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Plants (Basel, Switzerland) Jun 2024Ogura cytoplasmic male sterility (CMS) is considered the rapeseed ( L.) with the most potential to be utilized as a heterosis system worldwide, but it lacks sufficient...
Ogura cytoplasmic male sterility (CMS) is considered the rapeseed ( L.) with the most potential to be utilized as a heterosis system worldwide, but it lacks sufficient restorers. In this study, root tip cell (RTC) mitotic and pollen mother cell (PMC) meiosis observations were compared to ensure the number of chromosomes and the formation of a chromosomal bridge using restorer lines R2000, CLR650, and Zhehuhong (a new restorer) as the experimental material. Further, molecular markers of exogenous chromosomal fragments were detected and the sequence and expression differences of restorer genes in the three lines were determined to identify the distinctive characteristics of Zhehuhong. The results showed that the number of chromosomes in Zhehuhong was stable (2n = 38), indicating that the exogenous radish chromosome segment had been integrated into the chromosome of Zhehuhong. Molecular marker detection revealed that Zhehuhong was detected at most loci, with only the RMA05 locus being missed. The exogenous radish chromosome segment of Zhehuhong differed from R2000 and CLR650. The pollen mother cells of Zhehuhong showed chromosome lagging in the meiotic metaphase I, meiotic anaphase I, and meiotic anaphase II, which was consistent with R2000 and CLR650. The restorer gene in Zhehuhong had 85 SNPs compared with R2000 and 119 SNPs compared with CLR650, indicating the distinctive characteristic of in Zhehuhong. In terms of the spatial expression of , the highest level was detected in the anthers in the three restorer lines. In addition, in terms of temporal expression, the gene expression of Zhehuhong was highest at a bud length of 4 mm. Our results clearly indicated that Zhehuhong is a new restorer line for the Ogura CMS system, which can be used further in rapeseed heterosis utilization.
PubMed: 38931135
DOI: 10.3390/plants13121703 -
International Journal of Molecular... Jun 2024Paclitaxel induces multipolar spindles at clinically relevant doses but does not substantially increase mitotic indices. Paclitaxel's anti-cancer effects are... (Review)
Review
Suppressing Anaphase-Promoting Complex/Cyclosome-Cell Division Cycle 20 Activity to Enhance the Effectiveness of Anti-Cancer Drugs That Induce Multipolar Mitotic Spindles.
Paclitaxel induces multipolar spindles at clinically relevant doses but does not substantially increase mitotic indices. Paclitaxel's anti-cancer effects are hypothesized to occur by promoting chromosome mis-segregation on multipolar spindles leading to apoptosis, necrosis and cyclic-GMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) pathway activation in daughter cells, leading to secretion of type I interferon (IFN) and immunogenic cell death. Eribulin and vinorelbine have also been reported to cause increases in multipolar spindles in cancer cells. Recently, suppression of Anaphase-Promoting Complex/Cyclosome-Cell Division Cycle 20 (APC/C-CDC20) activity using CRISPR/Cas9 mutagenesis has been reported to increase sensitivity to Kinesin Family 18a (KIF18a) inhibition, which functions to suppress multipolar mitotic spindles in cancer cells. We propose that a way to enhance the effectiveness of anti-cancer agents that increase multipolar spindles is by suppressing the APC/C-CDC20 to delay, but not block, anaphase entry. Delaying anaphase entry in genomically unstable cells may enhance multipolar spindle-induced cell death. In genomically stable healthy human cells, delayed anaphase entry may suppress the level of multipolar spindles induced by anti-cancer drugs and lower mitotic cytotoxicity. We outline specific combinations of molecules to investigate that may achieve the goal of enhancing the effectiveness of anti-cancer agents.
Topics: Humans; Anaphase-Promoting Complex-Cyclosome; Antineoplastic Agents; Spindle Apparatus; Cdc20 Proteins; Neoplasms; Mitosis
PubMed: 38928036
DOI: 10.3390/ijms25126329 -
International Journal of Biological... 2024Cysteine-rich angiogenic inducer 61 (CYR61), also called CCN1, has long been characterized as a secretory protein. Nevertheless, the intracellular function of CYR61...
