The active form of the metabolic sensor AMP-activated protein kinase α (AMPKα) directly binds the mitotic apparatus and travels from centrosomes to the spindle midzone during mitosis and cytokinesis

The metabolic rheostat AMP-activated protein kinase (AMPK) is unexpectedly required for proper cell division and faithful chromosomal segregation during mitosis. Although it is conceptually attractive to assume that AMPK-interpreted microenvironmental bioenergetics may strictly engage cell’s energy status, cell grow, and cell division to avoid that energy stresses trigger cell death, the ultimate framework of AMPK activity towards chromosomal and cytoskeletal mitotic regulation is a question that remains unanswered. We herein reveal that the active form of the α-catalytic AMPK subunit (P-AMPKαThr172) -but not its total form (AMPKα)- transiently associates with several mitotic structures including centrosomes, spindle poles, the central spindle midzone and the midbody throughout all of the mitotic stages and cytokinesis in human cancer-derived epithelial cells. At prophase, P-AMPKαThr172 associates with the two asters of microtubules that begin to nucleate from mature centrosomes. The overlapping localization of P-AMPKαThr172 with the mitotic centrosomal Aurora-A kinase is also apparent on the microtubules near the spindle poles in metaphase and in early anaphase. This Aurora A-like centrosomal localization of P-AMPKαThr172 cannot be detected following chromatid separation following anaphase-telophase transition. Rather, toward the end of anaphase and in telophase P-AMPKαThr172 reactivity exhibited a similar but not identical localization to that occupied by the bona fide chromosomal passenger proteins INCENCP and Aurora-B. This localization of P-AMPKαThr172 at the central spindle and midbody persisted during the furrowing process and, at the completion of telophase, a prominent staining of P-AMPKαThr172 as doublet was apparent on either side of the midbody within the intercellular cytokinetic bridge. An identical mitotic geography of P-AMPKαThr172 was observed in cancer cells lacking the AMPK kinase LKB1, in non-cancerous human epithelial cells, and in mouse fibroblasts. The active form of AMPKα bound to the mitotic apparatus may physically tether the bioenergetic state of a cell to the four-dimensional regulation of the chromosomal and cytoskeletal mitotic events, thus suggesting a putative cytokinetic suppressor function. In this newly discovered scenario, we suggest a primordial mitotic role for the α catalytic AMPK subunit in the eukaryotic evolutionary process as it may ensure, at the cell level, an exquisite coordination between sensing of energy resources and the fundamental biological process of genome division.     

Polo-like kinase 1 directs the AMPK-mediated activation of myosin regulatory light chain at the cytokinetic cleavage furrow independently of energy balance

It has been recently proposed that AMP-activated protein kinase (AMPK) might indirectly promote the phosphorylation of MRLC (myosin II regulatory light chain) at Ser19 to regulate the transition from metaphase to anaphase and the completion of cytokinesis. Although these findings provide biochemical support for our earlier observations showing that the active form of the α catalytic AMPK subunit associates dynamically with essential mitotic regulators, several important issues remained unexplored. Does glucose starvation alter the ability of AMPK to bind to the mitotic apparatus and travel from centrosomes to the spindle midzone during mitosis and cytokinesis? Does AMPK activate MRLC exclusively at the cleavage furrow during cytokinesis? What is the mitosis-specific stimulus that activates the mito-cytokinetic AMPK/MRLC axis regardless of energy deprivation? First, we confirm that exogenous glucose deprivation fails to alter the previously described distribution of phospho-AMPKα^Thr172 in all of the mitotic phases and does not disrupt its apparent association with the mitotic spindle and other structures involved in cell division. Second, we establish for the first time that phospho-AMPKα^Thr172 colocalizes exclusively with Ser19-phosphorylated MRLC at the cleavage furrow of dividing cells, a previously unvisualized interaction between phospho-AMPKα^Thr172 and phospho-MRLC^Ser19 that occurs in cleavage furrows, intercellular bridges and the midbody during cell division that appears to occur irrespective of glucose availability. Third, we reveal for the first time that the inhibition of AMPK mitotic activity in response to PLK1 inhibition completely prevents the co-localization of phospho-AMPKα^Thr172 and phospho-MRLC^Ser19 during the final stages of cytokinesis and midbody ring formation. Because PLK1 inhibition efficiently suppresses the AMPK-mediated activation of MRLC at the cytokinetic cleavage furrow, we propose a previously unrecognized role for AMPK in ensuring that cytokinesis occurs at the proper place and time by establishing a molecular dialog between PLK1 and MRLC in an energy-independent manner.     

