Manuel Mendoza

The NoCut Pathway Links Completion of Cytokinesis to Spindle Midzone Function to Prevent Chromosome Breakage

During anaphase, spindle elongation pulls sister chromatids apart until each pair is fully separated. In turn, cytokinesis cleaves the cell between the separated chromosomes. What ensures that cytokinesis proceeds only after that all chromosome arms are pulled out of the cleavage plane was unknown. Here, we show that a signaling pathway, which we call NoCut, delays the completion of cytokinesis in cells with spindle-midzone defects. NoCut depends on the Aurora kinase Ipl1 and the anillin-related proteins Boi1 and Boi2, which localize to the site of cleavage in an Ipl1-dependent manner and act as abscission inhibitors. Inactivation of NoCut leads to premature abscission and chromosome breakage by the cytokinetic machinery and is lethal in cells with spindle-elongation defects. We propose that NoCut monitors clearance of chromatin from the midzone to ensure that cytokinesis completes only after all chromosomes have migrated to the poles.

A mechanism for chromosome segregation sensing by the NoCut checkpoint

In Saccharomyces cerevisiae and HeLa cells, the NoCut checkpoint, which involves the chromosome passenger kinase Aurora B, delays the completion of cytokinesis in response to anaphase defects. However, how NoCut monitors anaphase progression has not been clear. Here, we show that retention of chromatin in the plane of cleavage is sufficient to trigger NoCut, provided that Aurora/Ipl1 localizes properly to the spindle midzone, and that the ADA histone acetyltransferase complex is intact. Furthermore, forcing Aurora onto chromatin was sufficient to activate NoCut independently of anaphase defects. These findings provide the first evidence that NoCut is triggered by the interaction of acetylated chromatin with the passenger complex at the spindle midzone.

A Midzone-Based Ruler Adjusts Chromosome Compaction to Anaphase Spindle Length

The degree of chromosome condensation can be modulated within cells to ensure proper segregation. Partitioning of chromatids during mitosis requires that chromosome compaction and spindle length scale appropriately with each other. However, it is not clear whether chromosome condensation and spindle elongation are linked. Here, we find that yeast cells could cope with a 45% increase in the length of their longest chromosome arm by increasing its condensation. The spindle midzone, aurora/Ipl1 activity, and Ser10 of histone H3 mediated this response. Thus, the anaphase spindle may function as a ruler to adapt the condensation of chromatids, promoting their segregation regardless of chromosome or spindle length.

The Aurora-B-dependent NoCut checkpoint prevents damage of anaphase bridges after DNA replication stress

Anaphase chromatin bridges can lead to chromosome breakage if not properly resolved before completion of cytokinesis. The NoCut checkpoint, which depends on Aurora B at the spindle midzone, delays abscission in response to chromosome segregation defects in yeast and animal cells. How chromatin bridges are detected, and whether abscission inhibition prevents their damage, remain key unresolved questions. We find that bridges induced by DNA replication stress and by condensation or decatenation defects, but not dicentric chromosomes, delay abscission in a NoCut-dependent manner. Decatenation and condensation defects lead to spindle stabilization during cytokinesis, allowing bridge detection by Aurora B. NoCut does not prevent DNA damage following condensin or topoisomerasexa0II inactivation; however, it protects anaphase bridges and promotes cellular viability after replication stress. Therefore, the molecular origin of chromatin bridges is critical for activation of NoCut, which plays a key role in the maintenance of genome stability after replicativexa0stress.

Chromosome length and perinuclear attachment constrain resolution of DNA intertwines

Independent of the presence of rDNA repeats, topological constraints imposed by chromosome length and perinuclear attachment determine the efficiency with which sister chromatid intertwines are resolved by topoisomerase II and dynamic microtubules during anaphase.

Co-ordination of cytokinesis with chromosome segregation.

During anaphase, the spindle pulls the sister kinetochores apart until the sister chromatids are fully separated from each other. Subsequently, cytokinesis cleaves between the two separated chromosome masses to form two nucleated cells. Results from Schizosaccharomyces pombe suggested that cytokinesis and chromosome segregation are not co-ordinated with each other. However, recent studies indicate that, at least in budding yeast, a checkpoint called NoCut prevents abscission when spindle elongation is impaired, and might delay cytokinesis until all chromosomes are pulled out of the cleavage plane. Here, we discuss this possibility and summarize evidence suggesting that such a checkpoint is likely to be conserved in higher eukaryotes.

Daughter-cell-specific modulation of nuclear pore complexes controls cell cycle entry during asymmetric division

The acquisition of cellular identity is coupled to changes in the nuclear periphery and nuclear pore complexes (NPCs). Whether and how these changes determine cell fate remain unclear. We have uncovered a mechanism that regulates NPC acetylation to direct cell fate after asymmetric division in budding yeast. The lysine deacetylase Hos3 associates specifically with daughter cell NPCs during mitosis to delay cell cycle entry (Start). Hos3-dependent deacetylation of nuclear basket and central channel nucleoporins establishes daughter-cell-specific nuclear accumulation of the transcriptional repressor Whi5 during anaphase and perinuclear silencing of the G1/S cyclin gene CLN2 in the following G1 phase. Hos3-dependent coordination of both events restrains Start in daughter, but not in mother, cells. We propose that deacetylation modulates transport-dependent and transport-independent functions of NPCs, leading to differential cell cycle progression in mother and daughter cells. Similar mechanisms might regulate NPC functions in specific cell types and/or cell cycle stages in multicellular organisms.Kumar et al. discover a pathway that regulates asymmetric cell cycle entry in budding yeast through Hos3-mediated deacetylation of nucleoporins in daughter cells, which affects the localization of the cell cycle regulators Whi5 and Cln2.

