The E3 ubiquitin ligase HECTD1 contributes to cell cycle progression through an effect on mitosis

Mechanistic studies of how protein ubiquitylation regulates the cell cycle, in particular during mitosis, has provided unique insights which have contributed to the emergence of the Ubiquitin code. In contrast to RING E3 ubiquitin ligases such as the APC/c ligase complex, the contribution of other E3 ligase families during cell cycle progression remains less well understood. Similarly, the contribution of ubiquitin chain types beyond homotypic K48 chains in S-phase or branched K11/K48 chains assembled by APC/c during mitosis, also remains to be fully determined. Our recent findings that HECTD1 ubiquitin ligase activity assembles branched K29/K48 ubiquitin linkages prompted us to evaluate its function during the cell cycle. We used transient knockdown and genetic knockout to show that HECTD1 depletion in HEK293T and HeLa cells decreases cell proliferation and we established that this is mediated through loss of its ubiquitin ligase activity. Interestingly, we found that HECTD1 depletion increases the proportion of cells with aligned chromosomes (Prometa/Metaphase). We confirmed this molecularly using phospho-Histone H3 (Ser28) as a marker of mitosis. Time-lapse microscopy of NEBD to anaphase onset established that HECTD1-depleted cells take on average longer to go through mitosis. To explore the mechanisms involved, we used proteomics to explore the endogenous HECTD1 interactome in mitosis and validated the Mitosis Checkpoint Complex protein BUB3 as a novel HECTD1 interactor. In line with this, we found that HECTD1 depletion reduces the activity of the Spindle Assembly Checkpoint. Overall, our data suggests a novel role for HECTD1 ubiquitin ligase activity in mitosis.

The MAP She1 coordinates directional spindle positioning by spatially restricting dynein activity

Dynein motors move the mitotic spindle to the cell division plane in many cell types, including in budding yeast, in which dynein is assisted by numerous factors including the microtubule-associated protein (MAP) She1. Evidence suggests that She1 plays a role in polarizing dynein-mediated spindle movements toward the daughter cell; however, how She1 performs this function is unknown. We find that She1 assists dynein in maintaining the spindle in close proximity to the bud neck, such that at anaphase onset the chromosomes are segregated to mother and daughter cells. She1 does so by attenuating the initiation of dynein-mediated spindle movements within the mother cell, thus ensuring such movements are polarized toward the daughter cell. Our data indicate that this activity relies on She1 binding to the microtubule-bound conformation of the dynein microtubule-binding domain, and to astral microtubules within mother cells. Our findings reveal how an asymmetrically localized MAP directionally tunes dynein activity by attenuating motor activity in a spatially confined manner.

Alternative Cdc20 translational isoforms bypass the spindle assembly checkpoint to control mitotic arrest duration

Mitotic chromosome segregation defects activate the Spindle Assembly Checkpoint (SAC), which inhibits the APC/C co-activator Cdc20 to induce a prolonged cell cycle arrest. Once errors are corrected, the SAC is silenced thereby allowing anaphase onset and mitotic exit to proceed. However, in the presence of persistent, unresolvable errors, cells can undergo "mitotic slippage", exiting mitosis into a tetraploid G1 state and escaping the cell death that results from a prolonged arrest. The molecular logic that allows cells to balance these dueling mitotic arrest and slippage behaviors remains unclear. Here we demonstrate that human cells modulate their mitotic arrest duration through the presence of conserved, alternative Cdc20 translational isoforms. Translation initiation at downstream start sites results in truncated Cdc20 isoforms that are resistant to SAC-mediated inhibition and promote mitotic exit even in the presence of mitotic perturbations. Targeted molecular changes or naturally-occurring mutations in cancer cells that alter the relative Cdc20 isoform levels or its translational regulatory control modulate both mitotic arrest duration and anti-mitotic drug sensitivity. Our work reveals a critical role for the differential translational regulation of Cdc20 in mitotic arrest timing, with important implications for the diagnosis and treatment of human cancers.

