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Methods in Molecular Biology (Clifton,... 2022Bacillus subtilis is a widely used model bacterium to study cellular processes and development. The availability of an arrayed mutant library gave us the opportunity to...
Bacillus subtilis is a widely used model bacterium to study cellular processes and development. The availability of an arrayed mutant library gave us the opportunity to cytologically analyze every mutant and screen for new genes involved in cell shape determination, cell division, and chromosome segregation. Here we describe a high-throughput method to image arrayed B. subtilis mutant libraries using wide-field fluorescence microscopy. We provide a detailed description of growing the arrayed strain collection, preparing slides containing agarose pedestals, setting up the microscopy procedure, acquiring images, and analyzing the images.
Topics: Bacillus subtilis; Cell Division; Chromosome Segregation; Gene Library; Microscopy, Fluorescence
PubMed: 35635710
DOI: 10.1007/978-1-0716-2221-6_19 -
Cellular & Molecular Biology Letters Apr 2022Autophagy plays an essential role in maintaining cellular homeostasis and in the response to cellular stress. Autophagy is also involved in cell cycle progression, yet...
BACKGROUND
Autophagy plays an essential role in maintaining cellular homeostasis and in the response to cellular stress. Autophagy is also involved in cell cycle progression, yet the relationship between these processes is not clearly defined.
RESULTS
In exploring this relationship, we observed that the inhibition of autophagy impaired the G2/M phase-arresting activity of etoposide but enhanced the G1 phase-arresting activity of palbociclib. We further investigated the connection of basal autophagy and cell cycle by utilizing the autophagosome tracer dye Cyto-ID in two ways. First, we established a double-labeling flow-cytometric procedure with Cyto-ID and the DNA probe DRAQ5, permitting the cell cycle phase-specific determination of autophagy in live cells. This approach demonstrated that different cell cycle phases were associated with different autophagy levels: G1-phase cells had the lowest level, and G2/M-phase cells had the highest one. Second, we developed a flow-cytometric cell-sorting procedure based on Cyto-ID that separates cell populations into fractions with low, medium, and high autophagy. Cell cycle analysis of Cyto-ID-sorted cells confirmed that the high-autophagy fraction contained a much higher percentage of G2/M-phase cells than the low-autophagy fraction. In addition, Cyto-ID-based cell sorting also proved to be useful for assessing other autophagy-related processes: extracellular flux analysis revealed metabolic differences between the cell populations, with higher autophagy being associated with higher respiration, higher mitochondrial ATP production, and higher glycolysis.
CONCLUSION
This work provides clear evidence of high autophagy in G2/M-phase cells by establishing a novel cell sorting technique based on Cyto-ID.
Topics: Autophagy; Cell Cycle; Cell Division; G1 Phase; Humans; Leukemia
PubMed: 35382734
DOI: 10.1186/s11658-022-00334-8 -
Cytoskeleton (Hoboken, N.J.) Mar 2018Traditionally, we imagine that cell division gives rise to two identical daughter cells. Nevertheless, all cell divisions, to some degree, display asymmetry. Asymmetric... (Review)
Review
Traditionally, we imagine that cell division gives rise to two identical daughter cells. Nevertheless, all cell divisions, to some degree, display asymmetry. Asymmetric cell division is defined as the generation of two daughter cells with different physical content and/or developmental potential. Several organelles and cellular components including the centrosome, non-coding RNA, chromatin, and recycling endosomes are involved in the process of asymmetric cell division. Disruption of this important process is known to induce profound defects in development, the immune response, regeneration of tissues, aging, and cancer. Here, we discuss recent advances that expand our understanding of the mechanisms and consequences of asymmetric cell division in vertebrate organisms.
Topics: Animals; Asymmetric Cell Division; Humans; Mitosis; Stem Cells; Vertebrates
PubMed: 29360272
DOI: 10.1002/cm.21434 -
Philosophical Transactions of the Royal... Oct 2015Bacterial cells divide by targeting a transmembrane protein machine to the division site and regulating its assembly and disassembly so that cytokinesis occurs at the... (Review)
Review
Bacterial cells divide by targeting a transmembrane protein machine to the division site and regulating its assembly and disassembly so that cytokinesis occurs at the correct time in the cell cycle. The structure and dynamics of this machine (divisome) in bacterial model systems are coming more clearly into focus, thanks to incisive cell biology methods in combination with biochemical and genetic approaches. The main conserved structural element of the machine is the tubulin homologue FtsZ, which assembles into a circumferential ring at the division site that is stabilized and anchored to the inner surface of the cytoplasmic membrane by FtsZ-binding proteins. Once this ring is in place, it recruits a series of transmembrane proteins that ultimately trigger cytokinesis. This review will survey the methods used to characterize the structure of the bacterial divisome, focusing mainly on the Escherichia coli model system, as well as the challenges that remain. These methods include recent super-resolution microscopy, cryo-electron tomography and synthetic reconstitution.
