate SPS1, we utilized a histone phosphorylation defect of sps1 mutants to screen for genes with a similar phenotype and found that cdc15 shared this phenotype. CDC15 encodes a Hippo-like kinase that is part of the mitotic exit network. We find that Sps1 complexes with Cdc15, that Sps1 phosphorylation requires Cdc15, and that CDC15 is also required for timely prospore membrane closure. We also find that SPS1, like CDC15, is required for meiosis II spindle disassembly and sustained anaphase II release of Cdc14 in meiosis. However, the NDR-kinase complex encoded by DBF2/DBF20MOB1 which functions downstream of CDC15 in mitotic cells, does not appear to play a role in spindle disassembly, timely prospore membrane closure, or sustained anaphase II Cdc14 release. Taken together, our results suggest that the mitotic exit network is rewired for exit from meiosis II, such that SPS1 replaces the NDR-kinase complex downstream of CDC15.

A non-canonical Hippo pathway regulates spindle disassembly and cytokinesis during meiosis in Saccharomyces cerevisiae

ABSTRACTMeiosis in the budding yeast Saccharomyces cerevisiae is used to create haploid yeast spores from a diploid mother cell. During meiosis II, cytokinesis occurs by closure of the prospore membrane, a membrane that initiates at the spindle pole body and grows to surround each of the haploid meiotic products. Timely prospore membrane closure requires SPS1, which encodes a STE20-family GCKIII kinase. To identify genes that may activate SPS1, we utilized a histone phosphorylation defect of sps1 mutants to screen for genes with a similar phenotype and found that cdc15 shared this phenotype. CDC15 encodes a Hippo-like kinase that is part of the mitotic exit network. We find that Sps1 complexes with Cdc15, that Sps1 phosphorylation requires Cdc15, and that CDC15 is also required for timely prospore membrane closure. We also find that SPS1, like CDC15, is required for meiosis II spindle disassembly and sustained anaphase II release of Cdc14 in meiosis. However, the NDR-kinase complex encoded by DBF2/DBF20 MOB1 which functions downstream of CDC15 in mitotic cells, does not appear to play a role in spindle disassembly, timely prospore membrane closure, or sustained anaphase II Cdc14 release. Taken together, our results suggest that the mitotic exit network is rewired for exit from meiosis II, such that SPS1 replaces the NDR-kinase complex downstream of CDC15.

Mitotic spindle disassembly in human cells relies on CRIPT-bearing hierarchical redox signals

Swift and complete spindle disassembly is essential for cell survival, yet how it happens is largely unknown. Here we used real-time live-cell microscopy and biochemical assays to show that a cysteine-rich protein CRIPT dictates the spindle disassembly in a redox-dependent manner in human cells. This previously reported cytoplasmic protein was found to have a confined nuclear localization during interphase but was distributed to spindles and underwent redox modifications to form disulfides within CXXC pairs during mitosis. Then, it interacts with and transfers redox response to tubulin subunits to induce microtubule depolymerization. The mutants with any of cysteine substitution completely block the spindle disassembly generating two cell populations with long-lasting metaphase spindles or spindle remnants. The live cell recordings of a disease-relevant mutant (CRIPTC3Y) revealed that microtubule depolymerization at spindle ends during anaphase and the entire spindle dissolution during telophase may share a common CRIPT-bearing redox-controlled mechanism.

A role for liquid-liquid phase separation in ESCRT-mediated nuclear envelope reformation

At mitotic exit, microtubule arrays are dismantled in concert with the reformation of the nuclear envelope. We show how the inner nuclear membrane protein, LEM2, exploits liquid-liquid phase separation to direct microtubule remodeling and nuclear envelope sealing via the Endosomal Sorting Complexes Required for Transport (ESCRT) pathway. LEM2 tethers membrane to chromatin disks through direct binding between its LEM motif and the chromatin-associated barrier-to-autointegration factor (BAF). Concurrently, a low-complexity domain within LEM2 undergoes liquid-liquid phase separation to coat spindle microtubule bundles. Spatially restricted, LEM2’s winged helix (WH) domain activates the ESCRT-II/ESCRT-III hybrid protein, CHMP7. Together LEM2 and CHMP7 copolymerize around microtubule bundles to form a molecular “O-ring” that promotes nuclear compartmentalization and initiates downstream ESCRT factor recruitment. These results demonstrate how multivalent interactions of a transmembrane protein, including those that mediate phase separation, coordinate localized ESCRT polymerization, mitotic spindle disassembly, and membrane fusion. Defects in this pathway compromise spindle disassembly, nuclear integrity, and genome stability.