Cysteine-rich angiogenic inducer 61 (CYR61), also called CCN1, has long been characterized as a secretory protein. Nevertheless, the intracellular function of CYR61 remains unclear. Here, we found that CYR61 is important for proper cell cycle progression. Specifically, CYR61 interacts with microtubules and promotes microtubule polymerization to ensure mitotic entry. Moreover, CYR61 interacts with PLK1 and accumulates during the mitotic process, followed by degradation as mitosis concludes. The proteolysis of CYR61 requires the PLK1 kinase activity, which directly phosphorylates two conserved motifs on CYR61, enhancing its interaction with the SCF E3 complex subunit FBW7 and mediating its degradation by the proteasome. Mutations of phosphorylation sites of Ser167 and Ser188 greatly increase CYR61's stability, while deletion of CYR61 extends prophase and metaphase and delays anaphase onset. In summary, our findings highlight the precise control of the intracellular CYR61 by the PLK1-FBW7 pathway, accentuating its significance as a microtubule-associated protein during mitotic progression.
Topics: Protein Serine-Threonine Kinases; Humans; Polo-Like Kinase 1; Mitosis; Cell Cycle Proteins; Proto-Oncogene Proteins; Cysteine-Rich Protein 61; Microtubules; F-Box-WD Repeat-Containing Protein 7; HeLa Cells; Phosphorylation; Ubiquitin-Protein Ligases; Microtubule-Associated Proteins
PubMed: 38904029
DOI: 10.7150/ijbs.93335 -
The Journal of Biological Chemistry Jun 2024O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme that catalyzes all O-GlcNAcylation reactions intracellularly. Previous investigations...
O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme that catalyzes all O-GlcNAcylation reactions intracellularly. Previous investigations have found that OGT levels oscillate during the cell division process. Specifically, OGT abundance is downregulated during mitosis, but the underlying mechanism is lacking. Here we demonstrate that OGT is ubiquitinated by the ubiquitin E3 ligase, anaphase promoting complex/cyclosome (APC/C)-cell division cycle 20 (Cdc20). We show that APC/C interacts with OGT through a conserved destruction box (D-box): Arg-351/Leu-354, the abrogation of which stabilizes OGT. As APC/C-substrate binding is often preceded by a priming ubiquitination event, we also used mass spectrometry and mapped OGT Lys-352 to be a ubiquitination site, which is a prerequisite for OGT association with APC/C subunits. Interestingly in The Cancer Genome Atlas, R351C is a uterine carcinoma mutant, suggesting that mutations of the D-box are linked with tumorigenesis. Paradoxically, we found that both R351C and the D-box mutants (R351A/L354A) inhibit uterine carcinoma in mouse xenograft models, probably due to impaired cell division and proliferation. In sum, we propose a model where OGT Lys-352 ubiquitination primes its binding with APC/C, and then APC/C partners with OGT through the D-box for its mitotic destruction. Our work not only highlights the key mechanism that regulates OGT during the cell cycle, but also reveals the mutual coordination between glycosylation and the cell division machinery.
PubMed: 38844135
DOI: 10.1016/j.jbc.2024.107448 -
Cell Structure and Function Jun 2024In metazoans, the nuclear envelope (NE) disassembles during the prophase and reassembles around segregated chromatids during the telophase. The process of NE formation...
