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The Journal of Cell Biology Feb 2024Precise chromosome congression and segregation requires the proper assembly of a steady-state metaphase spindle, which is dynamic and maintained by continuous...
Precise chromosome congression and segregation requires the proper assembly of a steady-state metaphase spindle, which is dynamic and maintained by continuous microtubule flux. NuSAP is a microtubule-stabilizing and -bundling protein that promotes chromosome-dependent spindle assembly. However, its function in spindle dynamics remains unclear. Here, we demonstrate that NuSAP regulates the metaphase spindle length control. Mechanistically, NuSAP facilitates kinetochore capture and spindle assembly by promoting Eg5 binding to microtubules. It also prevents excessive microtubule depolymerization through interaction with Kif2A, which reduces Kif2A spindle-pole localization. NuSAP is phosphorylated by Aurora A at Ser-240 during mitosis, and this phosphorylation promotes its interaction with Kif2A on the spindle body and reduces its localization with the spindle poles, thus maintaining proper spindle microtubule flux. NuSAP knockout resulted in the formation of shorter spindles with faster microtubule flux and chromosome misalignment. Taken together, we uncover that NuSAP participates in spindle assembly, dynamics, and metaphase spindle length control through the regulation of microtubule flux and Kif2A localization.
Topics: Humans; Chromosome Segregation; HeLa Cells; Kinesins; Kinetochores; Microtubule-Associated Proteins; Microtubules; Mitosis; Spindle Apparatus
PubMed: 38117947
DOI: 10.1083/jcb.202108070 -
Seminars in Cell & Developmental Biology Sep 2021The mitotic spindle is a bipolar cellular structure, built from tubulin polymers, called microtubules, and interacting proteins. This macromolecular machine orchestrates... (Review)
Review
The mitotic spindle is a bipolar cellular structure, built from tubulin polymers, called microtubules, and interacting proteins. This macromolecular machine orchestrates chromosome segregation, thereby ensuring accurate distribution of genetic material into the two daughter cells during cell division. Powered by GTP hydrolysis upon tubulin polymerization, the microtubule ends exhibit a metastable behavior known as the dynamic instability, during which they stochastically switch between the growth and shrinkage phases. In the context of the mitotic spindle, dynamic instability is furthermore regulated by microtubule-associated proteins and motor proteins, which enables the spindle to undergo profound changes during mitosis. This highly dynamic behavior is essential for chromosome capture and congression in prometaphase, as well as for chromosome alignment to the spindle equator in metaphase and their segregation in anaphase. In this review we focus on the mechanisms underlying microtubule dynamics and sliding and their importance for the maintenance of shape, structure and dynamics of the metaphase spindle. We discuss how these spindle properties are related to the phenomenon of microtubule poleward flux, highlighting its highly cooperative molecular basis and role in keeping the metaphase spindle at a steady state.
Topics: Humans; Metaphase; Microtubules; Spindle Apparatus
PubMed: 34053864
DOI: 10.1016/j.semcdb.2021.05.016 -
Genes Jan 2022Oligo-fluorescence in situ hybridization (FISH) facilitates precise chromosome identification and comparative cytogenetic analysis. Detection of autosomal chromosomes of...
Oligo-fluorescence in situ hybridization (FISH) facilitates precise chromosome identification and comparative cytogenetic analysis. Detection of autosomal chromosomes of has not been achieved using oligonucleotide sequences. Here, the chromosomes of five taxa in the mitotic metaphase and mitotic metaphase to anaphase were detected using the oligo-FISH probes (AGT), 5S rDNA, and (TTG). In total, 24 small chromosomes were clearly observed in the mitotic metaphase (0.89-3.03 μm), whereas 24-48 small chromosomes were observed in the mitotic metaphase to anaphase (0.94-3.10 μm). The signal number and intensity of (AGT), 5S rDNA, and (TTG) in the mitotic metaphase to anaphase chromosomes were nearly consistent with those in the mitotic metaphase chromosomes when the two split chromosomes were integrated as one unit. Of note, 14 chromosomes (there is a high chance that sex chromosomes are included) were exclusively identified by (AGT), 5S rDNA, and (TTG). The other 10 also showed a terminal signal with (AGT). Moreover, these oligo-probes were able to distinguish one wild taxon from four taxa. These chromosome identification and taxa differentiation data will help in elucidating visual and elaborate physical mapping and guide breeders' utilization of wild resources of .
