-
ELife Apr 2023The trapping of Poly-ADP-ribose polymerase (PARP) on DNA caused by PARP inhibitors (PARPi) triggers acute DNA replication stress and synthetic lethality (SL) in...
The trapping of Poly-ADP-ribose polymerase (PARP) on DNA caused by PARP inhibitors (PARPi) triggers acute DNA replication stress and synthetic lethality (SL) in BRCA2-deficient cells. Hence, DNA damage is accepted as a prerequisite for SL in BRCA2-deficient cells. In contrast, here we show that inhibiting ROCK in BRCA2-deficient cells triggers SL independently from acute replication stress. Such SL is preceded by polyploidy and binucleation resulting from cytokinesis failure. Such initial mitosis abnormalities are followed by other M phase defects, including anaphase bridges and abnormal mitotic figures associated with multipolar spindles, supernumerary centrosomes and multinucleation. SL was also triggered by inhibiting Citron Rho-interacting kinase, another enzyme that, similarly to ROCK, regulates cytokinesis. Together, these observations demonstrate that cytokinesis failure triggers mitotic abnormalities and SL in BRCA2-deficient cells. Furthermore, the prevention of mitotic entry by depletion of Early mitotic inhibitor 1 (EMI1) augmented the survival of BRCA2-deficient cells treated with ROCK inhibitors, thus reinforcing the association between M phase and cell death in BRCA2-deficient cells. This novel SL differs from the one triggered by PARPi and uncovers mitosis as an Achilles heel of BRCA2-deficient cells.
Topics: Anaphase; DNA Damage; Mitosis; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Synthetic Lethal Mutations; rho-Associated Kinases; BRCA2 Protein; Humans
PubMed: 37073955
DOI: 10.7554/eLife.80254 -
The FEBS Journal Jul 2015In mitotically dividing cells, the cohesin complex tethers sister chromatids, the products of DNA replication, together from the time they are generated during S phase... (Review)
Review
In mitotically dividing cells, the cohesin complex tethers sister chromatids, the products of DNA replication, together from the time they are generated during S phase until anaphase. Cohesion between sister chromatids ensures accurate chromosome segregation, and promotes normal gene regulation and certain kinds of DNA repair. In somatic cells, the core cohesin complex is composed of four subunits: Smc1, Smc3, Rad21 and an SA subunit. During meiotic cell divisions meiosis-specific isoforms of several of the cohesin subunits are also expressed and incorporated into distinct meiotic cohesin complexes. The relative contributions of these meiosis-specific forms of cohesin to chromosome dynamics during meiotic progression have not been fully worked out. However, the localization of these proteins during chromosome pairing and synapsis, and their unique loss-of-function phenotypes, suggest non-overlapping roles in controlling meiotic chromosome behavior. Many of the proteins that regulate cohesin function during mitosis also appear to regulate cohesin during meiosis. Here we review how cohesin contributes to meiotic chromosome dynamics, and explore similarities and differences between cohesin regulation during the mitotic cell cycle and meiotic progression. A deeper understanding of the regulation and function of cohesin in meiosis will provide important new insights into how the cohesin complex is able to promote distinct kinds of chromosome interactions under diverse conditions.
Topics: Carrier Proteins; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; DNA Damage; DNA-Binding Proteins; Female; Humans; Male; Meiosis; Nuclear Proteins; Phosphoproteins; Phosphorylation; Proto-Oncogene Proteins; Sex Characteristics; Cohesins
PubMed: 25895170
DOI: 10.1111/febs.13301 -
Journal of Cell Science May 2022Proper mitotic spindle orientation depends on the correct anchorage of astral microtubules to the cortex. It relies on the remodeling of the cell cortex, a process not...
Proper mitotic spindle orientation depends on the correct anchorage of astral microtubules to the cortex. It relies on the remodeling of the cell cortex, a process not fully understood. Annexin A2 (Anx2; also known as ANXA2) is a protein known to be involved in cortical domain remodeling. Here, we report that in HeLa cell early mitosis, Anx2 recruits the scaffold protein Ahnak at the cell cortex facing spindle poles, and the distribution of both proteins is controlled by cell adhesion. Depletion of either protein or impaired cortical Ahnak localization result in delayed anaphase onset and unstable spindle anchoring, which leads to altered spindle orientation. We find that Ahnak is present in a complex with dynein-dynactin. Furthermore, Ahnak and Anx2 are required for correct dynein and NuMA (also known as NUMA1) cortical localization and dynamics. We propose that the Ahnak-Anx2 complex influences the cortical organization of the astral microtubule-anchoring complex, and thereby mitotic spindle positioning in human cells. This article has an associated First Person interview with the first author of the paper.
