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Time-restricted feeding (TRF) has recently gained interest as a potential anti-ageing treatment for organisms from Drosophila to humans1–5. TRF restricts food intake to specific hours of the day. Because TRF controls the timing of feeding, rather than nutrient or caloric content, TRF has been hypothesized to depend on circadian-regulated functions; the underlying molecular mechanisms of its effects remain unclear. Here, to exploit the genetic tools and well-characterized ageing markers of Drosophila, we developed an intermittent TRF (iTRF) dietary regimen that robustly extended fly lifespan and delayed the onset of ageing markers in the muscles and gut. We found that iTRF enhanced circadian-regulated transcription and that iTRF-mediated lifespan extension required both circadian regulation and autophagy, a conserved longevity pathway. Night-specific induction of autophagy was both necessary and sufficient to extend lifespan on an ad libitum diet and also prevented further iTRF-mediated lifespan extension. By contrast, day-specific induction of autophagy did not extend lifespan. Thus, these results identify circadian-regulated autophagy as a critical contributor to iTRF-mediated health benefits in Drosophila. Because both circadian regulation and autophagy are highly conserved processes in human ageing, this work highlights the possibility that behavioural or pharmaceutical interventions that stimulate circadian-regulated autophagy might provide people with similar health benefits, such as delayed ageing and lifespan extension. Circadian-regulated autophagy contributes to the health benefits of intermittent time-restricted feeding in Drosophila.

Because old age is associated with defects in circadian rhythm, loss of circadian regulation is thought to be pathogenic and contribute to mortality. We show instead that loss of specific circadian clock components Period (Per) and Timeless (Tim) in male Drosophila significantly extends lifespan. This lifespan extension is not mediated by canonical diet-restriction longevity pathways but is due to altered cellular respiration via increased mitochondrial uncoupling. Lifespan extension of per mutants depends on mitochondrial uncoupling in the intestine. Moreover, upregulated uncoupling protein UCP4C in intestinal stem cells and enteroblasts is sufficient to extend lifespan and preserve proliferative homeostasis in the gut with age. Consistent with inducing a metabolic state that prevents overproliferation, mitochondrial uncoupling drugs also extend lifespan and inhibit intestinal stem cell overproliferation due to aging or even tumorigenesis. These results demonstrate that circadian-regulated intestinal mitochondrial uncoupling controls longevity in Drosophila and suggest a new potential anti-aging therapeutic target., Disruption of different components of molecular circadian clocks has varying effects on health and lifespan of model organisms. Here the authors show that loss of period extends life in drosophila melanogaster.

Contractile ring constriction during cytokinesis is thought to compact central spindle microtubules to form the midbody, an antiparallel microtubule bundle at the intercellular bridge. In Caenorhabditis elegans, central spindle microtubule assembly requires targeting of the CLASP family protein CLS-2 to the kinetochores in metaphase and spindle midzone in anaphase. CLS-2 targeting is mediated by the CENP-F–like HCP-1/2, but their roles in cytokinesis and midbody assembly are not known. We found that although HCP-1 and HCP-2 mostly function cooperatively, HCP-1 plays a more primary role in promoting CLS-2–dependent central spindle microtubule assembly. HCP-1/2 codisrupted embryos did not form central spindles but completed cytokinesis and formed functional midbodies capable of supporting abscission. These central spindle–independent midbodies appeared to form via contractile ring constriction–driven bundling of astral microtubules at the furrow tip. This work suggests that, in the absence of a central spindle, astral microtubules can support midbody assembly and that midbody assembly is more predictive of successful cytokinesis than central spindle assembly.

SUMMARY The urothelium is an epithelia barrier lined by a luminal layer of binucleated, octoploid, superficial cells. Superficial cells are critical for production and transport of uroplakins, a family of proteins that assemble into a waterproof crystalline plaque that helps protect against infection and toxic substances. Adult urothelium is nearly quiescent, but rapidly regenerates in response to injury. Yet the mechanism by which binucleated, polyploid, superficial cells are produced remains unclear. Here, we show that superficial cells are likely to be derived from a population of binucleated intermediate cells, which are produced from mononucleated intermediate cells via incomplete cytokinesis. We show that binucleated intermediate and superficial cells increase DNA content via endoreplication, passing through S phase without entering mitosis. The urothelium can be permanently damaged by repetitive or chronic injury or disease. Identification of the mechanism by which superficial cells are produced may be important for developing strategies for urothelial repair., Graphical Abstract, In Brief Binucleated superficial cells are critical for urothelial barrier function. Wang et al. show that they derive from binucleated intermediate cells that form via incomplete cytokinesis. Both superficial and intermediate cells increase ploidy via endoreplication, a feature likely to be important for repair and response to environmental changes.