Topics: Chromosomes, Plant; DNA, Ribosomal; Hippophae; In Situ Hybridization, Fluorescence; RNA, Ribosomal, 5S
PubMed: 35205242
DOI: 10.3390/genes13020195 -
Methods in Molecular Biology (Clifton,... 2022During budding yeast mitosis, duplicated chromosomes are aligned at the center of the metaphase mitotic spindle, and the centromeres are stretched by forces generated...
During budding yeast mitosis, duplicated chromosomes are aligned at the center of the metaphase mitotic spindle, and the centromeres are stretched by forces generated within the mitotic spindle. In response to these stretching forces, mechanical tension builds up in the centromeric chromatin. The magnitude of this tension is detected by the cell to signal the attachment configuration of the sister chromosomes: a high tension signal would indicate that sister chromosomes are properly attached to opposite spindle poles, while a low tension signal could indicate the lack of a bipolar attachment. A low tension signal drives the cell to correct improper attachments in metaphase, thus preventing potential errors in anaphase chromosome segregation. In this paper, we describe a microscopy-based method to directly measure the magnitude of centromere tension in budding yeast metaphase spindles. The advantage of this method is that quantitative tension estimates are obtained without perturbing spindle and/or chromosome structure and as cells progress normally through mitosis.
Topics: Centromere; Kinetochores; Microtubules; Mitosis; Saccharomycetales; Spindle Apparatus
PubMed: 34972956
DOI: 10.1007/978-1-0716-1904-9_15 -
Frontiers in Oncology 2022FOSL1, a key component of the Activating protein-1 (AP-1) transcriptional complex, plays an important role in cancer cell migration, invasion, and proliferation....
BACKGROUND
FOSL1, a key component of the Activating protein-1 (AP-1) transcriptional complex, plays an important role in cancer cell migration, invasion, and proliferation. However, the impact of FOSL1 in ameloblastoma (AM) has not been clarified. Herein, we aimed to assess the expression of FOSL1 and investigate its functional role in AM.
METHODS
The expression of FOSL1 was examined based on an immunohistochemistry analysis of 96 AM samples. Cell proliferation, migration, invasion, and tumorigenesis were assessed using Cell Counting Kit-8 (CCK-8), colony formation, Transwell, and sphere formation assays. RNA sequencing (RNA-seq) was employed to investigate the molecular alterations of AM cells upon FOSL depletion. Microarrays of AMs were downloaded from the Gene Expression Omnibus (GEO) database for bioinformatics analysis. In addition, patient-derived AM organoids were used to evaluate the therapeutic value of the AP-1 inhibitor.
RESULTS
FOSL1 was detected in the nuclei of AMs and upregulated in conventional AMs compared to unicystic AMs and normal oral epithelium. Compared with primary AM, FOSL1 expression was significantly increased in recurrent AM. Genetic knockdown of FOSL1 suppressed the proliferation, migration, invasion, and sphere formation of AMs. Similar results were also observed by pharmacological inhibition of AP-1 activity. Moreover, the AP-1 inhibitor T5224 impeded the growth of organoids derived from AM patients. Mechanistically, our Ingenuity Pathway Analysis (IPA) and gene set enrichment analysis (GSEA) results revealed that depletion of FOSL1 inactivated kinetochore metaphase signaling and the epithelial-mesenchymal transition pathway and then impaired the aggressiveness of AM cells accordingly.
CONCLUSION
FOSL1 promotes tumor recurrence and invasive growth in AM by modulating kinetochore metaphase signaling and the epithelial-mesenchymal transition pathway; thus, it represents a promising therapeutic target for AM treatment.