Processive translocation of cohesive and non-cohesive cohesin in vivo

Cohesin is a central architectural element of chromosome structure that regulates numerous DNA-based events. The complex holds sister chromatids together until anaphase onset and organizes individual chromosomal DNAs into loops. In vitro, cohesin translocates along DNA and extrudes loops in an ATP-dependent fashion. In vivo, cohesin redistributes in response to transcription as if pushed by RNA polymerase. Direct evidence of processive genomic translocation by the complex, however, is lacking. Here, obstacles of increasing size were tethered to DNA in yeast to detect translocation. The obstacles were built from a GFP-lacI core fused to one or more mCherries. Cohesin translocation was initiated from an upstream gene. A chimera with four mCherries blocked cohesin passage in late G1. During M phase, the threshold barrier to passage depended on the state of cohesion: non-cohesive complexes were also blocked by four mCherries whereas cohesive complexes were blocked by only three mCherries. That synthetic barriers alter cohesin redistribution demonstrates that the complex translocates processively on chromatin in vivo. The approach provides a relative measure of the maximum size of the protein chamber(s) that embraces DNA during cohesin translocation. The data indicate that the cohesive embrace is more restrictive than the embrace of non-cohesive complexes.

The RAD51 recombinase protects mitotic chromatin in human cells

The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in maintaining genome stability are well-characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. In cells experiencing mild replication stress, MiDAS was detected irrespective of mitotically generated DNA damage. MiDAS broadly required de novo RAD51 recruitment to single-stranded DNA, which was supported by the phosphorylation of RAD51 by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of MiDAS delayed anaphase onset and induced centromere fragility, suggesting a mechanism that prevents the satisfaction of the spindle assembly checkpoint while chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting genomic stability in mitosis.

Cohesin is essential for the timely activation of condensin and anaphase segregation

Chromosome segregation requires the separation of sister chromatids and the sustained condensation of chromatids during anaphase. Yeast cohesin has a dual function on metaphase chromosomes, holding sister chromatids together and organising individual chromatids into loops. Cleavage by separase is thought to remove all cohesin from chromosomes at the anaphase onset. This fulfils the requirement for chromatid separation, but it is unclear how the structure of segregating chromatids is maintained following cleavage. Here we demonstrate that auxin-mediated degradation of cohesin′s kleisin in anaphase, causes catastrophic chromosome mis-segregation demonstrating that cohesin affects post-metaphase processes. We identify a pool of cohesin bound to anaphase/telophase chromosomes and demonstrate that its inactivation causes defects in the organisation of centromeric regions and condensin function. Our data thus uncovers an unsuspected role for cohesin on anaphase/telophase chromosomes that is essential for the fidelity of segregation.

BubR1 recruitment to the kinetochore via Bub1 enhances Spindle Assembly Checkpoint signaling

During mitosis, unattached kinetochores in a dividing cell generate the anaphase-inhibitory Mitotic Checkpoint Complex (MCC) to activate the Spindle Assembly Checkpoint (SAC) and delay anaphase onset. To generate MCC, these kinetochores recruit MCC constituent proteins including the protein BubR1. The increased local concentration of BubR1 resulting from this recruitment should enhance MCC generation, but prior studies found this not to be the case. We analyzed the contribution of two BubR1 recruitment pathways to MCC generation in human kinetochores. For these analyses, we isolated a subset of the MCC generation reactions to the cytosol using ectopic SAC activation systems. These analyses and mathematical modeling show that BubR1 binding to the SAC protein Bub1, but not to the 'KI' motifs in the kinetochore protein Knl1, significantly enhances the rate of Bub1-mediated MCC generation in the kinetochore. Our work also suggests that Bub1-BubR1 stoichiometry will strongly influence the dose-response characteristics of SAC signaling.