The roles of the Rho-family GAP CYK-4 and small GTPase Rac during cytokinesis are examined in Caenorhabditis elegans embryos. CYK-4 opposes Rac (and potentially Cdc42) activity during cytokinesis. There is no evidence that CYK-4 is upstream of Rho activity or that Rac disruption is a general suppressor of cytokinesis failure., Cytokinesis is driven by constriction of an actomyosin contractile ring that is controlled by Rho-family small GTPases. Rho, activated by the guanine-nucleotide exchange factor ECT-2, is upstream of both myosin-II activation and diaphanous formin-mediated filamentous actin (f-actin) assembly, which drive ring constriction. The role for Rac and its regulators is more controversial, but, based on the finding that Rac inactivation can rescue cytokinesis failure when the GTPase-activating protein (GAP) CYK-4 is disrupted, Rac activity was proposed to be inhibitory to contractile ring constriction and thus specifically inactivated by CYK-4 at the division plane. An alternative model proposes that Rac inactivation generally rescues cytokinesis failure by reducing cortical tension, thus making it easier for the cell to divide when ring constriction is compromised. In this alternative model, CYK-4 was instead proposed to activate Rho by binding ECT-2. Using a combination of time-lapse in vivo single-cell analysis and Caenorhabditis elegans genetics, our evidence does not support this alternative model. First, we found that Rac disruption does not generally rescue cytokinesis failure: inhibition of Rac specifically rescues cytokinesis failure due to disruption of CYK-4 or ECT-2 but does not rescue cytokinesis failure due to disruption of two other contractile ring components, the Rho effectors diaphanous formin and myosin-II. Second, if CYK-4 regulates cytokinesis through Rho rather than Rac, then CYK-4 inhibition should decrease levels of downstream targets of Rho. Inconsistent with this, we found no change in the levels of f-actin or myosin-II at the division plane when CYK-4 GAP activity was reduced, suggesting that CYK-4 is not upstream of ECT-2/Rho activation. Instead, we found that the rescue of cytokinesis in CYK-4 mutants by Rac inactivation was Cdc42 dependent. Together our data suggest that CYK-4 GAP activity opposes Rac (and perhaps Cdc42) during cytokinesis.

FLIRT (Fast Local InfraRed Thermogenetics) is a microscopy-based technology to locally and reversibly manipulate protein function during cellular behaviors while simultaneously monitoring the effects in vivo. FLIRT locally inactivates fast-acting temperature sensitive (ts) mutant proteins, using non-ts mutants as controls. We demonstrate that FLIRT can control ts proteins required for cell division, Delta-Notch cell fate signaling, and germline structure in C. elegans with cell-specific and even subcellular precision.

International audience; Accurate chromosome segregation relies on bioriented amphitelic attachments of chromosomes to microtubules of the mitotic spindle, in which sister chromatids are connected to opposite spindle poles. BUB-1 is a protein of the Spindle Assembly Checkpoint (SAC) that coordinates chromosome attachment with anaphase onset. BUB-1 is also required for accurate sister chromatid segregation independently of its SAC function, but the underlying mechanism remains unclear. Here we show that, in Caenorhabditis elegans embryos, BUB-1 accelerates the establishment of non-merotelic end-on kinetochore-microtubule attachments by recruiting the RZZ complex and its downstream partner dynein-dynactin at the kinetochore. In parallel, BUB-1 limits attachment maturation by the SKA complex. This activity opposes kinetochore-microtubule attachment stabilisation promoted by CLS-2 CLASP-dependent kinetochore-microtubule assembly. BUB-1 is therefore a SAC component that coordinates the function of multiple downstream kinetochore-associated proteins to ensure accurate chromosome segregation.

Cytokinesis, the physical division of one cell into two, is powered by constriction of an actomyosin contractile ring. It has long been assumed that all animal cells divide by a similar molecular mechanism, but growing evidence suggests that cytokinetic regulation in individual cell types has more variation than previously realized. In the four-cell Caenorhabditis elegans embryo, each blastomere has a distinct cell fate, specified by conserved pathways. Using fast-acting temperature-sensitive mutants and acute drug treatment, we identified cell-type-specific variation in the cytokinetic requirement for a robust forminCYK-1-dependent filamentous-actin (F-actin) cytoskeleton. In one cell (P2), this cytokinetic variation is cell-intrinsically regulated, whereas in another cell (EMS) this variation is cell-extrinsically regulated, dependent on both SrcSRC-1 signaling and direct contact with its neighbor cell, P2. Thus, both cell-intrinsic and -extrinsic mechanisms control cytokinetic variation in individual cell types and can protect against division failure when the contractile ring is weakened., eLife digest The successful division of one cell into two is essential for all organisms to live, grow and reproduce. For an animal cell, the nucleus – the compartment containing the genetic material – must divide before the surrounding material. The rest of the cell, called the cytoplasm, physically separates later in a process known as cytokinesis. Cytokinesis in animal cells is driven by the formation of a ring in the middle of the dividing cell. The ring is composed of myosin motor proteins and filaments made of a protein called actin. The movements of the motor proteins along the filaments cause the ring to contract and tighten. This pulls the cell membrane inward and physically pinches the cell into two. For a long time, the mechanism of cytokinesis was assumed to be same across different types of animal cell, but later evidence suggested otherwise. For example, in liver, heat and bone cells, cytokinesis naturally fails during development to create cells with two or more nuclei. If a similar ‘failure’ happened in other cell types, it could lead to diseases such as cancers or blood disorders. This raised the question: what are the molecular mechanisms that allow cytokinesis to happen differently in different cell types? Davies et al. investigated this question using embryos of the worm Caenorhabditis elegans at a stage in their development when they consist of just four cells. The proteins forming the contractile ring in this worm are the same as those in humans. However, in the worm, the contractile ring can easily be damaged using chemical inhibitors or by mutating the genes that encode its proteins. Davies et al. show that when the contractile ring was damaged, two of the four cells in the worm embryo still divided successfully. This result indicates the existence of new mechanisms to divide the cytoplasm that allow division even with a weak contractile ring. In a further experiment, the embryos were dissected to isolate each of the four cells. Davies et al. saw that one of the two dividing cells could still divide on its own, while the other cell could not. This shows that this new method of cytokinesis is regulated both by factors inherent to the dividing cell and by external signals from other cells. Moreover, one of these extrinsic signals was found to be a signaling protein that had previously been implicated in human cancers. Future work will determine if these variations in cytokinesis between the different cell types found in the worm apply to humans too; and, more importantly from a therapeutic standpoint, if these new mechanisms exist in human cancers.