In metazoans, the nuclear envelope (NE) disassembles during the prophase and reassembles around segregated chromatids during the telophase. The process of NE formation has been extensively studied using live-cell imaging. At the early step of NE reassembly in human cells, specific pattern-like localization of inner nuclear membrane (INM) proteins, connected to the nuclear pore complex (NPC), was observed in the so-called "core" region and "noncore" region on telophase chromosomes, which corresponded to the "pore-free" region and the "pore-rich" region, respectively, in the early G1 interphase nucleus. We refer to these phenomena as NE subdomain formation. To biochemically investigate this process, we aimed to develop an in vitro NE reconstitution system using digitonin-permeabilized semi-intact mitotic human cells coexpressing two INM proteins, emerin and lamin B receptor, which were labeled with fluorescent proteins. The targeting and accumulation of INM proteins to chromosomes before and after anaphase onset in semi-intact cells were observed using time-lapse imaging. Our in vitro NE reconstitution system recapitulated the formation of the NE subdomain, as in living cells, although chromosome segregation and cytokinesis were not observed. This in vitro NE reconstitution required the addition of a mitotic cytosolic fraction supplemented with a cyclin-dependent kinase inhibitor and energy sources. The cytoplasmic soluble factor(s) dependency of INM protein targeting differed among the segregation states of chromosomes. Furthermore, the NE reconstituted on segregated chromosomes exhibited active nucleocytoplasmic transport competency. These results indicate that the chromosome status changes after anaphase onset for recruiting NPC components.Key words: nuclear envelope reassembly, inner nuclear membrane protein, nuclear pore complex, semi-intact cell, in vitro reconstitution.
PubMed: 38839376
DOI: 10.1247/csf.24003 -
Nature Communications Jun 2024Persisting replication intermediates can confer mitotic catastrophe. Loss of the fission yeast telomere protein Taz1 (ortholog of mammalian TRF1/TRF2) causes telomeric...
Persisting replication intermediates can confer mitotic catastrophe. Loss of the fission yeast telomere protein Taz1 (ortholog of mammalian TRF1/TRF2) causes telomeric replication fork (RF) stalling and consequently, telomere entanglements that stretch between segregating mitotic chromosomes. At ≤20 °C, these entanglements fail to resolve, resulting in lethality. Rif1, a conserved DNA replication/repair protein, hinders the resolution of telomere entanglements without affecting their formation. At mitosis, local nuclear envelope (NE) breakdown occurs in the cell's midregion. Here we demonstrate that entanglement resolution occurs in the cytoplasm following this NE breakdown. However, in response to taz1Δ telomeric entanglements, Rif1 delays midregion NE breakdown at ≤20 °C, in turn disfavoring entanglement resolution. Moreover, Rif1 overexpression in an otherwise wild-type setting causes cold-specific NE defects and lethality, which are rescued by membrane fluidization. Hence, NE properties confer the cold-specificity of taz1Δ lethality, which stems from postponement of NE breakdown. We propose that such postponement promotes clearance of simple stalled RFs, but resolution of complex entanglements (involving strand invasion between nonsister telomeres) requires rapid exposure to the cytoplasm.
Topics: Nuclear Envelope; Schizosaccharomyces; Telomere; Schizosaccharomyces pombe Proteins; Telomere-Binding Proteins; Anaphase; DNA Replication
PubMed: 38830842
DOI: 10.1038/s41467-024-48382-2 -
Chronic spindle assembly checkpoint activation causes myelosuppression and gastrointestinal atrophy.EMBO Reports Jun 2024Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic...
Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor, CDC20, preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However, the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression, we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for, gastrointestinal atrophy is detrimental. Remarkably, deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome, while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect, identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast, only overexpression of anti-apoptotic BCL2, but none of the BH3-only protein deficiencies mentioned above, can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo.
Topics: Animals; Bcl-2-Like Protein 11; Mice; Mad2 Proteins; Apoptosis Regulatory Proteins; M Phase Cell Cycle Checkpoints; Proto-Oncogene Proteins c-bcl-2; Apoptosis; Atrophy; Proto-Oncogene Proteins; Mitosis; BH3 Interacting Domain Death Agonist Protein; Cdc20 Proteins; Bone Marrow; Membrane Proteins; Tumor Suppressor Proteins
PubMed: 38806674
DOI: 10.1038/s44319-024-00160-3 -
Open Biology May 2024The precise spatial and temporal control of histone phosphorylations is important for the ordered progression through the different phases of mitosis. The...
