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Journal of Microscopy May 2023A secondary ion mass spectrometry (SIMS)-based isotopic imaging technique of ion microscopy was used for observing calcium influx in single renal epithelial LLC-PK...
A secondary ion mass spectrometry (SIMS)-based isotopic imaging technique of ion microscopy was used for observing calcium influx in single renal epithelial LLC-PK cells. The CAMECA IMS-3f SIMS instrument, used in the study, is capable of producing isotopic images of single cells at 500 nm spatial resolution. Due to the high-vacuum requirements of the instrument the cells were prepared cryogenically with a freeze-fracture method and frozen freeze-dried cells were used for SIMS analysis. The influx of calcium was imaged directly by exposure of cells to Ca stable isotope in the extracellular buffer for 10 min. The Ca influx was measured at mass 44 and the distribution of endogenous calcium at mass 40 ( Ca) in the same cell. A direct comparison of interphase cells to cells undergoing division revealed that calcium influx is restricted in metaphase and post-metaphase stages of cell division. This restriction is lifted in late cytokinesis. The net influx of Ca in 10 min was approximately half under calcium influx restriction in comparison to interphase cells. Under calcium influx restriction the Ca concentration was the same between the mitotic chromosome and the cytoplasm. These observations indicate that the endoplasmic reticulum (ER) calcium uptake is compromised under calcium influx restriction in cells undergoing division.
Topics: Metaphase; Calcium; Spectrometry, Mass, Secondary Ion; Cell Division; Cytoplasm
PubMed: 36864642
DOI: 10.1111/jmi.13182 -
PloS One 2013Mitosis in the early syncytial Drosophila embryo is highly correlated in space and time, as manifested in mitotic wavefronts that propagate across the embryo. In this...
Mitosis in the early syncytial Drosophila embryo is highly correlated in space and time, as manifested in mitotic wavefronts that propagate across the embryo. In this paper we investigate the idea that the embryo can be considered a mechanically-excitable medium, and that mitotic wavefronts can be understood as nonlinear wavefronts that propagate through this medium. We study the wavefronts via both image analysis of confocal microscopy videos and theoretical models. We find that the mitotic waves travel across the embryo at a well-defined speed that decreases with replication cycle. We find two markers of the wavefront in each cycle, corresponding to the onsets of metaphase and anaphase. Each of these onsets is followed by displacements of the nuclei that obey the same wavefront pattern. To understand the mitotic wavefronts theoretically we analyze wavefront propagation in excitable media. We study two classes of models, one with biochemical signaling and one with mechanical signaling. We find that the dependence of wavefront speed on cycle number is most naturally explained by mechanical signaling, and that the entire process suggests a scenario in which biochemical and mechanical signaling are coupled.
Topics: Anaphase; Animals; Biomechanical Phenomena; Drosophila melanogaster; Embryo, Nonmammalian; Mechanotransduction, Cellular; Metaphase; Microscopy, Confocal; Models, Biological; Video Recording
PubMed: 24204774
DOI: 10.1371/journal.pone.0077216 -
Molecules and Cells Dec 2000Flow cytometric analysis was systematically performed to optimize the concentration and duration of hydroxyurea (DNA synthesis inhibitor) and trifluralin (metaphase...
Flow cytometric analysis was systematically performed to optimize the concentration and duration of hydroxyurea (DNA synthesis inhibitor) and trifluralin (metaphase blocking reagent) treatments for synchronizing the cell cycle and accumulating metaphase chromosomes in barley root tips. A high metaphase index (76.5% in the root tip meristematic area) was routinely achieved. Seedlings of about 1.0-cm length were treated with 1.25 mM hydroxyurea for 14 h to synchronize the root tip meristem cells at the S/G2 phase. After rinsing with hydroxyurea, the seedlings were incubated in a hydroxyurea-free solution for 2 h and were treated with 1 microM trifluralin for 4 h to accumulate mitotic cells in the metaphase. The consistent high metaphase index depended on the uniform germination of seeds prior to treatment. High-quality and high-quantity isolated metaphase chromosomes were suitable for flow cytometric analysis and sorting. Flow karyotypes of barley chromosomes were established via univariate and bivariate analysis. A variation of flow karyotypes was detected among barley lines. Two single chromosome types were identified and sorted. Bivariate analysis showed no variation among barley individual chromosomes in AT and GC content.
