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Cytoskeleton (Hoboken, N.J.) 2023Accurate placement of the cleavage furrow is crucial for successful cell division. Recent advancements have revealed that diverse mechanisms have evolved across...
Accurate placement of the cleavage furrow is crucial for successful cell division. Recent advancements have revealed that diverse mechanisms have evolved across different branches of the phylogenetic tree. Here, we employed Dictyostelium cells to validate previous models. We observed that during metaphase and early anaphase, mitotic spindles exhibited random rotary movements which ceased when the spindle elongated by approximately 7 μm. At this point, astral microtubules reached the polar cell cortex and fixed the spindle axis, causing cells to elongate by extending polar pseudopods and divide along the spindle axis. Therefore, the position of the furrow is determined when the spindle orientation is fixed. The distal ends of astral microtubules stimulate the extension of pseudopods at the polar cortex. One signal for pseudopod extension may be phosphatidylinositol trisphosphate in the cell membrane, but there appears to be another unknown signal. At the onset of polar pseudopod extension, cortical flow began from both poles toward the equator. We suggest that polar stimulation by astral microtubules determines the furrow position, induces polar pseudopod extension and cortical flow, and accumulates the elements necessary for the construction of the contractile ring.
Topics: Dictyostelium; Phylogeny; Cytokinesis; Microtubules; Spindle Apparatus; Anaphase
PubMed: 37650534
DOI: 10.1002/cm.21784 -
Frontiers in Genetics 2022To sustain genomic stability by correct DNA replication and mitosis, cell cycle progression is tightly controlled by the cyclic activity of cyclin-dependent kinases,... (Review)
Review
To sustain genomic stability by correct DNA replication and mitosis, cell cycle progression is tightly controlled by the cyclic activity of cyclin-dependent kinases, their binding to cyclins in the respective phase and the regulation of cyclin levels by ubiquitin-dependent proteolysis. The spindle assembly checkpoint plays an important role at the metaphase-anaphase transition to ensure a correct separation of sister chromatids before cytokinesis and to initiate mitotic exit, as an incorrect chromosome distribution may lead to genetically unstable cells and tumorigenesis. The ubiquitin ligase anaphase-promoting complex or cyclosome (APC/C) is essential for these processes by mediating the proteasomal destruction of cyclins and other important cell cycle regulators. To this end, it interacts with the two regulatory subunits Cdh1 and Cdc20. Both play a role in tumorigenesis with Cdh1 being a tumor suppressor and Cdc20 an oncogene. In this review, we summarize the current knowledge about the APC/C-regulators Cdh1 and Cdc20 in tumorigenesis and potential targeted therapeutic approaches.
PubMed: 35832196
DOI: 10.3389/fgene.2022.941565 -
Life Science Alliance Oct 2022Histone acetylation levels are reduced during mitosis. To study the mitotic regulation of H3K9ac, we used an array of inhibitors targeting specific histone deacetylases....
Histone acetylation levels are reduced during mitosis. To study the mitotic regulation of H3K9ac, we used an array of inhibitors targeting specific histone deacetylases. We evaluated the involvement of the targeted enzymes in regulating H3K9ac during all mitotic stages by immunofluorescence and immunoblots. We identified HDAC2, HDAC3, and SIRT1 as modulators of H3K9ac mitotic levels. HDAC2 inhibition increased H3K9ac levels in prophase, whereas HDAC3 or SIRT1 inhibition increased H3K9ac levels in metaphase. Next, we performed ChIP-seq on mitotic-arrested cells following targeted inhibition of these histone deacetylases. We found that both HDAC2 and HDAC3 have a similar impact on H3K9ac, and inhibiting either of these two HDACs substantially increases the levels of this histone acetylation in promoters, enhancers, and insulators. Altogether, our results support a model in which H3K9 deacetylation is a stepwise process-at prophase, HDAC2 modulates most transcription-associated H3K9ac-marked loci, and at metaphase, HDAC3 maintains the reduced acetylation, whereas SIRT1 potentially regulates H3K9ac by impacting HAT activity.