International audience; Successive cell divisions during embryonic cleavage create increasingly smaller cells, so intracellular structures must adapt accordingly. Mitotic spindle size correlates with cell size, but the mechanisms for this scaling remain unclear. Using live cell imaging, we analyzed spindle scaling during embryo cleavage in the nematode Caenorhabditis elegans and sea urchin Paracentrotus lividus. We reveal a common scaling mechanism, where the growth rate of spindle microtubules scales with cell volume, which explains spindle shortening. Spindle assembly timing is, however, constant throughout successive divisions. Analyses in silico suggest that controlling the microtubule growth rate is sufficient to scale spindle length and maintain a constant assembly timing. We tested our in silico predictions to demonstrate that modulating cell volume or microtubule growth rate in vivo induces a proportional spindle size change. Our results suggest that scalability of the microtubule growth rate when cell size varies adapts spindle length to cell volume.

Although sleep appears to be broadly conserved in animals, the physiological functions of sleep remain unclear. In this study, we sought to identify a physiological defect common to a diverse group of short-sleeping Drosophila mutants, which might provide insight into the function and regulation of sleep. We found that these short-sleeping mutants share a common phenotype of sensitivity to acute oxidative stress, exhibiting shorter survival times than controls. We further showed that increasing sleep in wild-type flies using genetic or pharmacological approaches increases survival after oxidative challenge. Moreover, reducing oxidative stress in the neurons of wild-type flies by overexpression of antioxidant genes reduces the amount of sleep. Together, these results support the hypothesis that a key function of sleep is to defend against oxidative stress and also point to a reciprocal role for reactive oxygen species (ROS) in neurons in the regulation of sleep., Author summary Most animals sleep; humans sleep nearly a third of their lives. Yet the fundamental functions of sleep remain unknown. Here, we used short-sleeping Drosophila mutants to uncover a role for sleep in resistance to oxidative stress. Oxidative stress is an imbalance of reactive oxygen species and antioxidant responses. Although these short-sleeping mutants have defects in diverse pathways, they all exhibit sensitivity to oxidative stress. Moreover, increasing sleep in wild-type flies increased resistance to oxidative stress. This suggests that one function of sleep is to defend against oxidative stress. Finally, reducing oxidative stress in neurons of wild-type flies reduces their sleep, suggesting that oxidative stress also regulates sleep. Taken together, our results support an intriguing hypothesis for a bidirectional relationship between sleep and oxidative stress: oxidative stress triggers sleep, which then acts as an antioxidant for both the body and the brain. These results have implications for human patients suffering from chronic sleep restriction and diseases associated with oxidative stress.

Fast-acting temperature-sensitive (ts) mutations are powerful conditional tools for studying transient cellular processes such as cytokinesis. Fast-acting ts cytokinesis-defective mutants are functional at the permissive temperature; yet show a fully penetrant loss-of-function cytokinesis failure phenotype when upshifted to the restrictive temperature. Fast-acting ts mutations thus allow functional tunability and rapid and reversible protein inactivation by simply shifting the temperature at precise times throughout cell division. In this chapter, we describe several techniques and discuss various approaches for harnessing the power of fast-acting ts mutants to study cytokinesis in Caenorhabditis elegans using both simple passive heat transfer and more advanced fluidic-based thermal control systems. We also provide detailed protocols for standard dissection, mounting, and imaging of early worm embryos.

Recent evidence suggests that Fragile X syndrome and other types of autism are associated with immune system defects. Here, O’Connor et al. find that Drosophila Fmr1 mutants, a model for Fragile X syndrome, exhibit defects in phagocytosis by innate immune cells in both the body and the brain., Fragile X syndrome, the most common known monogenic cause of autism, results from the loss of FMR1, a conserved, ubiquitously expressed RNA-binding protein. Recent evidence suggests that Fragile X syndrome and other types of autism are associated with immune system defects. We found that Drosophila melanogaster Fmr1 mutants exhibit increased sensitivity to bacterial infection and decreased phagocytosis of bacteria by systemic immune cells. Using tissue-specific RNAi-mediated knockdown, we showed that Fmr1 plays a cell-autonomous role in the phagocytosis of bacteria. Fmr1 mutants also exhibit delays in two processes that require phagocytosis by glial cells, the immune cells in the brain: neuronal clearance after injury in adults and the development of the mushroom body, a brain structure required for learning and memory. Delayed neuronal clearance is associated with reduced recruitment of activated glia to the site of injury. These results suggest a previously unrecognized role for Fmr1 in regulating the activation of phagocytic immune cells both in the body and the brain.

Cytokinesis is the physical division of one cell into two daughter cells that occurs at the end of the cell cycle.1 Cytokinesis is thought to be fundamentally similar in all metazoan divisions-that...

