The establishment, maintenance, and dissolution of sister chromatid cohesion are sequentially coordinated during the cell cycle to ensure faithful chromosome transmission. This cell-cycle-dependent ...regulation of cohesion is mediated, in part, by distinct posttranslational modifications of cohesin, a protein complex consisting of the Smc1-Smc3 ATPase, the Mcd1/Scc1 α-kleisin, and Scc3. Although cohesion is established in S phase, cohesins are not sufficient to maintain cohesion as cells progress from G2 to the metaphase-to-anaphase transition. Rather, the cohesin-associated factor Pds5 is also required to keep sisters paired until anaphase onset. How Pds5 maintains cohesion at the molecular level and whether this maintenance involves the regulation of cohesin modifications remains to be defined.
In pds5 mutants, we find that Mcd1 is extensively SUMOylated and that premature sister separation requires Siz2-dependent polySUMOylation. Moreover, abrogation of Pds5 function promotes the proteasome-dependent degradation of Mcd1 and a significant loss of cohesin from chromatin independently of anaphase onset. We further demonstrate that inactivation of the Slx5-Slx8 SUMO-targeted ubiquitin ligase, required for targeting polySUMOylated factors for proteasome-mediated destruction, limits Mcd1 turnover and restores both cell growth and cohesion in metaphase cells defective for Pds5 function.
We propose that Pds5 maintains cohesion, at least in part, by antagonizing the polySUMO-dependent degradation of cohesin.
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•SUMO both positively and negatively regulates sister chromatid cohesion•Siz2-dependent polySUMOylation promotes sister separation in pds5-1 cells•Pds5 prevents extensive SUMOylation and proteasome-mediated degradation of Mcd1•Slx5-Slx8 STUbL inactivation limits Mcd1 degradation to restore cohesion in pds5-1
Why sister chromatid cohesion established during S phase must be maintained by specialized factors as cells transit G2/metaphase has remained an enigma. D’Ambrosio and Lavoie find that the essential cohesion maintenance factor Pds5 prevents premature sister separation by protecting the cohesin complex from untimely SUMO-targeted degradation.
Abstract Cytokinesis is the final step of the cell division cycle that leads to the formation of two new cells. Successful cytokinesis requires significant remodelling of the plasma membrane by ...spatially distinct β- and γ-actin networks. These networks are generated by the formin family of actin nucleators, DIAPH3 and DIAPH1 respectively. Here we show that β- and γ-actin perform specialized and non-redundant roles in cytokinesis and cannot substitute for one another. Expression of hybrid DIAPH1 and DIAPH3 proteins with altered actin isoform specificity relocalized cytokinetic actin isoform networks within the cell, causing cytokinetic failure. Consistent with this we show that β-actin networks, but not γ-actin networks, are required for the maintenance of non-muscle myosin II and RhoA at the cytokinetic furrow. These data suggest that independent and spatially distinct actin isoform networks form scaffolds of unique interactors that facilitate localized biochemical activities to ensure successful cell division.
During cytokinesis, the actin cytoskeleton is partitioned into two spatially distinct actin isoform specific networks: a β-actin network that generates the equatorial contractile ring, and a γ-actin ...network that localizes to the cell cortex. Here we demonstrate that the opposing regulation of the β- and γ-actin networks is required for successful cytokinesis. While activation of the formin DIAPH3 at the cytokinetic furrow underlies β-actin filament production, we show that the γ-actin network is specifically depleted at the cell poles through the localized deactivation of the formin DIAPH1. During anaphase, CLIP170 is delivered by astral microtubules and displaces IQGAP1 from DIAPH1, leading to formin autoinhibition, a decrease in cortical stiffness and localized membrane blebbing. The contemporaneous production of a β-actin contractile ring at the cell equator and loss of γ-actin from the poles is required to generate a stable cytokinetic furrow and for the completion of cell division.
