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BioRxiv : the Preprint Server For... May 2024The eukaryotic cell division machinery must rapidly and reproducibly duplicate and partition the cell's chromosomes in a carefully coordinated process. However,...
The eukaryotic cell division machinery must rapidly and reproducibly duplicate and partition the cell's chromosomes in a carefully coordinated process. However, chromosome number varies dramatically between genomes, even on short evolutionary timescales. We sought to understand how the mitotic machinery senses and responds to karyotypic changes by using a series of budding yeast strains in which the native chromosomes have been successively fused. Using a combination of cell biological profiling, genetic engineering, and experimental evolution, we show that chromosome fusions are well tolerated up until a critical point. Cells with fewer than five centromeres lack the necessary number of kinetochore-microtubule attachments needed to counter outward forces in the metaphase spindle, triggering the spindle assembly checkpoint and prolonging metaphase. Our findings demonstrate that spindle architecture is a constraining factor for karyotype evolution.
PubMed: 37961714
DOI: 10.1101/2023.10.25.563899 -
Science Advances Mar 2024Canonical mitotic and meiotic cell divisions commence with replicated chromosomes consisting of two sister chromatids. Here, we developed and explored a model of...
Canonical mitotic and meiotic cell divisions commence with replicated chromosomes consisting of two sister chromatids. Here, we developed and explored a model of premature cell division, where nonreplicated, G/G-stage somatic cell nuclei are transplanted to the metaphase cytoplasm of mouse oocytes. Subsequent cell division generates daughter cells with reduced ploidy. Unexpectedly, genome sequencing analysis revealed proper segregation of homologous chromosomes, resulting in complete haploid genomes. We observed a high occurrence of somatic genome haploidization in nuclei from inbred genetic backgrounds but not in hybrids, emphasizing the importance of sequence homology between homologs. These findings suggest that premature cell division relies on mechanisms similar to meiosis I, where genome haploidization is facilitated by homologous chromosome interactions, recognition, and pairing. Unlike meiosis, no evidence of recombination between somatic cell homologs was detected. Our study offers an alternative in vitro gametogenesis approach by directly reprogramming diploid somatic cells into haploid oocytes.
Topics: Animals; Mice; Haploidy; Diploidy; Meiosis; Cell Nucleus; Chromatids
PubMed: 38457500
DOI: 10.1126/sciadv.adk9001 -
Stem Cell Research & Therapy Dec 2023Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in...
BACKGROUND
Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications.
METHODS
After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined.
RESULTS
Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype.
CONCLUSION
This manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.
Topics: Humans; Stem Cells; Limbal Stem Cells; Limbus Corneae; Epithelium, Corneal; Spindle Apparatus; Endotoxins
PubMed: 38093301
DOI: 10.1186/s13287-023-03586-z -
BioRxiv : the Preprint Server For... Apr 2024Accurate positioning of the mitotic spindle within the rounded cell body is critical to physiological maintenance. Adherent mitotic cells encounter confinement from...
Accurate positioning of the mitotic spindle within the rounded cell body is critical to physiological maintenance. Adherent mitotic cells encounter confinement from neighboring cells or the extracellular matrix (ECM), which can cause rotation of mitotic spindles and, consequently, titling of the metaphase plate (MP). To understand the positioning and orientation of mitotic spindles under confinement by fibers (ECM-confinement), we use flexible ECM-mimicking nanofibers that allow natural rounding of the cell body while confining it to differing levels. Rounded mitotic bodies are anchored in place by actin retraction fibers (RFs) originating from adhesion clusters on the ECM-mimicking fibers. We discover the extent of ECM-confinement patterns RFs in 3D: triangular and band-like at low and high confinement, respectively. A stochastic Monte-Carlo simulation of the centrosome (CS), chromosome (CH), membrane interactions, and 3D arrangement of RFs on the mitotic body recovers MP tilting trends observed experimentally. Our mechanistic analysis reveals that the 3D shape of RFs is the primary driver of the MP rotation. Under high ECM-confinement, the fibers can mechanically pinch the cortex, causing the MP to have localized deformations at contact sites with fibers. Interestingly, high ECM-confinement leads to low and high MP tilts, which mechanistically depend upon the extent of cortical deformation, RF patterning, and MP position. We identify that cortical deformation and RFs work in tandem to limit MP tilt, while asymmetric positioning of MP leads to high tilts. Overall, we provide fundamental insights into how mitosis may proceed in fibrous ECM-confining microenvironments in vivo.