In asymmetrically dividing C. elegans embryos, the core cortical PAR proteins are required to retain septin and anillin at the anterior cortex away from the contractile ring and to promote normal F-actin levels at the contractile ring and successful cytokinesis., Cytokinesis, the physical division of one cell into two, is thought to be fundamentally similar in most animal cell divisions and driven by the constriction of a contractile ring positioned and controlled solely by the mitotic spindle. During asymmetric cell divisions, the core polarity machinery (partitioning defective [PAR] proteins) controls the unequal inheritance of key cell fate determinants. Here, we show that in asymmetrically dividing Caenorhabditis elegans embryos, the cortical PAR proteins (including the small guanosine triphosphatase CDC-42) have an active role in regulating recruitment of a critical component of the contractile ring, filamentous actin (F-actin). We found that the cortical PAR proteins are required for the retention of anillin and septin in the anterior pole, which are cytokinesis proteins that our genetic data suggest act as inhibitors of F-actin at the contractile ring. Collectively, our results suggest that the cortical PAR proteins coordinate the establishment of cell polarity with the physical process of cytokinesis during asymmetric cell division to ensure the fidelity of daughter cell formation.

Dietary restriction (DR), or decreased consumption of nutrients such as protein, extends lifespan in multiple model organisms, including Drosophila (Fontana and Partridge, 2015). In a recent article in Cell Metabolism, Katewa et al. (2016) presented evidence indicating that arrhythmic Drosophila females lacking the circadian regulators period (per) or timeless (tim) exhibit truncated lifespan extension in response to DR (Katewa et al., 2016). In Drosophila, Per and Tim proteins are critical regulators of circadian rhythm, or daily oscillations in activity and physiological function.

Mitotic spindle positioning in the Caenorhabditis elegans zygote involves microtubule-dependent pulling forces applied to centrosomes. In this study, we investigate the role of actomyosin in centration, the movement of the nucleus–centrosome complex (NCC) to the cell center. We find that the rate of wild-type centration depends equally on the nonmuscle myosin II NMY-2 and the Gα proteins GOA-1/GPA-16. In centration- defective let-99(−) mutant zygotes, GOA-1/GPA-16 and NMY-2 act abnormally to oppose centration. This suggests that LET-99 determines the direction of a force on the NCC that is promoted by Gα signaling and actomyosin. During wild-type centration, NMY-2–GFP aggregates anterior to the NCC tend to move further anterior, suggesting that actomyosin contraction could pull the NCC. In GOA-1/GPA-16–depleted zygotes, NMY-2 aggregate displacement is reduced and largely randomized, whereas in a let-99(−) mutant, NMY-2 aggregates tend to make large posterior displacements. These results suggest that Gα signaling and LET-99 control centration by regulating polarized actomyosin contraction.

International audience; A critical structure poised to coordinate chromosome segregation with division plane specification is the central spindle that forms between separating chromosomes after anaphase onset. The central spindle acts as a signalling centre that concentrates proteins essential for division plane specification and contractile ring constriction. However, the molecular mechanisms that control the initial stages of central spindle assembly remain elusive. Using Caenorhabditis elegans zygotes, we found that the microtubule-bundling protein SPD-1(PRC1) and the motor ZEN-4(MKLP-1) are required for proper central spindle structure during its elongation. In contrast, we found that the kinetochore controls the initiation of central spindle assembly. Specifically, central spindle microtubule assembly is dependent on kinetochore recruitment of the scaffold protein KNL-1, as well as downstream partners BUB-1, HCP-1/2(CENP-F) and CLS-2(CLASP); and is negatively regulated by kinetochore-associated protein phosphatase 1 activity. This in turn promotes central spindle localization of CLS-2(CLASP) and initial central spindle microtubule assembly through its microtubule polymerase activity. Together, our results reveal an unexpected role for a conserved kinetochore protein network in coupling two critical events of cell division: chromosome segregation and cytokinesis.

Forces in the spindle that align and segregate chromosomes produce a steady poleward flux of kinetochore microtubules (MTs [kMTs]) in higher eukaryotes. In several nonmammalian systems, flux is driven by the tetrameric kinesin Eg5 (kinesin 5), which slides antiparallel MTs toward their minus ends. However, we find that the inhibition of kinesin 5 in mammalian cultured cells (PtK1) results in only minor reduction in the rate of kMT flux from approximately 0.7 to approximately 0.5 microm/min, the same rate measured in monopolar spindles that lack antiparallel MTs. These data reveal that the majority of poleward flux of kMTs in these cells is not driven by Eg5. Instead, we favor a polar "pulling-in" mechanism in which a depolymerase localized at kinetochore fiber minus ends makes a major contribution to poleward flux. One candidate, Kif2a (kinesin 13), was detected at minus ends of fluxing kinetochore fibers. Kif2a remains associated with the ends of K fibers upon disruption of the spindle by dynein/dynactin inhibition, and these K fibers flux.

Background: The PAR proteins are part of an ancient and widely conserved machinery for polarizing cells during animal development. Here we use a combination of genetics and live imaging methods in the model organism Caenorhabditis elegans to dissect the cellular mechanisms by which PAR proteins polarize cells. Results: We demonstrate two distinct mechanisms by which PAR proteins polarize the C. elegans zygote. First, we show that several components of the PAR pathway function in intracellular motility, producing a polarized movement of the cell cortex. We present evidence that this cortical motility may drive the movement of cellular components that must become asymmetrically distributed, including both germline-specific ribonucleoprotein complexes and cortical domains containing the PAR proteins themselves. Second, PAR-1 functions to refine the asymmetric localization of germline ribonucleoprotein complexes by selectively stabilizing only those complexes that reach the PAR-1-enriched posterior cell cortex during the period of cortical motility. Conclusions: These results identify two cellular mechanisms by which the PAR proteins polarize the C. elegans zygote, and they suggest mechanisms by which PAR proteins may polarize cells in diverse animal systems.

