Separase Protease Activity is Required for Cytokinesis in addition to Chromosome Segregation

2016 ◽  
Author(s):  
Xiaofei Bai ◽  
Joshua N. Bembenek
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Protease Activity ◽  
Vesicle Trafficking ◽  
Cortical Granule ◽  
Daughter Cells ◽  
Sister Chromatids ◽  
Vesicle Exocytosis ◽  
Substrate Cleavage ◽  
Cortical Granule Exocytosis

AbstractChromosomal segregation and cytokinesis are tightly regulated processes required for successful cell division. The cysteine protease separase cleaves a subunit of the cohesin complex to allow chromosome segregation at anaphase onset. Separase also regulates meiotic cortical granule exocytosis and vesicle trafficking during cytokinesis, both of which involve RAB-11. Separase has non-proteolytic signaling functions in addition to its role in substrate cleavage, and its mechanism in exocytosis is unknown. We sought to determine whether separase regulates RAB-11 vesicle exocytosis through a proteolytic or non-proteolytic mechanism. To address this question, we generated a protease-dead separase, SEP-1PD::GFP, and unexpectedly found that it is dominant negative. Consistent with its role in cohesin cleavage, SEP-1PD::GFP causes chromosome segregation defects. Depletion of the substrate subunit of cohesin rescues this defect, suggesting that SEP-1PD::GFP impairs cohesin cleavage by a substrate trapping mechanism. We investigated whether SEP-1PD::GFP also impairs RAB-11 vesicle trafficking. SEP-1PD::GFP causes a low rate of cytokinesis failure that is synergistically exacerbated by depletion of the core exocytic t-SNARE protein SYX-4. Interestingly, SEP-1PD::GFP causes an accumulation of RAB-11 vesicles at the cleavage furrow site and delayed the exocytosis of cortical granules during anaphase I. Depletion of syx-4 further enhanced RAB-11::mCherry and SEP-1PD::GFP plasma membrane accumulation during cytokinesis. These findings suggest that the protease activity of separase is required for the exocytosis of RAB-11 vesicles during cortical granule exocytosis and mitotic cytokinesis.Author SummaryThe defining event of cell division is the equal distribution of the genetic material to daughter cells. Once sister chromatids align on the metaphase plate, the cell releases the brakes to enter anaphase by activating the protease separase. Separase cleaves the cohesin glue holding duplicated sister chromatids together allowing chromosome segregation. Subsequently, the cell must orchestrate a complex series of anaphase events to equally partition the chromatids and the rest of the cellular components into two distinct daughter cells during cytokinesis. Separase has multiple functions during anaphase to help regulate several key events, including promoting vesicle exocytosis required for cytokinesis. Previous studies have shown that separase can exert control over different events either through substrate cleavage, or by triggering signaling pathways. Here we analyze the cellular functions of separase that are impacted by protease inactive separase. Our results show that separase cleaves cohesin to promote chromosome segregation and also cleaves another independent substrate to promote exocytosis. These findings provide a foundation for understanding the molecular control of separase in exocytosis and indicate that separase has multiple independent substrates that it must cleave to execute various functions. This mechanism may enable the cell to coordinate multiple anaphase events with chromosome segregation.

scholarly journals The structural basis for Ulp2 recruitment to the kinetochore

2021 ◽  
Author(s):  
Yun Quan ◽  
Stephen M. Hinshaw ◽  
Pang-Che Wang ◽  
Stephen C. Harrison ◽  
Huilin Zhou
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Chromosome Segregation ◽  
Structural Basis ◽  
Centromeric Dna ◽  
Step Process ◽  
Sumo Protease ◽  
Daughter Cells ◽  
Sister Chromatids ◽  
Spindle Microtubules

ABSTRACTThe step-by-step process of chromosome segregation defines the stages of the cell division cycle. In eukaryotes, signaling pathways that control these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to the centromere of each chromosome. Kinetochores control and adapt to major chromosomal transactions, including replication of centromeric DNA, biorientation of sister centromeres on the metaphase spindle, and transit of sister chromatids into daughter cells during anaphase. Although the mechanisms that ensure tight microtubule coupling at anaphase are at least partly understood, kinetochore adaptations that support other cell cycle transitions are not. We report here a mechanism that enables regulated control of kinetochore sumoylation. A conserved surface of the Ctf3/CENP-I kinetochore protein provides a binding site for the SUMO protease, Ulp2. Ctf3 mutations that disable Ulp2 recruitment cause elevated inner kinetochore sumoylation and defective chromosome segregation. The location of the site within the assembled kinetochore suggests coordination between sumoylation and other cell cycle-regulated processes.