PubMed: 36185257
DOI: 10.3389/fonc.2022.900108 -
Scientific Reports Jul 2020Recent advances in fluorescence super-resolution microscopy are providing important insights into details of cellular structures. To acquire three dimensional (3D)...
Recent advances in fluorescence super-resolution microscopy are providing important insights into details of cellular structures. To acquire three dimensional (3D) super-resolution images of DNA, we combined binding activated localization microscopy (BALM) using fluorescent double-stranded DNA intercalators and optical astigmatism. We quantitatively establish the advantage of bis- over mono-intercalators before demonstrating the approach by visualizing single DNA molecules stretched between microspheres at various heights. Finally, the approach is applied to the more complex environment of intact and damaged metaphase chromosomes, unravelling their structural features.
Topics: Chromosomes; DNA; Humans; Imaging, Three-Dimensional; Jurkat Cells; Kinetics; Metaphase; Microscopy, Fluorescence; Optical Imaging; Protein Binding
PubMed: 32719468
DOI: 10.1038/s41598-020-68892-5 -
Journal of Reproductive Immunology Nov 2021Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by generation of autoantibodies and severe damage of various organs. The...
Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by generation of autoantibodies and severe damage of various organs. The hormonal changes associated with pregnancy and especially estrogen might lead to damage of reproductive function and ovarian quality. We employed a pristane-induced lupus model of Balb/c mice which resembles human lupus in an attempt to follow oogenesis disruption during the disease progression. The integrity of cytoskeletal and chromatin structures was estimated in oocytes derived by hormonally stimulated ovulation in lupus mice and the results were compared with those from healthy mice. Chromatin, tubulin and actin structures in oocytes were detected by Hoechst 33258, anti-alpha-tubulin antibody and rhodamine-labeled phalloidin, respectively. All available meiotic spindles were analyzed - in immature (metaphase I) and mature oocytes (metaphase II). The total number of mature oocytes obtained from lupus mice was lower compared to healthy controls. The maturation rate was 9.8 % for lupus mice, 12.7 % for 7-month old controls, and 14.3 % for the young control mice (4 weeks old). Another major difference between the studied groups was the higher percentage of defective metaphase I spindles registered in oocytes derived from lupus mice (60 % normal spindles), while for the young and older controls this proportion was 86 % and 81 %, respectively. No such difference was registered for metaphase II spindles. For both metaphase I and metaphase II oocytes, the proportions of normal actin cap and chromosomal condensation were similar between the experimental groups.
Topics: Animals; Cell Differentiation; Cells, Cultured; Disease Models, Animal; Female; Humans; Lupus Erythematosus, Systemic; Metaphase; Mice; Mice, Inbred BALB C; Oogenesis; Pregnancy; Terpenes
PubMed: 34492566
DOI: 10.1016/j.jri.2021.103370 -
Nature Communications Apr 2021Telomere crisis contributes to cancer genome evolution, yet only a subset of cancers display breakage-fusion-bridge (BFB) cycles and chromothripsis, hallmarks of...
Telomere crisis contributes to cancer genome evolution, yet only a subset of cancers display breakage-fusion-bridge (BFB) cycles and chromothripsis, hallmarks of experimental telomere crisis identified in previous studies. We examine the spectrum of structural variants (SVs) instigated by natural telomere crisis. Eight spontaneous post-crisis clones did not show prominent patterns of BFB cycles or chromothripsis. Their crisis-induced genome rearrangements varied from infrequent simple SVs to more frequent and complex SVs. In contrast, BFB cycles and chromothripsis occurred in MRC5 fibroblast clones that escaped telomere crisis after CRISPR-controlled telomerase activation. This system revealed convergent evolutionary lineages altering one allele of chromosome 12p, where a short telomere likely predisposed to fusion. Remarkably, the 12p chromothripsis and BFB events were stabilized by independent fusions to chromosome 21. The data establish that telomere crisis can generate a wide spectrum of SVs implying that a lack of BFB patterns and chromothripsis in cancer genomes does not indicate absence of past telomere crisis.