Some of the basic principles of cytokinesis were established 40 or more years ago. Cytokinesis researchers since then have struggled with the confusions inherent in redundant and interdependent pathways. Now, however, enough molecular detail is accumulating to bring some order to thoughts on how one

Members of the Ndc80/Nuf2 complex have been shown in several systems to be important in formation of stable kinetochore-microtubule attachments and chromosome alignment in mitosis 1, 2, 3, 4, 5, 6, 7, 8 and 9. In HeLa cells, we have shown that depletion of Nuf2 by RNA interference (RNAi) results in a strong prometaphase block with an active spindle checkpoint, which correlates with low but detectable Mad2 at kinetochores that have no or few stable kinetochore microtubules [5]. Another RNAi study in HeLa cells reported that Hec1 (the human Ndc80 homolog) is required for Mad1 and Mad2 binding to kinetochores and that kinetochore bound Mad2 does not play a role in generating and maintaining the spindle assembly checkpoint [6]. Here, we show that depletion of either Nuf2 or Hec1 by RNAi in HeLa cells results in reduction of both proteins at kinetochores and in the cytoplasm. Mad1 and Mad2 concentrate at kinetochores in late prophase/early prometaphase but become depleted by 5-fold or more over the course of the prometaphase block, which is Mad2 dependent. The reduction of Mad1 and Mad2 is reversible upon spindle depolymerization. Our observations support a model in which Nuf2 and Hec1 function to prevent microtubule-dependent stripping of Mad1 and Mad2 from kinetochores that have not yet formed stable kinetochore-microtubule attachments.

Merotelic kinetochore orientation is an error that occurs when a single kinetochore becomes attached to microtubules from two spindle poles rather than just to one pole. We obtained the first evidence that merotelic kinetochore orientation occurs very frequently during early mitosis in mammalian tissue cells and that two different correction mechanisms are critical for accurate chromosome segregation in cells possessing bipolar spindles and unperturbed chromosomes. Our data show that about 30% of prometaphase PtK1 cells possess one or more merotelically oriented kinetochores. This frequency is increased to over 90% in cells recovering from a nocodazole-induced mitotic block. A delay in establishing spindle bipolarity is responsible for the high frequency of merotelic orientations seen in cells recovering from nocodazole, but not in untreated cells. The frequency of anaphase cells with merotelically oriented lagging chromosomes is 1% in untreated cells and 18% in cells recovering from nocodazole. Prolonging metaphase by 2 hours reduced the frequency of anaphase cells with lagging chromosomes both for untreated and for nocodazole-treated cells. Surprisingly, anaphase lagging chromosomes represented a very small fraction of merotelic kinetochore orientations present in late metaphase. Our data indicate that two correction mechanisms operate to prevent chromosome missegregation due to merotelic kinetochore orientation. The first, a pre-anaphase correction mechanism increases the ratio of kinetochore microtubules attached to the correct versus incorrect pole and might eventually result in kinetochore reorientation before anaphase onset. The increase in microtubule ratio to opposite poles is the groundwork for a second mechanism, active in anaphase, that promotes the segregation of merotelically oriented chromosomes to the correct pole.

Proper positioning of the cell division plane during mitosis is essential for determining the size and position of the two daughter cells--a critical step during development and cell differentiation. A bipolar microtubule array has been proposed to be a minimum requirement for furrow positioning in mammalian cells, with furrows forming at the site of microtubule plus-end overlap between the spindle poles. Observations in other species have suggested, however, that this may not be true. Here we show, by inducing mammalian tissue cells with monopolar spindles to enter anaphase, that furrow formation in cultured mammalian cells does not require a bipolar spindle. Unexpectedly, cytokinesis occurs at high frequency in monopolar cells. Division always occurs at a cortical position distal to the chromosomes. Analysis of microtubules during cytokinesis in cells with monopolar and bipolar spindles shows that a subpopulation of stable microtubules extends past chromosomes and binds to the cell cortex at the site of furrow formation. Our data are consistent with a model in which chromosomes supply microtubules with factors that promote microtubule stability and furrowing.

CYK4 activity as a GTPase-activating protein is required during anaphase to inhibit Rac1-dependent effector pathways associated with control of cell spreading and adhesion., In mitosis, animal cells lose their adhesion to the surrounding surfaces and become rounded. During mitotic exit, they reestablish these adhesions and at the same time physically contract and divide. How these competing processes are spatially segregated at the cell cortex remains mysterious. To address this question, we define the specific effector pathways used by RhoA and Rac1 in mitotic cells. We demonstrate that the MKlp1–CYK4 centralspindlin complex is a guanosine triphosphatase–activating protein (GAP) for Rac1 and not RhoA and that CYK4 negatively regulated Rac1 activity at the cell equator in anaphase. Cells expressing a CYK4 GAP mutant had defects in cytokinesis and showed elevated staining for the cell adhesion marker vinculin. These defects could be rescued by depletion of ARHGEF7 and p21-activated kinase, Rac1-specific effector proteins required for cell adhesion. Based on these findings, we propose that CYK4 GAP activity is required during anaphase to inhibit Rac1-dependent effector pathways associated with control of cell spreading and adhesion.