PubMed: 38659898
DOI: 10.1101/2024.04.12.589246 -
Research in Pharmaceutical Sciences 2023Previous studies highlighted that chemoprevention curcumin analog-1.1 (CCA-1.1) demonstrated an antitumor effect on breast, leukemia, and colorectal cancer cells. By...
Chemoprevention curcumin analog 1.1 promotes metaphase arrest and enhances intracellular reactive oxygen species levels on TNBC MDA-MB-231 and HER2-positive HCC1954 cells.
BACKGROUND AND PURPOSE
Previous studies highlighted that chemoprevention curcumin analog-1.1 (CCA-1.1) demonstrated an antitumor effect on breast, leukemia, and colorectal cancer cells. By utilizing immortalized MDA-MB-231 and HCC1954 cells, we evaluated the anticancer properties of CCA-1.1 and its mediated activity to promote cellular death.
EXPERIMENTAL APPROACH
Cytotoxicity and anti-proliferation were assayed using trypan blue exclusion. The cell cycle profile after CCA-1.1 treatment was established through flow cytometry. May-Grünwald-Giemsa and Hoechst staining were performed to determine the cell cycle arrest upon CCA-1.1 treatment. The involvement of CCA-1.1 in mitotic kinases (aurora A, p-aurora A, p-PLK1, and p-cyclin B1) expression was investigated by immunoblotting. CCA-1.1-treated cells were stained with the X-gal solution to examine the effect on senescence. ROS level and mitochondrial respiration were assessed by DCFDA assay and mitochondrial oxygen consumption rate, respectively.
FINDINGS/RESULTS
CCA-1.1 exerted cytotoxic activity and inhibited cell proliferation with an irreversible effect, and the flow cytometry analysis demonstrated that CCA-1.1 significantly halted during the G2/M phase, and further assessment revealed that CCA-1.1 caused metaphase arrest. Immunoblot assays confirmed CCA-1.1 suppressed aurora A kinase in MDA-MB-231 cells. The ROS level was elevated after treatment with CCA-1.1, which might promote cellular senescence and suppress basal mitochondrial respiration in MDA-MB-231 cells.
CONCLUSION AND IMPLICATIONS
Our data suggested the proof-of-concept that supports the involvement in cell cycle regulation and ROS generation as contributors to the effectiveness of CCA-1.1 in suppressing breast cancer cell growth.
PubMed: 37614620
DOI: 10.4103/1735-5362.378083 -
Cellular and Molecular Life Sciences :... Oct 2023Induction of DNA damage response (DDR) to ensure accurate duplication of genetic information is crucial for maintaining genome integrity during DNA replication. Cellular...