Nuclear envelope fission is linked to cytokinesis in budding yeast

We have investigated the relationship between nuclear envelope fission and cytokinesis during mitotic cell division in budding yeast. By carrying out time-lapse and optical sectioning video microscopy analysis of cells that express green fluorescent protein (GFP)-tagged nuclear envelope and actomyosin ring components, we found that nuclear division is temporally coupled to cytokinesis. Light and electron microscopy analysis also showed that nuclear envelope fission and the division of the nucleoplasm are severely delayed in cytokinesis mutants, resulting in discoupling between the nuclear division cycle and the budding cycle. These results suggest that homotypic membrane fusion may be activated by components or the mechanical action of cytokinetic structures and presents a mechanism for the equal partitioning of the nucleus and the temporal coordination of this event with chromosome segregation during mitosis.     

Ser2481-autophosphorylated mTOR colocalizes with chromosomal passenger proteins during mammalian cell cytokinesis

Energy- and nutrient-sensing proteins such as AMPK, mTOR and S6K1 are now recognized as novel regulators of mitotic completion in proliferating cells. We investigated the cellular distribution of the Ser2481 autophosphorylation of mTOR, which directly monitors mTORC-specific catalytic activity, during mammalian cell mitosis and cytokinesis. Automated immunofluorescence experiments in human carcinoma cell lines revealed that phospho-mTOR^Ser2481 exhibited profound spatial and temporal dynamics during cell division. Phospho-mTOR^Ser2481 was strikingly enriched in mitotic cells, and in prophase, bright phospho-mTOR^Ser2481 staining could be clearly observed among condensed chromosomes. Phospho-mTOR^Ser2481 then redistributes from diffuse cytosolic staining that partially colocalizes with the mitotic spindle during the early phases of mitosis to the furrow at the onset of cytokinesis. Like the bona fide chromosomal passenger proteins (CPPs) INCENP and Aurora B, phospho-mTOR^Ser2481 displayed noteworthy accumulation in the central spindle midzone and the midbody regions, which persisted during the furrowing process. Accordingly, double-staining experiments confirmed that phospho-mTOR^Ser2481 largely colocalized with CCPs in the midbodies. The CPP-like mitotic localization of phospho-mTOR^Ser2481 was fully prevented by the microtubule-depolymerizing drug nocodazole; mitotic traveling of phospho-mTOR^Ser2481 to the midbody during telophase and cytokinesis, where it appears to be integrated into the CPP-driven cytokinetic machinery, may therefore require dynamic microtubules. Although the Ser2448-phosphorylated form of mTOR was also found at high levels during M-phase in human cancer cells, we failed to observe a significant association of phospho-mTOR^Ser2448 with CCP-positive mitotic and cytokinetic structures. Our findings add phospho-mTOR^Ser2481 to the growing list of phospho-active forms of proteins belonging to the AMPK/mTOR/S6K1 signaling axis that reside at the mitotic and cytokinetic apparatus. Future studies should elucidate how the specific ability of phospho-mTOR^Ser2481 to spatially and temporally couple to the cleavage furrow and midbody region as a CPP-like protein can signal to or from adjacent signaling complexes and/or with the basic machinery of cell abscission.     

Ser2481-autophosphorylated mTOR colocalizes with chromosomal passenger proteins during mammalian cell cytokinesis