The spindle checkpoint monitors microtubule attachment and tension at kinetochores to ensure proper chromosome segregation. Previously, PtK1 cells in hypothermic conditions (23 degrees C) were shown to have a pronounced mitotic delay, despite having normal numbers of kinetochore microtubules. At 23 degrees C, we found that PtK1 cells remained in metaphase for an average of 101 min, compared with 21 min for cells at 37 degrees C. The metaphase delay at 23 degrees C was abrogated by injection of Mad2 inhibitors, showing that Mad2 and the spindle checkpoint were responsible for the prolonged metaphase. Live cell imaging showed that kinetochore Mad2 became undetectable soon after chromosome congression. Measurements of the stretch between sister kinetochores at metaphase found a 24% decrease in tension at 23 degrees C, and metaphase kinetochores at 23 degrees C exhibited higher levels of 3F3/2, Bub1, and BubR1 compared with 37 degrees C. Microinjection of anti-BubR1 antibody abolished the metaphase delay at 23 degrees C, indicating that the higher kinetochore levels of BubR1 may contribute to the delay. Disrupting both Mad2 and BubR1 function induced anaphase with the same timing as single inhibitions, suggesting that these checkpoint genes function in the same pathway. We conclude that reduced tension at kinetochores with a full complement of kinetochore microtubules induces a checkpoint dependent metaphase delay associated with elevated amounts of kinetochore 3F3/2, Bub1, and BubR1 labeling.

In most species, oocytes lack centrosomes. Accurate meiotic spindle assembly and chromosome segregation – essential to prevent miscarriage or developmental defects – thus occur through atypical mechanisms that are not well characterized. Using quantitative in vitro and in vivo functional assays in the C. elegans oocyte, we provide novel evidence that the kinesin-13 KLP-7 promotes destabilization of the whole cellular microtubule network. By counteracting ectopic microtubule assembly and disorganization of the microtubule network, this function is strictly required for spindle organization, chromosome segregation and cytokinesis in meiotic cells. Strikingly, when centrosome activity was experimentally reduced, the absence of KLP-7 or the mammalian kinesin-13 protein MCAK (KIF2C) also resulted in ectopic microtubule asters during mitosis in C. elegans zygotes or HeLa cells, respectively. Our results highlight the general function of kinesin-13 microtubule depolymerases in preventing ectopic, spontaneous microtubule assembly when centrosome activity is defective or absent, which would otherwise lead to spindle microtubule disorganization and aneuploidy.

SummaryHow might the extracellular matrix contribute to cytokinesis? In a recent report, evidence is presented that the conserved extracellular matrix protein hemicentinHIM-4 is required for cytokinesis in worms and mice.

International audience; To take full advantage of fast-acting temperature-sensitive mutations, thermal control must be extremely rapid. We developed the Therminator, a device capable of shifting sample temperature in ∼17 s while simultaneously imaging cell division in vivo. Applying this technology to six key regulators of cytokinesis, we found that each has a distinct temporal requirement in the Caenorhabditis elegans zygote. Specifically, myosin-II is required throughout cytokinesis until contractile ring closure. In contrast, formin-mediated actin nucleation is only required during assembly and early contractile ring constriction. Centralspindlin is required to maintain division after ring closure, although its GAP activity is only required until just prior to closure. Finally, the chromosomal passenger complex is required for cytokinesis only early in mitosis, but not during metaphase or cytokinesis. Together, our results provide a precise functional timeline for molecular regulators of cytokinesis using the Therminator, a powerful tool for ultra-rapid protein inactivation.

Female sea urchins store their gametes as haploid eggs. The zygote enters S-phase 1 h after fertilization, initiating a series of cell cycles that lack gap phases. We have cloned cyclin E from the sea urchin Strongylocentrotus purpuratus. Cyclin E is synthesized during oogenesis, is present in the germinal vesicle, and is released into the egg cytoplasm at oocyte maturation. Cyclin E synthesis is activated at fertilization, although there is no increase in cyclin E protein levels due to continuous turnover of the protein. Cyclin E protein levels decline in morula embryos, while cyclin E mRNA levels remain high. After the blastula stage, cyclin E mRNA and protein levels are very low, and cyclin E expression is predominant only in cells that are actively dividing. These include cells in the left coelomic pouch, which forms the adult rudiment in the embryo. The cyclin E present in the egg is complexed with a protein kinase. Activity of the cyclin E/cdk2 changes little during the initial cell cycles. In particular, cyclin E-cdk2 levels remain high during both S-phase and mitosis. Our results suggest that progression through the early embryonic cell cycles in the sea urchin does not require fluctuations in cyclin E kinase activity.

We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 “dynamitin” protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.

The SWI/SNF family of chromatin-remodeling complexes facilitates gene expression by helping transcription factors gain access to their targets in chromatin. SWI/SNF and Rsc are distinctive members of this family from yeast. They have similar protein components and catalytic activities but differ in biological function. Rsc is required for cell cycle progression through mitosis, whereas SWI/SNF is not. Human complexes of this family have also been identified, which have often been considered related to yeast SWI/SNF. However, all human subunits identified to date are equally similar to components of both SWI/SNF and Rsc, leaving open the possibility that some or all of the human complexes are rather related to Rsc. Here, we present evidence that the previously identified human SWI/SNF-B complex is indeed of the Rsc type. It contains six components conserved in both Rsc and SWI/SNF. Importantly, it has a unique subunit, BAF180, that harbors a distinctive set of structural motifs characteristic of three components of Rsc. Of the two mammalian ATPases known to be related to those in the yeast complexes, human SWI/SNF-B contains only the homolog that functions like Rsc during cell growth. Immunofluorescence studies with a BAF180 antibody revealed that SWI/SNF-B localizes at the kinetochores of chromosomes during mitosis. Our data suggest that SWI/SNF-B and Rsc represent a novel subfamily of chromatin-remodeling complexes conserved from yeast to human, and could participate in cell division at kinetochores of mitotic chromosomes.