Induction of DNA damage response (DDR) to ensure accurate duplication of genetic information is crucial for maintaining genome integrity during DNA replication. Cellular senescence is a DDR mechanism that prevents the proliferation of cells with damaged DNA to avoid mitotic anomalies and inheritance of the damage over cell generations. Human WWOX gene resides within a common fragile site FRA16D that is preferentially prone to form breaks on metaphase chromosome upon replication stress. We report here that primary Wwox knockout (Wwox) mouse embryonic fibroblasts (MEFs) and WWOX-knockdown human dermal fibroblasts failed to undergo replication-induced cellular senescence after multiple passages in vitro. Strikingly, by greater than 20 passages, accelerated cell cycle progression and increased apoptosis occurred in these late-passage Wwox MEFs. These cells exhibited γH2AX upregulation and microsatellite instability, indicating massive accumulation of nuclear DNA lesions. Ultraviolet radiation-induced premature senescence was also blocked by WWOX knockdown in human HEK293T cells. Mechanistically, overproduction of cytosolic reactive oxygen species caused p16 promoter hypermethylation, aberrant p53/p21 signaling axis and accelerated p27 protein degradation, thereby leading to the failure of senescence induction in Wwox-deficient cells after serial passage in culture. We determined that significantly reduced protein stability or loss-of-function A135P/V213G mutations in the DNA-binding domain of p53 caused defective induction of p21 in late-passage Wwox MEFs. Treatment of N-acetyl-L-cysteine prevented downregulation of cyclin-dependent kinase inhibitors and induced senescence in Wwox MEFs. Our findings support an important role for fragile WWOX gene in inducing cellular senescence for maintaining genome integrity during DDR through alleviating oxidative stress.
Topics: Animals; Humans; Mice; Cellular Senescence; DNA; Fibroblasts; Genomic Instability; HEK293 Cells; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Ultraviolet Rays; WW Domain-Containing Oxidoreductase
PubMed: 37897534
DOI: 10.1007/s00018-023-04950-1 -
Cell Cycle (Georgetown, Tex.) Jul 2023Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-phase transitions. Protein Phosphatase 1 (PP1) is one of these...
Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-phase transitions. Protein Phosphatase 1 (PP1) is one of these phosphatases, with oscillations in PP1 activity driving mitotic M-phase. Evidence from a variety of experimental systems also points to roles in meiosis. Here, we report that PP1 is important for M-phase transitions through mouse oocyte meiosis. We employed a unique small-molecule approach to inhibit or activate PP1 at distinct phases of mouse oocyte meiosis. These studies show that temporal control of PP1 activity is essential for the G2/M transition, metaphase I/anaphase I transition, and the formation of a normal metaphase II oocyte. Our data also reveal that inappropriate activation of PP1 is more deleterious at the G2/M transition than at prometaphase I-to-metaphase I, and that an active pool of PP1 during prometaphase is vital for metaphase I/anaphase I transition and metaphase II chromosome alignment. Taken together, these results establish that loss of oscillations in PP1 activity causes a range of severe meiotic defects, pointing to essential roles for PP1 in female fertility, and more broadly, M-phase regulation.
Topics: Female; Mice; Animals; Meiosis; Oocytes; Metaphase; Anaphase; Mitosis; Protein Phosphatase 1; Mammals
PubMed: 37340734
DOI: 10.1080/15384101.2023.2225924 -
BMC Biology Mar 2024Mitogen-activated protein kinases (MAPKs) preserve cell homeostasis by transducing physicochemical fluctuations of the environment into multiple adaptive responses....
BACKGROUND
Mitogen-activated protein kinases (MAPKs) preserve cell homeostasis by transducing physicochemical fluctuations of the environment into multiple adaptive responses. These responses involve transcriptional rewiring and the regulation of cell cycle transitions, among others. However, how stress conditions impinge mitotic progression is largely unknown. The mitotic checkpoint is a surveillance mechanism that inhibits mitotic exit in situations of defective chromosome capture, thus preventing the generation of aneuploidies. In this study, we investigate the role of MAPK Pmk1 in the regulation of mitotic exit upon stress.
RESULTS
We show that Schizosaccharomyces pombe cells lacking Pmk1, the MAP kinase effector of the cell integrity pathway (CIP), are hypersensitive to microtubule damage and defective in maintaining a metaphase arrest. Epistasis analysis suggests that Pmk1 is involved in maintaining spindle assembly checkpoint (SAC) signaling, and its deletion is additive to the lack of core SAC components such as Mad2 and Mad3. Strikingly, pmk1Δ cells show up to twofold increased levels of the anaphase-promoting complex (APC/C) activator Cdc20 during unperturbed growth. We demonstrate that Pmk1 physically interacts with Cdc20 N-terminus through a canonical MAPK docking site. Most important, the Cdc20 pool is rapidly degraded in stressed cells undergoing mitosis through a mechanism that requires MAPK activity, Mad3, and the proteasome, thus resulting in a delayed mitotic exit.