Topics: Acetylation; Histones; Mitosis; Protein Processing, Post-Translational; Sirtuin 1
PubMed: 35981887
DOI: 10.26508/lsa.202201433 -
Seminars in Cell & Developmental Biology Sep 2021The ultimate goal of cell division is to generate two identical daughter cells that resemble the mother cell from which they derived. Once all the proper attachments to... (Review)
Review
The ultimate goal of cell division is to generate two identical daughter cells that resemble the mother cell from which they derived. Once all the proper attachments to the spindle have occurred, the chromosomes have aligned at the metaphase plate and the spindle assembly checkpoint (a surveillance mechanism that halts cells form progressing in the cell cycle in case of spindle - microtubule attachment errors) has been satisfied, mitotic exit will occur. Mitotic exit has the purpose of completing the separation of the genomic material but also to rebuild the cellular structures necessary for the new cell cycle. This stage of mitosis received little attention until a decade ago, therefore our knowledge is much patchier than the molecular details we now have for the early stages of mitosis. However, it is emerging that mitotic exit is not just the simple reverse of mitotic entry and it is highly regulated in space and time. In this review I will discuss the main advances in the field that provided us with a better understanding on the key role of protein phosphorylation/de-phosphorylation in this transition together with the concept of their spatial regulation. As this field is much younger, I will highlight general consensus, contrasting views together with the outstanding questions awaiting for answers.
Topics: Chromosome Segregation; Humans; Mitosis
PubMed: 33810980
DOI: 10.1016/j.semcdb.2021.03.010 -
Frontiers in Cell and Developmental... 2023Accurate chromosome segregation is vital for cell and organismal viability. The mitotic spindle, a bipolar macromolecular machine composed largely of dynamic... (Review)
Review
Accurate chromosome segregation is vital for cell and organismal viability. The mitotic spindle, a bipolar macromolecular machine composed largely of dynamic microtubules, is responsible for chromosome segregation during each cell replication cycle. Prior to anaphase, a bipolar metaphase spindle must be formed in which each pair of chromatids is attached to microtubules from opposite spindle poles. In this bipolar configuration pulling forces from the dynamic microtubules can generate tension across the sister kinetochores. The tension status acts as a signal that can destabilize aberrant kinetochore-microtubule attachments and reinforces correct, bipolar connections. Historically it has been challenging to isolate the specific role of tension in mitotic processes due to the interdependency of attachment and tension status at kinetochores. Recent technical and experimental advances have revealed new insights into how tension functions during mitosis. Here we summarize the evidence that tension serves as a biophysical signal that unifies multiple aspects of kinetochore and centromere function to ensure accurate chromosome segregation.
PubMed: 36755973
DOI: 10.3389/fcell.2023.1096333 -
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 -
PLoS Biology Jul 2022Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division...
Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and nondividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the centrosomes, the bipolar spindle, the spindle midzone, the central spindle, and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and orientation, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the centrosomes and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provide new insight into nonciliary defects that underlie ciliopathies.
Topics: Anaphase; Centrosome; Ciliopathies; Cytokinesis; Eye Proteins; Humans; Microtubule-Associated Proteins; Microtubules; Mitosis; Spindle Apparatus
PubMed: 35849559
DOI: 10.1371/journal.pbio.3001708 -
Cytogenetic and Genome Research 2019An experimental approach using monoclonal anti-5-methylcytosine antibodies and indirect immunofluorescence was elaborated for detecting 5-methylcytosine-rich chromosome...