Energy- and nutrient-sensing proteins such as AMPK, mTOR and S6K1 are now recognized as novel regulators of mitotic completion in proliferating cells. We investigated the cellular distribution of the Ser2481 autophosphorylation of mTOR, which directly monitors mTORC-specific catalytic activity, during mammalian cell mitosis and cytokinesis. Automated immunofluorescence experiments in human carcinoma cell lines revealed that phospho-mTOR^Ser2481 exhibited profound spatial and temporal dynamics during cell division. Phospho-mTOR^Ser2481 was strikingly enriched in mitotic cells, and in prophase, bright phospho-mTOR^Ser2481 staining could be clearly observed among condensed chromosomes. Phospho-mTOR^Ser2481 then redistributes from diffuse cytosolic staining that partially colocalizes with the mitotic spindle during the early phases of mitosis to the furrow at the onset of cytokinesis. Like the bona fide chromosomal passenger proteins (CPPs) INCENP and Aurora B, phospho-mTOR^Ser2481 displayed noteworthy accumulation in the central spindle midzone and the midbody regions, which persisted during the furrowing process. Accordingly, double-staining experiments confirmed that phospho-mTOR^Ser2481 largely colocalized with CCPs in the midbodies. The CPP-like mitotic localization of phospho-mTOR^Ser2481 was fully prevented by the microtubule-depolymerizing drug nocodazole; mitotic traveling of phospho-mTOR^Ser2481 to the midbody during telophase and cytokinesis, where it appears to be integrated into the CPP-driven cytokinetic machinery, may therefore require dynamic microtubules. Although the Ser2448-phosphorylated form of mTOR was also found at high levels during M-phase in human cancer cells, we failed to observe a significant association of phospho-mTOR^Ser2448 with CCP-positive mitotic and cytokinetic structures. Our findings add phospho-mTOR^Ser2481 to the growing list of phospho-active forms of proteins belonging to the AMPK/mTOR/S6K1 signaling axis that reside at the mitotic and cytokinetic apparatus. Future studies should elucidate how the specific ability of phospho-mTOR^Ser2481 to spatially and temporally couple to the cleavage furrow and midbody region as a CPP-like protein can signal to or from adjacent signaling complexes and/or with the basic machinery of cell abscission.     

A cancer-associated mutation inactivates a region of the high-mobility group protein HMG20b essential for cytokinesis

Defects in the completion of cell division by cytokinesis have long been proposed to foster carcinogenesis by engendering chromosome instability, but few tumor suppressor mechanisms controlling this process have so far been identified. Here, we identify a carboxyl (C)-terminal region of the high-mobility group protein HMG20b that is essential for cytokinesis, and report that it is inactivated by a cancer-associated mutation. We find that a C-terminal region of HMG20b spanning residues 173–317 is necessary and sufficient not only for its localization to cytokinetic structures, but also for its interaction with the tumor suppressor BRCA2, implicated in the abscission step of cytokinesis. Indeed, expression of this C-terminal HMG20b region suffices to restore cytokinesis in HMG20b-depleted cells. The non-conservative substitution of HMG20b residue Ala247 with Pro, reported in human lung cancer, disrupts these activities of HMG20b, impairing cytokinesis in a trans-dominant manner. Our findings provide fresh insight into the mechanism by which the HMG20b-BRCA2 complex controls mitotic cell division, and implicate heterozygous HMG20b mutations affecting cytokinesis regulation in the genesis of human cancers.     

CCAR2 controls mitotic progression through spatiotemporal regulation of Aurora B

CCAR2 (cell cycle and apoptosis regulator 2) is a multifaceted protein involved in cell survival and death following cytotoxic stress. However, little is known about the physiological functions of CCAR2 in regulating cell proliferation in the absence of external stimuli. The present study shows that CCAR2-deficient cells possess multilobulated nuclei, suggesting a defect in cell division. In particular, the duration of mitotic phase was perturbed. This disturbance of mitotic progression resulted from premature loss of cohesion with the centromere, and inactivation of the spindle assembly checkpoint during prometaphase and metaphase. It resulted in the formation of lagging chromosomes during anaphase, leading ultimately to the activation of the abscission checkpoint to halt cytokinesis. The CCAR2-dependent mitotic progression was related to spatiotemporal regulation of active Aurora B. In conclusion, the results suggest that CCAR2 governs mitotic events, including proper chromosome segregation and cytokinetic division, to maintain chromosomal stability.Subject terms: Mitosis, Checkpoints     

Midbody localization of vinexin recruits rhotekin to facilitate cytokinetic abscission

Vinexin is a SH3 domain-containing adaptor protein that has diverse roles in cell adhesion, signal transduction, gene regulation and stress granule assembly. In this study, we found that vinexin localizes at the midbody during cell division and facilitates cytokinesis. Knockdown of vinexin in HeLa cells delayed the mitotic cell cycle progression and increased the time of cell abscission and the failure to resolve the cytoplasmic bridge. Midbody-localized vinexin is essential for recruiting rhotekin to this structure for cytokinesis because overexpression of a vinexin mutant without a rhotekin-binding motif or knockdown of rhotekin also impaired cytokinetic abscission and increased the number of cells arrested at the midbody stage. Aberrant expression of vinexin and rhotekin in various cancers has been implicated to promote metastasis because of their functions in cell adhesion and signaling. Our findings reveal a novel role of vinexin and rhotekin in cytokinetic abscission and provide another perspective of how both molecules may affect oncogenic transformation via this fundamental cell cycle process.     