Time-Lapse Fluorescence Microscopy of Budding Yeast Cells.

The discovery of green fluorescent protein (GFP) allowed visualization of a wide variety of processes within living cells. Thanks to the development of differently colored fluorophores, it is now possible to simultaneously follow distinct subcellular events at the single cell level. Here, we describe a basic method to visualize multiple events during cytokinesis by time-lapse fluorescence microscopy in the budding yeast Saccharomyces cerevisiae. In this organism, contraction of an actomyosin-based ring drives ingression of the plasma membrane at the mother-bud division site to partition the cytoplasm of the dividing cell. Simultaneous visualization of distinct cytokinesis steps in living cells, such as ring contraction and membrane ingression, will facilitate a complete understanding of the mechanisms of eukaryotic cell division.

Distinct roles of the polarity factors Boi1 and Boi2 in the control of exocytosis and abscission in budding yeast

The budding yeast cortical proteins Boi1 and Boi2 were previously implicated in polarized growth and in inhibition of cytokinesis as part of the NoCut abscission checkpoint. This report establishes an essential role of Boi1 and Boi2 in vesicle exocytosis during bud growth, whereas Boi2 (but not Boi1) is required for abscission inhibition.

DNA Replication Stress: NoCut to the rescue

It is well known that checkpoint systems operate in the cell to inhibit progression through mitosis until errors detected in previous stages have been properly completed. Less is known, however, about mechanisms protecting cells against errors in chromosome segregation after anaphase has begun. Yet late segregation errors, such as lagging chromosomes and anaphase bridges, are not rare in normal conditions, and are frequent in tumors. Late-segregating DNA is thus exposed to potential damage by the cell division machinery. How do cells react to such a threat? We previously proposed that in budding yeast, an Aurora-Bdependent monitoring system, termed the NoCut checkpoint, responds to late DNA segregation errors by inhibiting completion of cytokinesis until chromatin is cleared from the cleavage plane. An Aurora-B-dependent abscission checkpoint homologous to NoCut was subsequently identified in human cells. However, it remained unclear under what physiological conditions NoCut is activated, and equally importantly, under what conditions the checkpoint prevents DNA bridge breakage. In a recent study, we determined how budding yeast cells respond to various types of chromatin bridges. Using time-lapse fluorescence microscopy and electron tomography, we found that the Aurora-B-dependent NoCut checkpoint delays abscission in response to chromatin bridges caused by inactivation of condensin and Topoisomerase II function. However, DNA breaks eventually occur in these 2 cases, probably linked to the fact that bridges cannot be resolved in the absence of condensin or Topoisomerase II, which are essential for chromosome segregation. This raised the question of whether the NoCut checkpoint can prevent DNA damage during cytokinesis. We found that in the presence of bridges caused by DNA replication stress after exposure to hydroxyurea, wild type cells delay abscission in a manner dependent on Aurora B, and that in this case NoCut does prevent DNA damage and ensures cell survival. We identified 2 essential requirements for abscission inhibition in response to DNA bridges. Firstly, Aurora-B kinase must be active and associated with microtubules of the spindle midzone during cytokinesis. These findings suggest that Aurora B acts as a sensor monitoring the presence of chromatin around the spindle midzone. Secondly, anaphase spindles must be stabilized during cytokinesis to allow NoCut function. Indeed, under normal conditions anaphase spindles disassemble before completion of cytokinesis, but we observed a delay in spindle disassembly in cells with chromatin bridges. Specifically, spindle factors that are normally degraded at the end of mitosis by the Anaphase Promoting Complex (APC) are stabilized in cells with catenated, decondensed, and replication stress-induced anaphase bridges. Spindle stabilization is essential for NoCut, because treatment of anaphase cells with the microtubule depolymerizing drug nocodazole allows completion of cytokinesis in the presence of catenated DNA bridges. These findings suggest that after DNA replication stress and/or defects in chromatin condensation and decatenation, degradation of spindle-stabilizing factors is delayed, thereby stabilizing the spindle during cytokinesis and allowing for detection of lagging chromatin by Aurora B at the spindle midzone. This in turn delays abscission and prolongs the lifetime of the chromatin bridge, allowing time for its final resolution (Fig. 1). Strikingly, a particular type of chromatin bridge failed to inhibit abscission. We found that activation of a conditionally dicentric chromosome led to dicentric bridges across the cell division plane, yet abscission proceeded normally. What is the basis for this differential response to chromatin bridges? Notably, spindle stabilization during cytokinesis did not occur in cells with dicentric bridges, offering a potential explanation for their lack of abscission defect. Indeed, impairment of the APC activator Cdh1 delayed spindle disassembly and restored NoCut function in the presence of dicentric bridges. These data suggest that inhibition of APC-dependent spindle disassembly might be required for efficient detection of chromatin bridges by Aurora B. We hypothesize that a chromatin-based damage signal is essential for the NoCut response upstream of Aurora B. The signal might be common to replication stress, and to condensation and decatenation defects; and be absent from the dicentric bridge due to its normal replication, condensation and decatenation. Whatever its nature, this signal would