Topics: Anaphase; Annexin A2; Cell Cycle Proteins; Dynactin Complex; Dyneins; HeLa Cells; Humans; Membrane Proteins; Microtubule-Associated Proteins; Microtubules; Mitosis; Neoplasm Proteins; Spindle Apparatus
PubMed: 35362526
DOI: 10.1242/jcs.259344 -
Aging Aug 2020The Anaphase Promoting Complex (APC), a multi-subunit ubiquitin ligase, facilitates mitotic and G1 progression, and is now recognized to play a role in maintaining... (Review)
Review
The Anaphase Promoting Complex (APC), a multi-subunit ubiquitin ligase, facilitates mitotic and G1 progression, and is now recognized to play a role in maintaining genomic stability. Many APC substrates have been observed overexpressed in multiple cancer types, such as CDC20, the Aurora A and B kinases, and Forkhead box M1 (FOXM1), suggesting APC activity is important for cell health. We performed BioGRID analyses of the APC coactivators CDC20 and CDH1, which revealed that at least 69 proteins serve as APC substrates, with 60 of them identified as playing a role in tumor promotion and 9 involved in tumor suppression. While these substrates and their association with malignancies have been studied in isolation, the possibility exists that generalized APC dysfunction could result in the inappropriate stabilization of multiple APC targets, thereby changing tumor behavior and treatment responsiveness. It is also possible that the APC itself plays a crucial role in tumorigenesis through its regulation of mitotic progression. In this review the connections between APC activity and dysregulation will be discussed with regards to cell cycle dysfunction and chromosome instability in cancer, along with the individual roles that the accumulation of various APC substrates may play in cancer progression.
Topics: Anaphase-Promoting Complex-Cyclosome; Animals; Cell Cycle; Cell Transformation, Neoplastic; Chromosomal Instability; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Mitosis; Neoplasms; Signal Transduction; Substrate Specificity
PubMed: 32805721
DOI: 10.18632/aging.103792 -
Methods in Molecular Biology (Clifton,... 2016The anaphase-promoting complex/cyclosome (APC/C) is a 1.2 MDa ubiquitin ligase complex with important functions in both proliferating and post-mitotic differentiated...
The anaphase-promoting complex/cyclosome (APC/C) is a 1.2 MDa ubiquitin ligase complex with important functions in both proliferating and post-mitotic differentiated cells. In proliferating cells, APC/C controls cell cycle progression by targeting inhibitors of chromosome segregation and mitotic exit for degradation by the 26S proteasome. To understand how APC/C recruits and ubiquitylates its substrate proteins and how these processes are controlled, it is essential to analyze APC/C activity in vitro. In the past, such experiments have been limited by the fact that large quantities of purified APC/C were difficult to obtain and that mutated versions of the APC/C could not be easily generated. In this chapter we review recent advances in generating and purifying recombinant forms of the human APC/C and its co-activators, using methods that are scalable and compatible with mutagenesis. We also describe a method that allows the quantitative analysis of APC/C activity using fluorescently labeled substrate proteins.
Topics: Anaphase-Promoting Complex-Cyclosome; Animals; Cdc20 Proteins; Cdh1 Proteins; Cyclin B; Humans; Recombinant Proteins; Sf9 Cells; Spodoptera; Ubiquitin; Ubiquitin-Activating Enzymes; Ubiquitin-Conjugating Enzymes; Ubiquitination
PubMed: 26254932
DOI: 10.1007/978-1-4939-2957-3_18 -
Communications Biology Jan 2023Profilin 1-encoded by PFN1-is a small actin-binding protein with a tumour suppressive role in various adenocarcinomas and pagetic osteosarcomas. However, its...
Profilin 1-encoded by PFN1-is a small actin-binding protein with a tumour suppressive role in various adenocarcinomas and pagetic osteosarcomas. However, its contribution to tumour development is not fully understood. Using fix and live cell imaging, we report that Profilin 1 inactivation results in multiple mitotic defects, manifested prominently by anaphase bridges, multipolar spindles, misaligned and lagging chromosomes, and cytokinesis failures. Accordingly, next-generation sequencing technologies highlighted that Profilin 1 knock-out cells display extensive copy-number alterations, which are associated with complex genome rearrangements and chromothripsis events in primary pagetic osteosarcomas with Profilin 1 inactivation. Mechanistically, we show that Profilin 1 is recruited to the spindle midzone at anaphase, and its deficiency reduces the supply of actin filaments to the cleavage furrow during cytokinesis. The mitotic defects are also observed in mouse embryonic fibroblasts and mesenchymal cells deriving from a newly generated knock-in mouse model harbouring a Pfn1 loss-of-function mutation. Furthermore, nuclear atypia is also detected in histological sections of mutant femurs. Thus, our results indicate that Profilin 1 has a role in regulating cell division, and its inactivation triggers mitotic defects, one of the major mechanisms through which tumour cells acquire chromosomal instability.