Subunit composition of AMPK trimers present in the cytokinetic apparatus: Implications for drug target identification

AMP-activated protein kinase has been shown to be a key regulator of energy homeostasis; it has also been identified as a tumor suppressor and is required for correct cell division and chromosome segregation during mitosis. The enzyme is a heterotrimer, with each subunit having more than one isoform, each encoded by a separate gene (two α, two β and three γ isoforms). In human endothelial cells, the activated kinase subunit of AMPK in the cytokinetic apparatus is α2, the minority α subunit, which co-localizes with β2 and γ2. This is the first demonstration of a trimeric complex of AMPK containing the γ2 regulatory subunit becoming selectively activated and being linked to mitotic processes. We also show that α1 and γ1, the predominant AMPK subunits, are almost exclusively localized in the cytoskeleton, while α2 and γ2 are present in all subcellular fractions, including the nuclei. These data suggest that pharmacological interventions targeted to specific AMPK subunit isoforms have the potential to modify selective functions of AMPK.     

Concentration of caveolin-1 in the cleavage furrow as revealed by time-lapse analysis

Caveolin-1 is a major component of caveolae. Recent studies have suggested a possible role of caveolin-1 in cell transformation and normal cell proliferation. To observe the behavior of caveolin-1 in living mitotic cells, we prepared cDNA constructs encoding the chimeric protein of alpha- or beta-caveolin-1 and green fluorescent protein (GFP) and transfected culture cells with them. Correct targeting of the chimera to the caveolae was confirmed by colocalization with the caveolar inositol 1,4,5-trisphosphate receptor-like protein. By time-lapse observation of mitotic MDCKII cells, the GFP-caveolin-1 chimeras were seen throughout the plasma membrane before cell division, but became markedly concentrated at the cleavage furrow during cytokinesis. Accumulation around the spindle poles was also observed at late telophase. The result showed that caveolin-1 undergoes a drastic distributional change during cell division and suggested that the protein may be involved in the cytokinetic process.     

A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis

Coordination of mitotic exit with timely initiation of cytokinesis is critical to ensure completion of mitotic events before cell division. The Saccharomyces cerevisiae polo kinase Cdc5 functions in a pathway leading to the degradation of mitotic cyclin Clb2, thereby permitting mitotic exit. Here we provide evidence that Cdc5 also plays a role in regulating cytokinesis and that an intact polo-box, a conserved motif in the noncatalytic COOH-terminal domain of Cdc5, is required for this event. Depletion of Cdc5 function leads to an arrest in cytokinesis. Overexpression of the COOH-terminal domain of Cdc5 (cdc5DeltaN), but not the corresponding polo-box mutant, resulted in connected cells. These cells shared cytoplasms with incomplete septa, and possessed aberrant septin ring structures. Provision of additional copies of endogenous CDC5 remedied this phenotype, suggesting a dominant-negative inhibition of cytokinesis. The polo-box-dependent interactions between Cdc5 and septins (Cdc11 and Cdc12) and genetic interactions between the dominant-negative cdc5DeltaN and Cyk2/Hof1 or Myo1 suggest that direct interactions between cdc5DeltaN and septins resulted in inhibition of Cyk2/Hof1- and Myo1-mediated cytokinetic pathways. Thus, we propose that Cdc5 may coordinate mitotic exit with cytokinesis by participating in both anaphase promoting complex activation and a polo-box-dependent cytokinetic pathway.     

Cdk1 phosphorylation of fission yeast paxillin inhibits its cytokinetic ring localization

Divisions of the genetic material and cytoplasm are coordinated spatially and temporally to ensure genome integrity. This coordination is mediated in part by the major cell cycle regulator cyclin-dependent kinase (Cdk1). Cdk1 activity peaks during mitosis, but during mitotic exit/cytokinesis Cdk1 activity is reduced, and phosphorylation of its substrates is reversed by various phosphatases including Cdc14, PP1, PP2A, and PP2B. Cdk1 is known to phosphorylate several components of the actin- and myosin-based cytokinetic ring (CR) that mediates division of yeast and animal cells. Here we show that Cdk1 also phosphorylates the Schizosaccharomyces pombe CR component paxillin Pxl1. We determined that both the Cdc14 phosphatase Clp1 and the PP1 phosphatase Dis2 contribute to Pxl1 dephosphorylation at mitotic exit, but PP2B/calcineurin does not. Preventing Pxl1 phosphorylation by Cdk1 results in increased Pxl1 levels, precocious Pxl1 recruitment to the division site, and increased duration of CR constriction. In vitro Cdk1-mediated phosphorylation of Pxl1 inhibits its interaction with the F-BAR domain of the cytokinetic scaffold Cdc15, thereby disrupting a major mechanism of Pxl1 recruitment. Thus, Pxl1 is a novel substrate through which S. pombe Cdk1 and opposing phosphatases coordinate mitosis and cytokinesis.     