The precise spatial and temporal control of histone phosphorylations is important for the ordered progression through the different phases of mitosis. The phosphorylation of H2B at S6 (H2B S6ph), which is crucial for chromosome segregation, reaches its maximum level during metaphase and is limited to the inner centromere. We discovered that the temporal and spatial regulation of this modification, as well as its intensity, are governed by the scaffold protein RepoMan and its associated catalytically active phosphatases, PP1α and PP1γ. Phosphatase activity is inhibited at the area of maximal H2B S6 phosphorylation at the inner centromere by site-specific Aurora B-mediated inactivation of the PP1/RepoMan complex. The motor protein Mklp2 contributes to the relocalization of Aurora B from chromatin to the mitotic spindle during anaphase, thus alleviating Aurora B-dependent repression of the PP1/RepoMan complex and enabling dephosphorylation of H2B S6. Accordingly, dysregulation of Mklp2 levels, as commonly observed in tumour cells, leads to the lack of H2B S6 dephosphorylation during early anaphase, which might contribute to chromosomal instability.
Topics: Aurora Kinase B; Phosphorylation; Humans; Histones; Mitosis; Protein Phosphatase 1; Cell Cycle Proteins; HeLa Cells; Spindle Apparatus; Centromere; Nuclear Proteins
PubMed: 38806145
DOI: 10.1098/rsob.230460 -
BioRxiv : the Preprint Server For... May 2024In the budding yeast , exit from mitosis is coupled to spindle position to ensure successful genome partitioning between mother and daughter cell. This coupling occurs...
In the budding yeast , exit from mitosis is coupled to spindle position to ensure successful genome partitioning between mother and daughter cell. This coupling occurs through a GTPase signaling cascade known as the mitotic exit network (MEN). The MEN senses spindle position via a Ras-like GTPase Tem1 which primarily localizes to the spindle pole bodies (SPBs, yeast equivalent of centrosomes) during anaphase. How Tem1 couples the status of spindle position to MEN activation is not fully understood. Here, we show that Tem1 does not function as a molecular switch as its nucleotide state does not change upon MEN activation. Instead, Tem1's nucleotide state regulates its SPB localization to establish a concentration difference in the cell in response to spindle position. By artificially tethering Tem1 to the SPB, we demonstrate that the essential function of Tem1 is to localize Tem1 to the SPB. Tem1 localization to the SPB primarily functions to generate a high effective concentration of Tem1 and MEN signaling can be initiated by concentrating Tem1 in the cytoplasm with genetically encoded multimeric nanoparticles. This localization/concentration-based GTPase signaling mechanism for Tem1 differs from the canonical Ras-like GTPase signaling paradigm and is likely relevant to other localization-based signaling scenarios.
PubMed: 38798491
DOI: 10.1101/2024.05.16.594582 -
Mathematical Biosciences May 2024This paper develops a theory for anaphase in cells. After a brief description of microtubules, the mitotic spindle and the centrosome, a mathematical model for anaphase...
This paper develops a theory for anaphase in cells. After a brief description of microtubules, the mitotic spindle and the centrosome, a mathematical model for anaphase is introduced and developed in the context of the cell cytoplasm and liquid crystalline structures. Prophase, prometaphase and metaphase are then briefly described in order to focus on anaphase, which is the main study of this paper. The entities involved are modelled in terms of liquid crystal defects and microtubules are represented as defect flux lines. The mathematical techniques employed make extensive use of energy considerations based on the work that was developed by Dafermos (1970) from the classical Frank-Oseen nematic liquid crystal energy (Frank, 1958; Oseen, 1933). With regard to liquid crystal theory we introduce the concept of regions of influence for defects which it is believed have important implications beyond the subject of this paper. The results of this paper align with observed biochemical phenomena and are explored in application to HeLa cells and Caenorhabditis elegans. This unified approach offers the possibility of gaining insight into various consequences of mitotic abnormalities which may result in Down syndrome, Hodgkin lymphoma, breast, prostate and various other types of cancer.
PubMed: 38795952
DOI: 10.1016/j.mbs.2024.109219