scholarly journals Recombination and chromosome segregation

2004 ◽  
Vol 359 (1441) ◽  
pp. 61-69 ◽  
Author(s):  
David J. Sherratt ◽  
Britta Søballe ◽  
François–Xavier Barre ◽  
Sergio Filipe ◽  
Ivy Lau ◽  
...  
Keyword(s):  
Cell Division ◽  
Homologous Recombination ◽  
Chromosome Segregation ◽  
Atp Hydrolysis ◽  
Daughter Cells ◽  
Recombination Proteins ◽  
Recombination System ◽  
Associated Proteins ◽  
Stalled Replication Forks ◽  
Circular Chromosomes

The duplication of DNA and faithful segregation of newly replicated chromosomes at cell division is frequently dependent on recombinational processes. The rebuilding of broken or stalled replication forks is universally dependent on homologous recombination proteins. In bacteria with circular chromosomes, crossing over by homologous recombination can generate dimeric chromosomes, which cannot be segregated to daughter cells unless they are converted to monomers before cell division by the conserved Xer site–specific recombination system. Dimer resolution also requires FtsK, a division septum–located protein, which coordinates chromosome segregation with cell division, and uses the energy of ATP hydrolysis to activate the dimer resolution reaction. FtsK can also translocate DNA, facilitate synapsis of sister chromosomes and minimize entanglement and catenation of newly replicated sister chromosomes. The visualization of the replication/recombination–associated proteins, RecQ and RarA, and specific genes within living Escherichia coli cells, reveals further aspects of the processes that link replication with recombination, chromosome segregation and cell division, and provides new insight into how these may be coordinated.

scholarly journals Anaphase Bridges: Not All Natural Fibers Are Healthy

Genes ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 902
Author(s):  
Alice Finardi ◽  
Lucia F. Massari ◽  
Rosella Visintin
Keyword(s):  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Potential Role ◽  
Genome Instability ◽  
Natural Fibers ◽  
S Phase ◽  
Dna Lesions ◽  
Anaphase Bridges ◽  
Daughter Cells ◽  
Sister Chromatids

At each round of cell division, the DNA must be correctly duplicated and distributed between the two daughter cells to maintain genome identity. In order to achieve proper chromosome replication and segregation, sister chromatids must be recognized as such and kept together until their separation. This process of cohesion is mainly achieved through proteinaceous linkages of cohesin complexes, which are loaded on the sister chromatids as they are generated during S phase. Cohesion between sister chromatids must be fully removed at anaphase to allow chromosome segregation. Other (non-proteinaceous) sources of cohesion between sister chromatids consist of DNA linkages or sister chromatid intertwines. DNA linkages are a natural consequence of DNA replication, but must be timely resolved before chromosome segregation to avoid the arising of DNA lesions and genome instability, a hallmark of cancer development. As complete resolution of sister chromatid intertwines only occurs during chromosome segregation, it is not clear whether DNA linkages that persist in mitosis are simply an unwanted leftover or whether they have a functional role. In this review, we provide an overview of DNA linkages between sister chromatids, from their origin to their resolution, and we discuss the consequences of a failure in their detection and processing and speculate on their potential role.

scholarly journals Pds5 regulators segregate cohesion and condensation pathways in Saccharomyces cerevisiae

2015 ◽  
Vol 112 (22) ◽  
pp. 7021-7026 ◽  
Author(s):  
Kevin Tong ◽  
Robert V. Skibbens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Temperature Sensitivity ◽  
Mutant Cell ◽  
Important Advance ◽  
Daughter Cells ◽  
Sister Chromatids ◽  
Discrete Structures ◽  
Cell Condensation ◽  
Mutant Cells