CONCLUSIONS
Our data reveal a novel function of MAPK in preventing mitotic exit and activation of cytokinesis in response to stress. The regulation of Cdc20 turnover by MAPK Pmk1 provides a key mechanism by which the timing of mitotic exit can be adjusted relative to environmental conditions.
Topics: Schizosaccharomyces; Anaphase-Promoting Complex-Cyclosome; Schizosaccharomyces pombe Proteins; Cdc20 Proteins; Cell Cycle Proteins; Mitosis; Spindle Apparatus
PubMed: 38523261
DOI: 10.1186/s12915-024-01865-6 -
Cell Reports Mar 2024Condensin shapes mitotic chromosomes by folding chromatin into loops, but whether it does so by DNA-loop extrusion remains speculative. Although loop-extruding cohesin...
Condensin shapes mitotic chromosomes by folding chromatin into loops, but whether it does so by DNA-loop extrusion remains speculative. Although loop-extruding cohesin is stalled by transcription, the impact of transcription on condensin, which is enriched at highly expressed genes in many species, remains unclear. Using degrons of Rpb1 or the torpedo nuclease Dhp1 to either deplete or displace RNAPII on chromatin in fission yeast metaphase cells, we show that RNAPII does not load condensin on DNA. Instead, RNAPII retains condensin in cis and hinders its ability to fold mitotic chromatin and to support chromosome segregation, consistent with the stalling of a loop extruder. Transcription termination by Dhp1 limits such a hindrance. Our results shed light on the integrated functioning of condensin, and we argue that a tight control of transcription underlies mitotic chromosome assembly by loop-extruding condensin.
Topics: Chromosome Segregation; DNA-Binding Proteins; Chromatin; Chromosomes; DNA; Schizosaccharomyces; RNA Polymerase II; Mitosis; Cell Cycle Proteins; Adenosine Triphosphatases; Multiprotein Complexes
PubMed: 38446663
DOI: 10.1016/j.celrep.2024.113901 -
Bio-protocol Sep 2023Studies on chromosomal status are a fundamental aspect of plant cytogenetics and breeding because changes in number, size, and shape of chromosomes determine plant...
Studies on chromosomal status are a fundamental aspect of plant cytogenetics and breeding because changes in number, size, and shape of chromosomes determine plant physiology/performance. Despite its significance, the classical cytogenetic study is now frequently avoided because of its tedious job. In general, root meristems are used to study the mitotic chromosome number, even though the use of root tips was restricted because of sample availability, processing, and lack of standard protocols. Moreover, to date, a protocol using shoot tips to estimate chromosome number has not yet been achieved for tree species' germplasm with a large number of accessions, like mulberry (spp.). Here, we provide a step-by-step, economically feasible protocol for the pretreatment, fixation, enzymatic treatment, staining, and squashing of meristematic shoot tips. The protocol is validated with worldwide collections of 200 core set accessions with a higher level of ploidy variation, namely diploid (2n = 2x = 28), triploid (2n = 3x = 42), tetraploid (2n = 4x = 56), hexaploid (2n = 6x = 84), and decosaploid (2n = 22x = 308) belonging to nine species of Morus spp. Furthermore, accession from each ploidy group was subjected to flow cytometry (FCM) analysis for confirmation. The present protocol will help to optimize metaphase plate preparation and estimation of chromosome number using meristematic shoot tips of tree species regardless of their sex, location, and/or resources.
PubMed: 38273895
DOI: 10.21769/BioProtoc.4643