The actin cytoskeleton is a dynamic array of filaments that undergoes rapid remodeling to drive many cellular processes. An essential feature of filament remodeling is the spatio-temporal regulation ...of actin filament nucleation. One family of actin filament nucleators, the Diaphanous-related formins, is activated by the binding of small G-proteins such as RhoA. However, RhoA only partially activates formins, suggesting that additional factors are required to fully activate the formin. Here we identify one such factor, IQ motif containing GTPase activating protein-1 (IQGAP1), which enhances RhoA-mediated activation of the Diaphanous-related formin (DIAPH1) and targets DIAPH1 to the plasma membrane. We find that the inhibitory intramolecular interaction within DIAPH1 is disrupted by the sequential binding of RhoA and IQGAP1. Binding of RhoA and IQGAP1 robustly stimulates DIAPH1-mediated actin filament nucleation in vitro. In contrast, the actin capping protein Flightless-I, in conjunction with RhoA, only weakly stimulates DIAPH1 activity. IQGAP1, but not Flightless-I, is required to recruit DIAPH1 to the plasma membrane where actin filaments are generated. These results indicate that IQGAP1 enhances RhoA-mediated activation of DIAPH1 in vivo. Collectively these data support a model where the combined action of RhoA and an enhancer ensures the spatio-temporal regulation of actin nucleation to stimulate robust and localized actin filament production in vivo.
The final step of cytokinesis is abscission when the intercellular bridge (ICB) linking the two new daughter cells is broken. Correct construction of the ICB is crucial for the assembly of factors ...involved in abscission, a failure in which results in aneuploidy. Using live imaging and subdiffraction microscopy, we identify new anillin–septin cytoskeleton-dependent stages in ICB formation and maturation. We show that after the formation of an initial ICB, septin filaments drive ICB elongation during which tubules containing anillin–septin rings are extruded from the ICB. Septins then generate sites of further constriction within the mature ICB from which they are subsequently removed. The action of the anillin–septin complex during ICB maturation also primes the ICB for the future assembly of the ESCRT III component Chmp4B at the abscission site. These studies suggest that the sequential action of distinct contractile machineries coordinates the formation of the abscission site and the successful completion of cytokinesis.
Rigorous spatiotemporal regulation of cell division is required to maintain genome stability. The final stage in cell division, when the cells physically separate (abscission), is tightly regulated ...to ensure that it occurs after cytokinetic events such as chromosome segregation. A key regulator of abscission timing is Aurora B kinase activity, which inhibits abscission and forms the major activity of the abscission checkpoint. This checkpoint prevents abscission until chromosomes have been cleared from the cytokinetic machinery. Here we demonstrate that the mitosis-specific CDK11p58 kinase specifically forms a complex with cyclin L1β that, in late cytokinesis, localizes to the stem body, a structure in the middle of the intercellular bridge that forms between two dividing cells. Depletion of CDK11 inhibits abscission, and rescue of this phenotype requires CDK11p58 kinase activity or inhibition of Aurora B kinase activity. Furthermore, CDK11p58 kinase activity is required for formation of endosomal sorting complex required for transport III filaments at the site of abscission. Combined, these data suggest that CDK11p58 kinase activity opposes Aurora B activity to enable abscission to proceed and result in successful completion of cytokinesis.
Background & Aims The 5-hydroxytryptamine receptor 4 (5-HT4R or HTR4) is expressed in the colonic epithelium but little is known about its functions there. We examined whether activation of colonic ...epithelial 5-HT4R protects colons of mice from inflammation. Methods The 5-HT4R agonist tegaserod (1 mg/kg), the 5-HT4R antagonist GR113808 (1 mg/kg), or vehicle (control) were delivered by enema to wild-type or 5-HT4R knockout mice at the onset of, or during, active colitis, induced by administration of dextran sodium sulfate or trinitrobenzene sulfonic acid. Inflammation was measured using the colitis disease activity index and by histologic analysis of intestinal tissues. Epithelial proliferation, wound healing, and resistance to oxidative stress-induced apoptosis were assessed, as was colonic motility. Results Rectal administration of tegaserod reduced the severity of colitis compared with mice given vehicle, and accelerated recovery from active colitis. Rectal tegaserod did not improve colitis in 5-HT4R knockout mice, and intraperitoneally administered tegaserod did not protect wild-type mice from colitis. Tegaserod increased proliferation of crypt epithelial cells. Stimulation of 5-HT4R increased Caco-2 cell migration and reduced oxidative stress-induced apoptosis; these actions were blocked by co-administration of the 5-HT4R antagonist GR113808. In noninflamed colons of wild-type mice not receiving tegaserod, inhibition of 5-HT4Rs resulted in signs of colitis within 3 days. In these mice, epithelial proliferation decreased and bacterial translocation to the liver and spleen was detected. Daily administration of tegaserod increased motility in inflamed colons of guinea pigs and mice, whereas administration of GR113808 disrupted motility in animals without colitis. Conclusions 5-HT4R activation maintains motility in healthy colons of mice and guinea pigs, and reduces inflammation in colons of mice with colitis. Agonists might be developed as treatments for patients with inflammatory bowel diseases.