Cell division inCarteria crucifera (chlorophyta): The role of the endomembrane system and phycoplast

Summary Phycoplast-mediated cytokinesis in the primitive green algal flagellate,Carteria crucifera, has been examined by electron microscopy. The key developmental foci during cell division are mobile centriole-MTOCs which control mitotic spindle formation, the establishment of the plane of cytokinesis, the initiation of the cytokinetic furrow, the formation of the phycoplast and the formation of morphogenetic microtubular arrays. The cytokinetic cleavage mechanism entails an ingressive furrowing closely associated with a prolific network of internuclear endoplasmic reticulum. Dictyosome activity is limited to the cleavage initiation zone and is responsible for the production of wall precursor-containing vesicles. Dictyosome materials do not contribute directly to the growing furrow edge. Potassium antimonate staining patterns reveal the cytokinetic ER as a storage/control site for calcium during cytokinesis. Discussion of possible models concerning this cytokinetic mechanism is presented.     

Mechanisms controlling division-plane positioning

A critical and irreversible step in the cell division cycle is cytokinesis which physically separates the two daughter cells. This event is consequently subject to tight spatial and temporal regulation. This review focuses on the spatial regulatory mechanisms controlling the position of the division plane. Studies performed in prokaryotic and eukaryotic systems have revealed that various signal-emitting spatial cues – mitotic spindle, nucleus, nucleoid or cell tips – can favour or inhibit the assembly of the cytokinetic apparatus in their vicinity. Most often, several mechanisms operate in parallel to integrate spatial information and promote faithful genome segregation as well as proper cytoplasmic division. We primarily describe the spatial regulatory mechanisms operating in the fission yeast model system, where a detailed molecular understanding of cytokinesis has been achieved. In this system, spatial regulations target a major factor controlling the position of the division plane, the anillin-like protein Mid1. These mechanisms are then compared to spatial regulatory mechanisms prevailing in animal cells and rod-shaped bacteria.     

Systematic epistatic mapping of cellular processes

Genetic screens have identified many novel components of various biological processes, such as components required for cell cycle and cell division. While forward genetic screens typically generate unstructured ‘hit’ lists, genetic interaction mapping approaches can identify functional relations in a systematic fashion. Here, we discuss a recent study by our group demonstrating a two-step approach to first screen for regulators of the mitotic cell cycle, and subsequently guide hypothesis generation by using genetic interaction analysis. The screen used a high-content microscopy assay and automated image analysis to capture defects during mitotic progression and cytokinesis. Genetic interaction networks derived from process-specific features generate a snapshot of functional gene relations in those processes, which follow a temporal order during the cell cycle. This complements a recently published approach, which inferred directional genetic interactions reconstructing hierarchical relationships between genes across different phases during mitotic progression. In conclusion, this strategy leverages unbiased, genome-wide, yet highly sensitive and process-focused functional screening in cells.     

Variable pathways for developmental changes of mitosis and cytokinesis in Physarum polycephalum