Cohesins are required both for the tethering together of sister chromatids (termed cohesion) and subsequent condensation into discrete structures—processes fundamental for faithful chromosome segregation into daughter cells. Differentiating between cohesin roles in cohesion and condensation would provide an important advance in studying chromatin metabolism. Pds5 is a cohesin-associated factor that is essential for both cohesion maintenance and condensation. Recent studies revealed that ELG1 deletion suppresses the temperature sensitivity of pds5 mutant cells. However, the mechanisms through which Elg1 may regulate cohesion and condensation remain unknown. Here, we report that ELG1 deletion from pds5-1 mutant cells results in a significant rescue of cohesion, but not condensation, defects. Based on evidence that Elg1 unloads the DNA replication clamp PCNA from DNA, we tested whether PCNA overexpression would similarly rescue pds5-1 mutant cell cohesion defects. The results indeed reveal that elevated levels of PCNA rescue pds5-1 temperature sensitivity and cohesion defects, but do not rescue pds5-1 mutant cell condensation defects. In contrast, RAD61 deletion rescues the condensation defect, but importantly, neither the temperature sensitivity nor cohesion defects exhibited by pds5-1 mutant cells. In combination, these findings reveal that cohesion and condensation are separable pathways and regulated in nonredundant mechanisms. These results are discussed in terms of a new model through which cohesion and condensation are spatially regulated.

scholarly journals Mechanical Mechanisms of Chromosome Segregation

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 465
Author(s):  
Maya I. Anjur-Dietrich ◽  
Colm P. Kelleher ◽  
Daniel J. Needleman
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Genetic Material ◽  
Evolutionary Genetics ◽  
Force Generation ◽  
Coarse Grained ◽  
Subcellular Structure ◽  
Daughter Cells ◽  
The Past ◽  
The Future

Chromosome segregation—the partitioning of genetic material into two daughter cells—is one of the most crucial processes in cell division. In all Eukaryotes, chromosome segregation is driven by the spindle, a microtubule-based, self-organizing subcellular structure. Extensive research performed over the past 150 years has identified numerous commonalities and contrasts between spindles in different systems. In this review, we use simple coarse-grained models to organize and integrate previous studies of chromosome segregation. We discuss sites of force generation in spindles and fundamental mechanical principles that any understanding of chromosome segregation must be based upon. We argue that conserved sites of force generation may interact differently in different spindles, leading to distinct mechanical mechanisms of chromosome segregation. We suggest experiments to determine which mechanical mechanism is operative in a particular spindle under study. Finally, we propose that combining biophysical experiments, coarse-grained theories, and evolutionary genetics will be a productive approach to enhance our understanding of chromosome segregation in the future.

scholarly journals Chromosome segregation drives division site selection inStreptococcus pneumoniae

2016 ◽  
Author(s):  
Renske van Raaphorst ◽  
Morten Kjos ◽  
Jan-Willem Veening
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Site Selection ◽  
Bacterial Cell Division ◽  
Division Plane ◽  
Canonical Systems ◽  
Daughter Cells ◽  
Division Site ◽  
Additional Protein ◽  
Site Control

AbstractAccurate spatial and temporal positioning of the tubulin-like protein FtsZ is key for proper bacterial cell division.Streptococcus pneumoniae(pneumococcus) is an oval-shaped, symmetrically dividing human pathogen lacking the canonical systems for division site control (nucleoid occlusion and the Min-system). Recently, the early division protein MapZ was identified and implicated in pneumococcal division site selection. We show that MapZ is important for proper division plane selection; thus the question remains what drives pneumococcal division site selection. By mapping the cell cycle in detail, we show that directly after replication both chromosomal origin regions localize to the future cell division sites, prior to FtsZ. Perturbing the longitudinal chromosomal organization by mutating the condensin SMC, by CRISPR/Cas9-mediated chromosome cutting or by poisoning DNA decatenation resulted in mistiming of MapZ and FtsZ positioning and subsequent cell elongation. Together, we demonstrate an intimate relationship between DNA replication, chromosome segregation and division site selection in the pneumococcus, providing a simple way to ensure equally sized daughter cells without the necessity for additional protein factors.

scholarly journals Dynamics of Escherichia coli Chromosome Segregation during Multifork Replication

2007 ◽  
Vol 189 (23) ◽  
pp. 8660-8666 ◽  
Author(s):  
Henrik J. Nielsen ◽  
Brenda Youngren ◽  
Flemming G. Hansen ◽  
Stuart Austin
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Growth Rate ◽  
Cell Division ◽  
Chromosome Segregation ◽  
Replication Forks ◽  
Daughter Cells ◽  
Initiation Of Replication ◽  
Replication Terminus ◽  
Multiple Replication

ABSTRACT Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division, the chromosomes contain multiple replication forks and must be segregated while this complex pattern of replication is still ongoing. Here, we show that replication and segregation continue in step, starting at the origin and progressing to the replication terminus. Thus, early-replicated markers on the multiple-branched chromosomes continue to separate soon after replication to form separate protonucleoids, even though they are not segregated into different daughter cells until later generations. The segregation pattern follows the pattern of chromosome replication and does not follow the cell division cycle. No extensive cohesion of sister DNA regions was seen at any growth rate. We conclude that segregation is driven by the progression of the replication forks.