The cytokinesis phase, or C phase, of the cell cycle results in the separation of one cell into two daughter cells after the completion of mitosis. Although it is known that microtubules are required for proper positioning of the cytokinetic furrow [1] [2], the role of pre-anaphase microtubules in cytokinesis has not been clearly defined for three key reasons. First, inducing microtubule depolymerization or stabilization before the onset of anaphase blocks entry into anaphase and cytokinesis via the spindle checkpoint [3]. Second, microtubule organization changes rapidly at anaphase onset as the mitotic kinase, Cdc2-cyclin B, is inactivated [4]. Third, the time between the onset of anaphase and the initiation of cytokinesis is very short, making it difficult to unambiguously alter microtubule polymer levels before cytokinesis, but after inactivation of the spindle checkpoint. Here, we have taken advantage of the discovery that microinjection of antibodies to the spindle checkpoint protein Mad2 (mitotic arrest deficient) in prometaphase abrogates the spindle checkpoint, producing premature chromosome separation, segregation, and normal cytokinesis [5] [6]. To test the role of pre-anaphase microtubules in cytokinesis, microtubules were disassembled in prophase and prometaphase cells, the cells were then injected with anti-Mad2 antibodies and recorded through C phase. The results show that exit from mitosis in the absence of microtubules triggered a 50 minute period of cortical contractility that was independent of microtubules. Furthermore, upon microtubule reassembly during this contractile C-phase period, approximately 30% of the cells underwent chromosome poleward movement, formed a midzone microtubule complex, and completed cytokinesis.

Division plane specification in animal cells has long been presumed to involve direct contact between microtubules of the anaphase mitotic spindle and the cell cortex. In this issue, von Dassow et al. (von Dassow et al. 2009. J. Cell. Biol. doi:10.1083/jcb.200907090) challenge this assumption by showing that spindle microtubules can effectively position the division plane at a distance from the cell cortex.

Rho GTPases are molecular switches that elicit distinct effects on the actomyosin cytoskeleton to accurately promote cytokinesis. Although they represent less than 1% of the human genome, Rho GTPases exert disproportionate control over cell division. Crucial to this master regulatory role is their localized occupation of specific domains of the cell to ensure the assembly of a contractile ring at the proper time and place. RhoA occupies the division plane and is the central positive Rho family regulator of cytokinesis. Rac1 is a negative regulator of cytokinesis and is inactivated within the division plane while active Rac1 occupies the cell poles. Cdc42 regulation during cytokinesis is less studied, but thus far a clear role has only been shown during polar body emission. Here we review what is known about the function of Rho family GTPases during cell division, as well as their upstream regulators and known downstream cytokinetic effectors. © 2012 Wiley Periodicals, Inc

During cytokinesis, the guanosine triphosphatase (GTPase) RhoA orchestrates contractile ring assembly and constriction. RhoA signaling is controlled by the central spindle, a set of microtubule bundles that forms between the separating chromosomes. Centralspindlin, a protein complex consisting of the kinesin-6 ZEN-4 and the Rho family GTPase activating protein (GAP) CYK-4, is required for central spindle assembly and cytokinesis in Caenorhabditis elegans . However, the importance of the CYK-4 GAP activity and whether it regulates RhoA remain unclear. We found that two separation-of-function mutations in the GAP domain of CYK-4 lead to cytokinesis defects that mimic centralspindlin loss of function. These defects could be rescued by depletion of the GTPase Rac or its effectors, but not by depletion of RhoA. Thus, inactivation of Rac by centralspindlin functions in parallel with RhoA activation to drive contractile ring constriction during cytokinesis.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications (e.g., brightfield, phase-contrast, differential interference contrast, and fluorescence epi-illumination), and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The principles of microscopy and step-by-step alignment and adjustment procedures are described in this unit.

Nat. Cell Biol. 17, 697–705 (2015); published online 13 April 2015; corrected after print 8 June 2015 In the version of this Letter originally published, the following sentence was omitted from the Acknowledgements: 'B.L. is supported by a post-doctoral fellowship from FRM (ARF20140129055).' This has been corrected in the online versions of the Letter.

How microtubules act to position the plane of cell division during cytokinesis is a topic of much debate. Recently, we showed that a subpopulation of stable microtubules extends past chromosomes and interacts with the cell cortex at the site of furrowing, suggesting that these stabilized microtubules may stimulate contractility. To test the hypothesis that stable microtubules can position furrows, we used taxol to rapidly suppress microtubule dynamics during various stages of mitosis in PtK1 cells. Cells with stabilized prometaphase or metaphase microtubule arrays were able to initiate furrowing when induced into anaphase by inhibition of the spindle checkpoint. In these cells, few microtubules contacted the cortex. Furrows formed later than usual, were often aberrant, and did not progress to completion. Images showed that furrowing correlated with the presence of one or a few stable spindle microtubule plus ends at the cortex. Actin, myosin II, and anillin were all concentrated in these furrows, demonstrating that components of the contractile ring can be localized by stable microtubules. Inner centromere protein (INCENP) was not found in these ingressions, confirming that INCENP is dispensable for furrow positioning. Taxol-stabilization of the numerous microtubule-cortex interactions after anaphase onset delayed furrow initiation but did not perturb furrow positioning. We conclude that taxol-stabilized microtubules can act to position the furrow and that loss of microtubule dynamics delays the timing of furrow onset and prevents completion. We discuss our findings relative to models for cleavage stimulation.