Topics: Cell Line; Chromosomal Instability; Chromothripsis; Fibroblasts; Genome; Genomic Instability; Humans; Lung; Metaphase; Models, Biological; Neoplasms; Telomere
PubMed: 33828097
DOI: 10.1038/s41467-021-21933-7 -
3 Biotech Mar 2023An efficient in vitro protocol for high-frequency polyploidization for the first time in gerbera hybrid (BGC-2019-01) was developed in the present study. Two-week-old in...
An efficient in vitro protocol for high-frequency polyploidization for the first time in gerbera hybrid (BGC-2019-01) was developed in the present study. Two-week-old in vitro-developed shoots (tips) were treated individually with 0.1%, 0.25% and 0.5% (/) colchicine solutions for 4, 6, 8, and 12 h. The colchicine-treated shoot tips were then inoculated on Murashige and Skoog (MS) medium fortified with 1.5 mg/l -Topolin for multiple shoot proliferation and later transferred into 1.5 mg/l indole-3-acetic acid-fortified MS medium for rooting of shoots. The ploidy levels of the colchicine-treated and regenerated plantlets along with the non-treated ones were confirmed via flow cytometry analysis and metaphasic chromosome count. The highest frequency of tetraploid plantlets (50%) were obtained when shoot tips were treated with 0.1% colchicine for 4 h. Morphological observations revealed that induced tetraploid plantlets exhibited delayed fresh shoot initiation, fewer but longer shoots, as well as fewer but broader leaves. Likewise, the study of stomata revealed that in comparison to their diploid counterparts, the tetraploid plantlets exhibited less frequent yet significantly larger stomata, and higher number of chloroplasts. The tetraploids were recorded with significantly higher chlorophyll, carotenoid, and anthocyanin content during the photosynthetic pigment analyses. During ex vitro acclimatization and field growth, the tetraploid plants exhibited delayed proliferation but with higher vigor and thickened broad leaves. The genetic uniformity among the diploid and the tetraploid plants was confirmed using conserved DNA-derived polymorphism (CDDP), directed amplification of minisatellite-region DNA (DAMD), inter simple sequence repeats (ISSR), and start codon targeted (SCoT) polymorphism marker systems. The tetraploids developed in the present study would be of immense importance for the genetic improvement of gerbera as far as its ornamental values are concerned.
PubMed: 36748015
DOI: 10.1007/s13205-022-03457-z -
Cell Cycle (Georgetown, Tex.) 2022Mammalian oocytes undergo two rounds of developmental arrest during maturation: at the diplotene of the first meiotic prophase and metaphase of the second meiosis. These... (Review)
Review
Mammalian oocytes undergo two rounds of developmental arrest during maturation: at the diplotene of the first meiotic prophase and metaphase of the second meiosis. These arrests are strictly regulated by follicular cells temporally producing the secondary messengers, cAMP and cGMP, and other factors to regulate maturation promoting factor (composed of cyclin B1 and cyclin-dependent kinase 1) levels in the oocytes. Out of these normally appearing developmental arrests, permanent arrests may occur in the oocytes at germinal vesicle (GV), metaphase I (MI), or metaphase II (MII) stage. This issue may arise from absence or altered expression of the oocyte-related genes playing key roles in nuclear and cytoplasmic maturation. Additionally, the assisted reproductive technology (ART) applications such as ovarian stimulation and culture conditions both of which harbor various types of chemical agents may contribute to forming the permanent arrests. In this review, the molecular determinants of developmental and permanent arrests occurring in the mammalian oocytes are comprehensively evaluated in the light of current knowledge. As number of permanently arrested oocytes at different stages is increasing in ART centers, potential approaches for inducing permanent arrests to obtain competent oocytes are discussed.
Topics: Animals; Mammals; Meiosis; Meiotic Prophase I; Metaphase; Oocytes
PubMed: 35072590
DOI: 10.1080/15384101.2022.2026704