Topics: Bacteria; Bacterial Proteins; Cell Division; Cell Membrane Structures; Cytokinesis; Cytoskeletal Proteins; Escherichia coli; Fluorescence Resonance Energy Transfer; Protein Interaction Mapping
PubMed: 26370940
DOI: 10.1098/rstb.2015.0028 -
Cells Jun 2020The study of oocytes has made enormous contributions to the understanding of the G/M transition. The complementarity of investigations carried out on various model... (Review)
Review
The study of oocytes has made enormous contributions to the understanding of the G/M transition. The complementarity of investigations carried out on various model organisms has led to the identification of the M-phase promoting factor (MPF) and to unravel the basis of cell cycle regulation. Thanks to the power of biochemical approaches offered by frog oocytes, this model has allowed to identify the core signaling components involved in the regulation of M-phase. A central emerging layer of regulation of cell division regards protein translation. Oocytes are a unique model to tackle this question as they accumulate large quantities of dormant mRNAs to be used during meiosis resumption and progression, as well as the cell divisions during early embryogenesis. Since these events occur in the absence of transcription, they require cascades of successive unmasking, translation, and discarding of these mRNAs, implying a fine regulation of the timing of specific translation. In the last years, the genome has been sequenced and annotated, enabling the development of omics techniques in this model and starting its transition into the genomic era. This review has critically described how the different phases of meiosis are orchestrated by changes in gene expression. The physiological states of the oocyte have been described together with the molecular mechanisms that control the critical transitions during meiosis progression, highlighting the connection between translation control and meiosis dynamics.
Topics: Animals; Gene Expression Regulation, Developmental; Genomics; Meiosis; Oocytes; Signal Transduction; Xenopus laevis
PubMed: 32575604
DOI: 10.3390/cells9061502 -
Current Opinion in Microbiology Dec 2014Bacteria face the problem of segregating their gigantic chromosomes without a segregation period restricted in time and space, as Eukaryotes do. Segregation thus... (Review)
Review
Bacteria face the problem of segregating their gigantic chromosomes without a segregation period restricted in time and space, as Eukaryotes do. Segregation thus involves multiple activities, general or specific of a chromosome region and differentially controlled. Recent advances show that these various mechanisms conform to a “pair and release” rule, which appears as a general rule in DNA segregation. We describe the latest advances in segregation of bacterial chromosomes with emphasis on the different pair and release mechanisms.
Topics: Bacteria; Cell Division; Chromosome Segregation; Chromosomes, Bacterial; Plasmids; Replication Origin
PubMed: 25460797
DOI: 10.1016/j.mib.2014.09.013 -
Current Opinion in Cell Biology Jun 2021Subversion of genome integrity fuels cellular adaptation and is a prerequisite for organismal evolution, yet genomic lesions are also the harmful driving force of cancer... (Review)
Review
Subversion of genome integrity fuels cellular adaptation and is a prerequisite for organismal evolution, yet genomic lesions are also the harmful driving force of cancer and other age-related human diseases. Genome integrity maintenance is inherently linked to genome organization and nuclear architecture, which are substantially remodeled during the cell cycle. Here we discuss recent findings on how actively dividing cells cope with endogenous genomic lesions that occur frequently at repetitive, heterochromatic, and late replicating regions as byproducts of genome duplication. We discuss how such lesions, rather than being resolved immediately when they occur, are dealt with in subsequent cell cycle phases, and even after mitotic cell division, and how this in turn affects genome organization, stability, and function.
Topics: Cell Cycle; Cell Division; DNA; DNA Replication; Genomic Instability; Humans; Mitosis
PubMed: 33310228
DOI: 10.1016/j.ceb.2020.11.001 -
Journal of Molecular Recognition : JMR Jul 2023Cell mechanics is a factor that determines cell growth, migration, proliferation, or differentiation, as well as trafficking inside the cytoplasm and organization of...