Topics: Animals; Humans; Mice; Anaphase; Cytokinesis; Fibroblasts; Genomic Instability; Mitosis; Profilins; Osteosarcoma
PubMed: 36599901
DOI: 10.1038/s42003-022-04392-8 -
Molecular & Cellular Oncology 2021At the metaphase-to-anaphase transition, phosphatase activity feeds back to reverse early mitotic phosphorylation events. Our recent work indicates that the pseudokinase...
At the metaphase-to-anaphase transition, phosphatase activity feeds back to reverse early mitotic phosphorylation events. Our recent work indicates that the pseudokinase domain of the spindle checkpoint protein BUB1 (Budding Uninhibited by Benzimidazoles 1) mitotic checkpoint serine/threonine kinase B (BUB1B, BUBR1) maintains kinase-phosphatase balance at the outer kinetochore during mitotic exit.
PubMed: 33860079
DOI: 10.1080/23723556.2021.1876511 -
Chromosome Research : An International... Jul 2023Micronuclei, small DNA-containing structures separate from the main nucleus, were used for decades as an indicator of genotoxic damage. Micronuclei containing whole... (Review)
Review
Micronuclei, small DNA-containing structures separate from the main nucleus, were used for decades as an indicator of genotoxic damage. Micronuclei containing whole chromosomes were considered a biomarker of aneuploidy and were believed to form, upon mitotic exit, from chromosomes that lagged behind in anaphase as all other chromosomes segregated to the poles of the mitotic spindle. However, the mechanism responsible for inducing anaphase lagging chromosomes remained unknown until just over twenty years ago. Here, I summarize what preceded and what followed this discovery, highlighting some of the open questions and opportunities for future investigation.
Topics: Humans; Kinetochores; Mitosis; Spindle Apparatus; Aneuploidy; Chromosome Segregation; Microtubules
PubMed: 37466740
DOI: 10.1007/s10577-023-09727-7 -
Frontiers in Cell and Developmental... 2016GTPases of the RAB family are key regulators of multiple steps of membrane trafficking. Several members of the RAB GTPase family have been implicated in mitotic... (Review)
Review
GTPases of the RAB family are key regulators of multiple steps of membrane trafficking. Several members of the RAB GTPase family have been implicated in mitotic progression. In this review, we will first focus on the function of endosome-associated RAB GTPases reported in early steps of mitosis, spindle pole maturation, and during cytokinesis. Second, we will discuss the role of Golgi-associated RAB GTPases at the metaphase/anaphase transition and during cytokinesis.
PubMed: 26925400
DOI: 10.3389/fcell.2016.00002 -
Cell Biochemistry and Function Jun 2020ZW10 interactor (Zwint-1) is an important component of the centromere and can recruit the dynamic protein kinase and dynein to promote chromosome movement and regulate...
ZW10 interactor (Zwint-1) is an important component of the centromere and can recruit the dynamic protein kinase and dynein to promote chromosome movement and regulate the spindle assembly checkpoint (SAC). Zwint-1 activity is tightly regulated during the cell cycle. However, how the stability of Zwint-1 is regulated has not been clarified. Here, we show that the relative levels of Zwint-1 expression gradually decreased with the progression of cell cycling and decline sharply during mitotic exit. Treatment with cycloheximide reduced the levels of Zwint-1 while treatment with MG132 to inhibit endogenous ubiquitin-proteasome elevated the levels of Zwint-1 in HEK293T cells or Hela cells. Such data suggest that Zwint-1 may be degraded by endogenous ubiquitin-proteasome. Furthermore, induction of cell-division cycle protein 20 (Cdc20) overexpression decreased the levels of Zwint-1, which was abrogated by MG132 treatment. In contrast, Cdc20 silencing promoted the accumulation of Zwint-1. in vivo ubiquitination assay revealed that Cdc20 promoted the formation of Zwint-1 and ubiquitin-proteasome conjugates. Cotransfection with Cdc20 and wild-type Zwint-1, but not Zwint-1 , reduced the levels of Zwint-1. Immunoprecipitation and western blot analyses showed that Cdc20 interacted with wild-type Zwint-1, but not Zwint-1 although both Zwint-1 and Zwint-1 overexpression did not induce mitotic arrest. Collectively, our data indicated that Zwint-1 was ubiquitinated by anaphase-promoting complex/cyclosome (APC/C)-Cdc20 in a D-box-dependent manner. Therefore, the APC/C-Cdc20 controls the stability of Zwint-1, ensuring accurate regulation of the spindle assembly during the cell cycling in HEK293T cells.
Topics: Anaphase-Promoting Complex-Cyclosome; Cdc20 Proteins; HEK293 Cells; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Nuclear Proteins; Proteasome Endopeptidase Complex; Proteolysis; Ubiquitin
PubMed: 31945194
DOI: 10.1002/cbf.3499