An experimental approach using monoclonal anti-5-methylcytosine antibodies and indirect immunofluorescence was elaborated for detecting 5-methylcytosine-rich chromosome regions in reptilian chromosomes. This technique was applied to conventionally prepared mitotic metaphases of 2 turtle species and 12 squamate species from 8 families. The hypermethylation patterns were compared with C-banding patterns obtained by conventional banding techniques. The hypermethylated DNA sequences are species-specific and are located in constitutive heterochromatin. They are highly reproducible and often found in centromeric, pericentromeric, and interstitial positions of the chromosomes. Heterochromatic regions in differentiated sex chromosomes are particularly hypermethylated.
Topics: 5-Methylcytosine; Animals; Centromere; Chromosomes; DNA Methylation; Heterochromatin; Karyotype; Karyotyping; Male; Reptiles; Sex Chromosomes; Species Specificity
PubMed: 30641524
DOI: 10.1159/000495893 -
Methods in Enzymology 2018Our conventional understanding of the process of DNA replication is that it occurs in the S-phase of the cell division cycle. However, during investigations into the... (Review)
Review
Our conventional understanding of the process of DNA replication is that it occurs in the S-phase of the cell division cycle. However, during investigations into the mechanism by which common fragile sites (CFSs) drive genome instability, we observed that some DNA synthesis was still occurring in early mitosis at these loci. This curious phenomenon of mitotic DNA synthesis (which we now term "MiDAS") appears to be a form of break-induced DNA replication (BIR), a DNA repair process based on homologous recombination that has been characterized in detail only in lower eukaryotes. During MiDAS, it is proposed that parts of the human genome that are not fully replicated when cells enter mitotic prophase complete their replicative cycle at that point. To date, the loci that most depend upon this process are those whose replication can be affected by oncogene-induced DNA replication stress (RS), most notably, CFSs. From our studies, it is clear that the successful completion of MiDAS at CFSs can minimize chromosome missegregation and nondisjunction. Nevertheless, it is still not clear which loci that can undergo MiDAS, whether MiDAS is associated with mutations or genome rearrangements, or whether MiDAS really is a form of BIR. In this review, we describe methods for detecting MiDAS both in prometaphase cells and directly on isolated metaphase chromosomes. In addition, we have included methods for combining MiDAS detection either with immunofluorescence (IF) detection of proteins that are recruited to the MiDAS loci, or with fluorescence in situ hybridization using probes that target specific genomic loci.
Topics: Cell Line; DNA; Genetic Techniques; Humans; Metaphase; Mitosis; Prometaphase; Recombinational DNA Repair
PubMed: 29523241
DOI: 10.1016/bs.mie.2017.11.025 -
Methods in Molecular Biology (Clifton,... 2023After DNAs are damaged, DNA repair proteins accumulate and are activated at the DNA damaged site. These accumulated proteins are visualized as foci by fluorescent...
After DNAs are damaged, DNA repair proteins accumulate and are activated at the DNA damaged site. These accumulated proteins are visualized as foci by fluorescent immunocytochemistry technique. This allows the DNA damage responses in interphase nuclei to be detected; it was earlier times difficult to analyze DNA damage in situ. In order to analyze DNA damage in interphase cells, either DNA is extracted to assay breaks biochemically, or premature chromosome condensation is conducted to observe as chromatin breaks. Although DNA damage-induced foci are typically analyzed in interphase cells, these foci can be also visualized on mitotic chromosomes. The foci where the repair proteins accumulate at the damage site is observed as mitotic chromosome break site. Since mitotic cells attach loosely or not attached to cell culture vessels, it is difficult to analyze foci on chromosomes in culture vessels under a microscope, so metaphase chromosome spread must be prepared for accurate analysis. The cytocentrifuge system is an ideal method to adhere mitotic cells to microscope slides for the fluorescent immunocytochemistry. This chapter introduces cytocentrifuge method to prepare metaphase spread for DNA damage foci analysis.
Topics: Chromosomes; DNA; DNA Damage; Interphase; Metaphase
PubMed: 36066713
DOI: 10.1007/978-1-0716-2433-3_10