Topoisomerase II deficiency leads to a postreplicative structural shift in all Saccharomyces cerevisiae chromosomes

The key role of Topoisomerase II (Top2) is the removal of topological intertwines between sister chromatids. In yeast, inactivation of Top2 brings about distinct cell cycle responses. In the case of the conditional top2-5 allele, interphase and mitosis progress on schedule but cells suffer from a chromosome segregation catastrophe. We here show that top2-5 chromosomes fail to enter a Pulsed-Field Gel Electrophoresis (PFGE) in the first cell cycle, a behavior traditionally linked to the presence of replication and recombination intermediates. We distinguished two classes of affected chromosomes: the rDNA-bearing chromosome XII, which fails to enter a PFGE at the beginning of S-phase, and all the other chromosomes, which fail at a postreplicative stage. In synchronously cycling cells, this late PFGE retention is observed in anaphase; however, we demonstrate that this behavior is independent of cytokinesis, stabilization of anaphase bridges, spindle pulling forces and, probably, anaphase onset. Strikingly, once the PFGE retention has occurred it becomes refractory to Top2 re-activation. DNA combing, two-dimensional electrophoresis, genetic analyses, and GFP-tagged DNA damage markers suggest that neither recombination intermediates nor unfinished replication account for the postreplicative PFGE shift, which is further supported by the fact that the shift does not trigger the G2/M checkpoint. We propose that the absence of Top2 activity leads to a general chromosome structural/topological change in mitosis.

Yeast Fin1-PP1 dephosphorylates an Ipl1 substrate, Ndc80, to remove Bub1-Bub3 checkpoint proteins from the kinetochore during anaphase

The spindle assembly checkpoint (SAC) prevents anaphase onset in response to chromosome attachment defects, and SAC silencing is essential for anaphase onset. Following anaphase onset, activated Cdc14 phosphatase dephosphorylates the substrates of cyclin-dependent kinase to facilitate anaphase progression and mitotic exit. In budding yeast, Cdc14 dephosphorylates Fin1, a regulatory subunit of protein phosphatase 1 (PP1), to enable kinetochore localization of Fin1-PP1. We previously showed that kinetochore-localized Fin1-PP1 promotes the removal of the SAC protein Bub1 from the kinetochore during anaphase. We report here that Fin1-PP1 also promotes kinetochore removal of Bub3, the Bub1 partner, but has no effect on another SAC protein Mad1. Moreover, the kinetochore localization of Bub1-Bub3 during anaphase requires Aurora B/Ipl1 kinase activity. We further showed that Fin1-PP1 facilitates the dephosphorylation of kinetochore protein Ndc80, a known Ipl1 substrate. This dephosphorylation reduces kinetochore association of Bub1-Bub3 during anaphase. In addition, we found that untimely Ndc80 dephosphorylation causes viability loss in response to tensionless chromosome attachments. These results suggest that timely localization of Fin1-PP1 to the kinetochore controls the functional window of SAC and is therefore critical for faithful chromosome segregation.

SUMOylation stabilizes sister kinetochore biorientation to allow timely anaphase

During mitosis, sister chromatids attach to microtubules from opposite poles, called biorientation. Sister chromatid cohesion resists microtubule forces, generating tension, which provides the signal that biorientation has occurred. How tension silences the surveillance pathways that prevent cell cycle progression and correct erroneous kinetochore–microtubule attachments remains unclear. Here we show that SUMOylation dampens error correction to allow stable sister kinetochore biorientation and timely anaphase onset. The Siz1/Siz2 SUMO ligases modify the pericentromere-localized shugoshin (Sgo1) protein before its tension-dependent release from chromatin. Sgo1 SUMOylation reduces its binding to protein phosphatase 2A (PP2A), and weakening of this interaction is important for stable biorientation. Unstable biorientation in SUMO-deficient cells is associated with persistence of the chromosome passenger complex (CPC) at centromeres, and SUMOylation of CPC subunit Bir1 also contributes to timely anaphase onset. We propose that SUMOylation acts in a combinatorial manner to facilitate dismantling of the error correction machinery within pericentromeres and thereby sharpen the metaphase–anaphase transition.