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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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.