The development of a uninucleate ameba into a multinucleate, syncytial plasmodium in myxomycetes involves a change from the open, astral mitosis of the ameba to the intranuclear, anastral mitosis of the plasmodium, and the omission of cytokinesis from the cell cycle. We describe immunofluorescence microscopic studies of the amebal-plasmodial transition (APT) in Physarum polycephalum. We demonstrate that the reorganization of mitotic spindles commences in uninucleate cells after commitment to plasmodium formation, is completed by the binucleate stage, and occurs via different routes in individual developing cells. Most uninucleate developing cells formed mitotic spindles characteristic either of amebae or of plasmodia. However, chimeric mitotic figures exhibiting features of both amebal and plasmodial mitoses, and a novel star microtubular array were also observed. The loss of the ameba-specific alpha 3-tubulin and the accumulation of the plasmodium-specific beta 2-tubulin isotypes during development were not sufficient to explain the changes in the organization of mitotic spindles. The majority of uninucleate developing cells undergoing astral mitoses (amebal and chimeric) exhibited cytokinetic furrows, whereas cells with the anastral plasmodial mitosis exhibited no furrows. Thus, the transition from astral to anastral mitosis during the APT could be sufficient for the omission of cytokinesis from the cell cycle. However, astral mitosis may not ensure cytokinesis: some cells undergoing amebal or chimeric mitosis contained unilateral cytokinetic furrows or no furrow at all. These cells would, most probably, fail to divide. We suggest that a uninucleate committed cell undergoing amebal or chimeric mitosis can either divide or else form a binucleate cell. In contrast, a uninucleate cell with a mitotic spindle of the plasmodial type gives rise only to a binucleate cells. Further, the decision to enter mitosis after commitment to the APT is independent of the developmental changes in the organization of the mitotic spindle and cytokinesis.     

The novel function of JADE1S in cytokinesis of epithelial cells

JADE1 belongs to a small family of PHD zinc finger proteins that interacts with histone acetyl transferase (HAT) HBO1 and is associated with chromatin. We recently reported JADE1 chromatin shuttling and phosphorylation during G2/M to G1 transition, which was sensitive to Aurora A inhibition. In the current study we examined mechanisms of the cell cycle regulation by the small isoform of JADE1 protein, JADE1S, and report data showing that JADE1S has a novel function in the regulation of cytokinesis. Using FACS assays, we show that, JADE1S depletion facilitated rates of G1-cells accumulation in synchronously dividing HeLa cell cultures. Depletion of JADE1S protein in asynchronously dividing cells decreased the proportion of cytokinetic cells, and increased the proportion of multi-nuclear cells, indicative of premature and failed cytokinesis. In contrast, moderate overexpression of JADE1S increased the number of cytokinetic cells in time- and dose- dependent manner, indicating cytokinetic delay. Pharmacological inhibition of Aurora B kinase resulted in the release of JADE1S-mediated cytokinetic delay and allowed progression of abscission in cells over-expressing JADE1S. Finally, we show that JADE1S protein localized to centrosomes in interphase and mitotic cells, while during cytokinesis JADE1S localized to the midbody. Neither JADE1L nor partner of JADE1, HAT HBO1 was localized to the centrosomes or midbodies. Our study identifies the novel role for JADE1S in regulation of cytokinesis and suggests function in Aurora B kinase-mediated cytokinesis checkpoint.     

The ciliopathy protein CCDC66 controls mitotic progression and cytokinesis by promoting microtubule nucleation and organization

Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and nondividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the centrosomes, the bipolar spindle, the spindle midzone, the central spindle, and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and orientation, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the centrosomes and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provide new insight into nonciliary defects that underlie ciliopathies.

The ciliopathy-linked protein CCDC66 is only known for its ciliary functions. This study reveals that CCDC66 also has extensive non-ciliary functions, localizing to the spindle poles, spindle midzone, central spindle and midbody throughout cell division, where it regulates mitosis and cytokinesis by promoting microtubule nucleation and organization.     

Unpicking neurodegeneration in a dish with human pluripotent stem cells

Eukaryotic cell division is an orderly and timely process involving the error-free segregation of chromosomes and cytoplasmic components to give rise to two separate daughter cells. Defects in genome maintenance mechanisms such as cell cycle checkpoints and DNA repair can impact the segregation of the genome during mitosis leading to multiple chromosomal imbalances. In mammals, the DNA damage checkpoint effector Checkpoint Kinase 1 (Chk1) is essential for responses to DNA replication errors, external DNA damage, and chromatin breaks. We reported recently that Chk1 also was essential for chromosome segregation and completion of cytokinesis to prevent genomic instability. Our studies demonstrated that Chk1 deficiency in mitotic cells causes chromosome mis-alignment, lagging chromosomes, chromosome mis- segregation, cytokinetic regression, and binucleation. In addition, abrogation of Chk1 resulted in aberrant localization of mitotic Aurora B kinase at the metaphase plate, anaphase spindle midzone, and cytokinetic midbody as studied both in various cell lines and in a mouse model. Therefore, inappropriate regulation of Chk1 levels during cell cycle progression will result in failed cell division and enhanced genomic instability.