Background: The spindle assembly checkpoint (SAC) imparts fidelity to chromosome segregation by delaying anaphase until all sister chromatid pairs have become bipolarly attached. Mad2 is a component of the SAC effector complex that sequesters Cdc20 to halt anaphase. In prometaphase, Mad2 is recruited to kinetochores with the help of Mad1, and it is activated to bind Cdc20. These events are linked to the existence of two distinct conformers of Mad2: a closed conformer bound to its kinetochore receptor Mad1 or its target in the checkpoint Cdc20 and an open conformer unbound to these ligands. Results: We investigated the mechanism of Mad2 recruitment to the kinetochore during checkpoint activation and subsequent transfer to Cdc20. We report that a closed conformer of Mad2 constitutively bound to Mad1, rather than Mad1 itself, is the kinetochore receptor for cytosolic open Mad2 and show that the interaction of open and closed Mad2 conformers is essential to sustain the SAC. Conclusions: We propose that closed Mad2 bound to Mad1 represents a template for the conversion of open Mad2 into closed Mad2 bound to Cdc20. This simple model, which we have named the "Mad2 template" model, predicts a mechanism for cytosolic propagation of the spindle checkpoint signal away from kinetochores.

Microtubule polymerization dynamics at kinetochores is coupled to chromosome movements, but its regulation there is poorly understood. The plus end tracking protein EB1 is required both for regulating microtubule dynamics and for maintaining a euploid genome. To address the role of EB1 in aneuploidy, we visualized its targeting in mitotic PtK1 cells. Fluorescent EB1, which localized to polymerizing ends of astral and spindle microtubules, was used to track their polymerization. EB1 also associated with a subset of attached kinetochores in late prometaphase and metaphase, and rarely in anaphase. Localization occurred in a narrow crescent, concave toward the centromere, consistent with targeting to the microtubule plus end–kinetochore interface. EB1 did not localize to kinetochores lacking attached kinetochore microtubules in prophase or early prometaphase, or upon nocodazole treatment. By time lapse, EB1 specifically targeted to kinetochores moving antipoleward, coupled to microtubule plus end polymerization, and not during plus end depolymerization. It localized independently of spindle bipolarity, the spindle checkpoint, and dynein/dynactin function. EB1 is the first protein whose targeting reflects kinetochore directionality, unlike other plus end tracking proteins that show enhanced kinetochore binding in the absence of microtubules. Our results suggest EB1 may modulate kinetochore microtubule polymerization and/or attachment.

The spindle checkpoint, which prevents anaphase onset upon spindle damage or incorrect chromosome alignment, presents a problem for experimental analysis of protein function in anaphase and cytokinesis. This is because the functional disruption of many proteins before anaphase onset can activate this checkpoint, preventing anaphase and subsequent cell cycle events. This paper compares new and old methods of overriding the spindle checkpoint in prometaphase mammalian tissue culture cells. Cell Motil. Cytoskeleton 52:61–65, 2002. © 2002 Wiley-Liss, Inc.

Spindle checkpoint proteins, such as Mad2 and BubR1, and the motors dynein/dynactin and CENP-E usually leave kinetochores prior to anaphase onset by microtubule-dependent mechanisms. Likewise, `chromosome passenger proteins'including INCENP are depleted from the centromeres after anaphase onset and then move to the midzone complex, an event that is essential for cytokinesis. Here we test whether the cell cycle changes that occur at anaphase onset require or contribute to the depletion of kinetochore and centromere proteins independent of microtubules. This required the development of a novel non-antibody method to induce precocious anaphase onset in vivo by using a bacterially expressed fragment of the spindle checkpoint protein Mad1 capable of activating the APC/C, called GST-Mad1F10. By injecting PtK1 cells in nocodazole with GST-Mad1F10 and processing the cells for immunofluorescence microscopy after anaphase sister chromatid separation in nocodazole we found that Mad2, BubR1, cytoplasmic dynein, CENP-E and the 3F3/2 phosphoepitope remain on kinetochores. Thus depletion of these proteins (or phosphoepitope)at kinetochores is not required for anaphase onset and anaphase onset does not produce their depletion independent of microtubules. In contrast, both microtubules and anaphase onset are required for depletion of the `chromosome passenger' protein INCENP from centromeres, as INCENP does not leave the chromosomes prior to anaphase onset in the presence or absence of microtubules, but does leave the centromeres after anaphase onset in the presence of microtubules.

Several cell motility processes including cytokinesis and cell locomotion are dependent on the interplay of the microtubule and actomyosin cytoskeletons. However, because such processes are essentially visual phenomena, interactions between the two cytoskeletal systems have been difficult to study quantitatively. To overcome this difficulty, we have developed the Xenopus oocyte as an inducible, quantitative model system for actomyosin-based cortical flow and then exploited the strengths of this system to assess the relationship between microtubules and cortical flow. As in other systems, oocyte cortical flow entails: (1) redistribution of cortical filamentous actin (f-actin); (2) a requirement for actomyosin; (3) redistribution of cell surface proteins; (4) a requirement for cell surface protein mobility; and (5) directed movement of cortical organelles. Cortical flow rate in the oocyte system is inversely proportional to the level of polymeric tubulin and microinjection of free tubulin has no effect on the rate of cortical flow. Enhancement of microtubule polymerization inhibits cortical f-actin cable formation during cortical flow. The effects of microtubule depolymerization on cortical flow are rapid, independent of transcription or translation, independent of effects on the oocyte intermediate filament system, and independent of the upstream stimulus for cortical flow. The results show that the microtubules themselves, or a factor associated with them, suppress cortical flow, either by mechanically resisting flow, or by modulating the actomyosin cytoskeleton.