Cell mechanics is a factor that determines cell growth, migration, proliferation, or differentiation, as well as trafficking inside the cytoplasm and organization of organelles. Knowledge about cell mechanics is critical to gaining insight into these biological processes. Here, we used atomic force microscopy to examine the elasticity, an important parameter of cell mechanics, of non-adherent Jurkat leukemic T-cells in both interphase and mitotic phases. We found that the elasticity of an individual cell does not significantly change at interphase. When a cell starts to divide, its elasticity increases in the transition from metaphase to telophase during normal division while the cell is stiffened right after it enters mitosis during abnormal division. At the end of the division, the cell elasticity gradually returned to the value of the mother cell. These changes may originate from the changes in cell surface tension during modulating actomyosin at the cleavage furrow, redistributing cell organelles, and constricting the contractile ring to sever mother cell to form daughters. The difference in elasticity patterns suggests that there is a discrepancy in the redistribution of the cell organelles during normal and abnormal division.
Topics: Mitosis; Cell Cycle; Telophase; Interphase; T-Lymphocytes
PubMed: 37061787
DOI: 10.1002/jmr.3019 -
Cellular and Molecular Life Sciences :... Mar 2022Mammalian oocytes are particularly susceptible to accumulating DNA damage. However, unlike mitotic cells in which DNA damage induces G2 arrest by activating the...
Mammalian oocytes are particularly susceptible to accumulating DNA damage. However, unlike mitotic cells in which DNA damage induces G2 arrest by activating the ATM-Chk1/2-Cdc25 pathway, oocytes readily enter M-phase immediately following DNA damage. This implies a lack of a robust canonical G2/M DNA damage checkpoint in oocytes. Here we show that MDC1 plays a non-canonical role in controlling G2/M transition by regulating APC/C-Cdh1-mediated cyclin B1 degradation in response to DNA damage in mouse oocytes. Depletion of MDC1 impaired M-phase entry by decreasing cyclin B1 levels via the APC/C-Cdh1 pathway. Notably, the APC/C-Cdh1 regulation mediated by MDC1 was achieved by a direct interaction between MDC1 and APC/C-Cdh1. This interaction was transiently disrupted after DNA damage with a concomitant increase in Cdh1 levels, which, in turn, decreased cyclin B1 levels and delayed M-phase entry. Moreover, MDC1 depletion impaired spindle assembly by decreasing the integrity of microtubule organizing centers (MTOCs). Therefore, our results demonstrate that MDC1 is an essential molecule in regulating G2/M transition in response to DNA damage and in regulating spindle assembly in mouse oocytes. These results provide new insights into the regulation of the G2/M DNA damage checkpoint and cell cycle control in oocytes.
Topics: Animals; Cell Cycle; Cell Cycle Proteins; Cell Division; DNA Damage; Mammals; Mice; Oocytes
PubMed: 35320416
DOI: 10.1007/s00018-022-04241-1 -
Critical Reviews in Oncology/hematology Mar 2018All cancer registries constantly show striking differences in cancer incidence by age and among tissues. For example, lung cancer is diagnosed hundreds of times more... (Review)
Review
All cancer registries constantly show striking differences in cancer incidence by age and among tissues. For example, lung cancer is diagnosed hundreds of times more often at age 70 than at age 20, and lung cancer in nonsmokers occurs thousands of times more frequently than heart cancer in smokers. An analysis of these differences using basic concepts in cell biology indicates that cancer is the end-result of the accumulation of cell divisions in stem cells. In other words, the main determinant of carcinogenesis is the number of cell divisions that the DNA of a stem cell has accumulated in any type of cell from the zygote. Cell division, process by which a cell copies and separates its cellular components to finally split into two cells, is necessary to produce the large number of cells required for living. However, cell division can lead to a variety of cancer-promoting errors, such as mutations and epigenetic mistakes occurring during DNA replication, chromosome aberrations arising during mitosis, errors in the distribution of cell-fate determinants between the daughter cells, and failures to restore physical interactions with other tissue components. Some of these errors are spontaneous, others are promoted by endogenous DNA damage occurring during quiescence, and others are influenced by pathological and environmental factors. The cell divisions required for carcinogenesis are primarily caused by multiple local and systemic physiological signals rather than by errors in the DNA of the cells. As carcinogenesis progresses, the accumulation of DNA errors promotes cell division and eventually triggers cell division under permissive extracellular environments. The accumulation of cell divisions in stem cells drives not only the accumulation of the DNA alterations required for carcinogenesis, but also the formation and growth of the abnormal cell populations that characterize the disease. This model of carcinogenesis provides a new framework for understanding the disease and has important implications for cancer prevention and therapy.
Topics: Carcinogenesis; Cell Differentiation; Cell Division; Epigenesis, Genetic; Humans; Models, Theoretical; Mutation; Neoplasms; Neoplastic Stem Cells; Stem Cells
PubMed: 29482784
DOI: 10.1016/j.critrevonc.2018.01.010