The compartmentalization of cell cycle regulators is a common mechanism to ensure the precise temporal control of key cell cycle events. For instance, many mitotic spindle assembly factors are known ...to be sequestered in the nucleus prior to mitotic onset. Similarly, the essential cytokinetic factor anillin, which functions at the cell membrane to promote the physical separation of daughter cells at the end of mitosis, is sequestered in the nucleus during interphase. To address the mechanism and role of anillin targeting to the nucleus in interphase, we identified the nuclear targeting motif. Here, we show that anillin is targeted to the nucleus by importin β2 in a Ran-dependent manner through an atypical basic patch PY nuclear localization signal motif. We show that although importin β2 binding does not regulate anillin's function in mitosis, it is required to prevent the cytosolic accumulation of anillin, which disrupts cellular architecture during interphase. The nuclear sequestration of anillin during interphase serves to restrict anillin's function at the cell membrane to mitosis and allows anillin to be rapidly available when the nuclear envelope breaks down to remodel the cellular architecture necessary for successful cell division.
Background: Anillin is an evolutionarily conserved protein essential for cytokinesis.
Results: Importin β2 targets anillin to the nucleus during interphase.
Conclusion: Anillin is spatio-temporally regulated during the cell cycle to prevent disruption of the interphase cellular architecture.
Significance: Sequestering proteins with key mitotic functions in the nucleus is a vital part of their cell cycle regulation.
Abstract
Background
Fecal microbiota transplantation (FMT) is a promising new strategy in the treatment of Inflammatory Bowel Disease, but long-term delivery systems are lacking. This randomized ...study was designed as a safety and feasibility study of long-term FMT in subjects with mild to moderate UC using frozen, encapsulated oral FMT (cFMT).
Methods
Subjects were randomized 1:1 to receive FMT induction by colonoscopy, followed by 12 weeks of daily oral administration of frozen encapsulated cFMT or sham therpay. Subjects were followed for 36 weeks and longitudenal clinical assessments included multiple subjective and objective markers of disease severity. Ribosomal 16S bacterial sequencing was used to assess donor-induced changes in the gut microbiota. Changes in T regulatory (Treg) and mucosal associated invariant T (MAIT) cell populations were evaluated by flow cytometry as an exploratory endpoint.
Results
Twelve subjects with active UC were randomized: 6 subjects completed the full 12-week course of FMT plus cFMT, and 6 subjects received sham treatment by colonic installation and longitudinal oral placebo capules. Chronic administration of cFMT was found to be safe and well-tolerated but home storage concerns exist. Protocol adherence was high, and none of the study subjects experienced FMT-associated treatment emergent adverse events. Two subjects that received cFMT achieved clinical remission versus none in the placebo group (95% CI = 0.38-infinity,
p
= 0.45). cFMT was associated with sustained donor-induced shifts in fecal microbial composition. Changes in MAIT cell cytokine production were observed in cFMT recipients and correlated with treatment response.
Conclusion
These pilot data suggest that daily encapsulated cFMT may extend the durability of index FMT-induced changes in gut bacterial community structure and that an association between MAIT cell cytokine production and clinical response to FMT may exist in UC populations. Oral frozen encapsulated cFMT is a promising FMT delivery system and may be preferred for longterm treatment strategies in UC and other chronic diseases but further evaluations will have to address home storage concerns. Larger trials should be done to explore the benefits of cFMT and to determine its long-term impacts on the colonic microbiome. Trial registration: ClinicalTrials.gov (NCT02390726). Registered 17 March 2015,
https://clinicaltrials.gov/ct2/show/NCT02390726?term=NCT02390726&draw=2&rank=1
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