scholarly journals The Saccharomyces cerevisiae Centromere Protein Slk19p Is Required for Two Successive Divisions During Meiosis

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 577-587 ◽  
Author(s):  
Xuemei Zeng ◽  
William S Saunders
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Chromosome Segregation ◽  
Meiotic Cell ◽  
Centromere Region ◽  
Sister Chromatids ◽  
Centromere Protein ◽  
Diploid Cells ◽  
Meiosis I

Abstract Meiotic cell division includes two separate and distinct types of chromosome segregation. In the first segregational event the sister chromatids remain attached at the centromere; in the second the chromatids are separated. The factors that control the order of chromosome segregation during meiosis have not yet been identified but are thought to be confined to the centromere region. We showed that the centromere protein Slk19p is required for the proper execution of meiosis in Saccharomyces cerevisiae. In its absence diploid cells skip meiosis I and execute meiosis II division. Inhibiting recombination does not correct this phenotype. Surprisingly, the initiation of recombination is apparently required for meiosis II division. Thus Slk19p appears to be part of the mechanism by which the centromere controls the order of meiotic divisions.

scholarly journals Pericentromere tension is self-regulated by spindle structure in metaphase

2014 ◽  
Vol 205 (3) ◽  
pp. 313-324 ◽  
Author(s):  
Jeremy M. Chacón ◽  
Soumya Mukherjee ◽  
Breanna M. Schuster ◽  
Duncan J. Clarke ◽  
Melissa K. Gardner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Chromosome Structure ◽  
Wild Type ◽  
Checkpoint Signaling ◽  
Daughter Cells ◽  
Fluorescent Markers ◽  
Spindle Structure

During cell division, a mitotic spindle is built by the cell and acts to align and stretch duplicated sister chromosomes before their ultimate segregation into daughter cells. Stretching of the pericentromeric chromatin during metaphase is thought to generate a tension-based signal that promotes proper chromosome segregation. However, it is not known whether the mitotic spindle actively maintains a set point tension magnitude for properly attached sister chromosomes to facilitate robust mechanochemical checkpoint signaling. By imaging and tracking the thermal movements of pericentromeric fluorescent markers in Saccharomyces cerevisiae, we measured pericentromere stiffness and then used the stiffness measurements to quantitatively evaluate the tension generated by pericentromere stretch during metaphase in wild-type cells and in mutants with disrupted chromosome structure. We found that pericentromere tension in yeast is substantial (4–6 pN) and is tightly self-regulated by the mitotic spindle: through adjustments in spindle structure, the cell maintains wild-type tension magnitudes even when pericentromere stiffness is disrupted.

scholarly journals An unbiased, quantitative and versatile method for determining misaligned and lagging chromosome during mitosis

2020 ◽  
Author(s):  
Luciano Gama Braga ◽  
Diogjena Katerina Prifti ◽  
Chantal Garand ◽  
Pawan Kumar Saini ◽  
Sabine Elowe
Keyword(s):  
Chromosome Segregation ◽  
Hallmarks Of Cancer ◽  
Analytical Geometry ◽  
Daughter Cells ◽  
Chromosome Missegregation ◽  
Microtubule Attachment ◽  
Sister Chromatids ◽  
Chromosome Congression ◽  
Chromosome Alignment ◽  
Lagging Chromosome

ABSTRACTAccurate chromosome alignment at metaphase facilitates the equal segregation of sister chromatids to each of the nascent daughter cells. Lack of proper metaphase alignment is an indicator of defective chromosome congression and aberrant kinetochore-microtubule attachments which in turn promotes chromosome missegregation and aneuploidy, hallmarks of cancer. Therefore, tools to sensitively and quantitatively measure chromosome alignment at metaphase will facilitate understanding of how changes in the composition and regulation of the microtubule attachment machinery impinge on this process. In this work, we have developed and validated a method based on analytical geometry to measure several indicators of chromosome misalignment. We generated semi-automated and flexible ImageJ2/Fiji pipelines to quantify kinetochore misalignment at metaphase plates as well as lagging chromosomes at anaphase. These tools will ultimately allow sensitive, unbiased, and systematic quantitation of these chromosome segregation defects in cells undergoing mitosis.

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