Topics: Analysis of Variance; Cell Cycle; Cell Separation; Chromosomes; DNA, Plant; Flow Cytometry; Hordeum; Hydroxyurea; Karyotyping; Metaphase; Plant Roots; Trifluralin
PubMed: 11211865
DOI: 10.1007/s10059-000-0619-y -
Molecular Cell Apr 2020As cells enter mitosis, the genome is restructured to facilitate chromosome segregation, accompanied by dramatic changes in gene expression. However, the mechanisms that...
As cells enter mitosis, the genome is restructured to facilitate chromosome segregation, accompanied by dramatic changes in gene expression. However, the mechanisms that underlie mitotic transcriptional regulation are unclear. In contrast to transcribed genes, centromere regions retain transcriptionally active RNA polymerase II (Pol II) in mitosis. Here, we demonstrate that chromatin-bound cohesin is necessary to retain elongating Pol II at centromeres. We find that WAPL-mediated removal of cohesin from chromosome arms during prophase is required for the dissociation of Pol II and nascent transcripts, and failure of this process dramatically alters mitotic gene expression. Removal of cohesin/Pol II from chromosome arms in prophase is important for accurate chromosome segregation and normal activation of gene expression in G1. We propose that prophase cohesin removal is a key step in reprogramming gene expression as cells transition from G2 through mitosis to G1.
Topics: Anaphase; Animals; Aurora Kinase B; Cell Cycle; Cell Cycle Proteins; Cell Line; Centromere; Chromosomal Proteins, Non-Histone; Chromosome Segregation; G1 Phase; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation; Humans; Metaphase; Mitosis; Prophase; RNA Polymerase II; Transcription, Genetic; Xenopus laevis; Cohesins
PubMed: 32035037
DOI: 10.1016/j.molcel.2020.01.023 -
ELife Jul 2022During cell division, kinetochore microtubules (KMTs) provide a physical linkage between the chromosomes and the rest of the spindle. KMTs in mammalian cells are...
During cell division, kinetochore microtubules (KMTs) provide a physical linkage between the chromosomes and the rest of the spindle. KMTs in mammalian cells are organized into bundles, so-called kinetochore-fibers (k-fibers), but the ultrastructure of these fibers is currently not well characterized. Here, we show by large-scale electron tomography that each k-fiber in HeLa cells in metaphase is composed of approximately nine KMTs, only half of which reach the spindle pole. Our comprehensive reconstructions allowed us to analyze the three-dimensional (3D) morphology of k-fibers and their surrounding MTs in detail. We found that k-fibers exhibit remarkable variation in circumference and KMT density along their length, with the pole-proximal side showing a broadening. Extending our structural analysis then to other MTs in the spindle, we further observed that the association of KMTs with non-KMTs predominantly occurs in the spindle pole regions. Our 3D reconstructions have implications for KMT growth and k-fiber self-organization models as covered in a parallel publication applying complementary live-cell imaging in combination with biophysical modeling (Conway et al., 2022). Finally, we also introduce a new visualization tool allowing an interactive display of our 3D spindle data that will serve as a resource for further structural studies on mitosis in human cells.
Topics: Animals; Chromosomes; HeLa Cells; Humans; Kinetochores; Mammals; Metaphase; Microtubules; Spindle Apparatus
PubMed: 35894209
DOI: 10.7554/eLife.75459 -
Methods in Cell Biology 2018Cell division involves mechanical processes, such as chromosome transport and centrosome separation. Quantitative micromanipulation-based approaches have been central to...
Cell division involves mechanical processes, such as chromosome transport and centrosome separation. Quantitative micromanipulation-based approaches have been central to dissecting the forces driving these processes. We highlight two biophysical assays that can be employed for such analyses. First, an in vitro "mini-spindle" assay is described that can be used to examine the collective mechanics of mitotic motor proteins cross-linking two microtubules. In the spindle, motor proteins (e.g., kinesin-5, kinesin-14, and dynein) can localize to overlapping microtubules that slide relative to each other, work as an ensemble, and equilibrate between cytoplasm and the microtubules. The "mini-spindle" assay can recapitulate these features and allows measurements of forces generated between adjacent microtubules and their dependence on filament orientation, sliding speed, overlap length, and motor protein density. Second, we describe a force-calibrated microneedle-based "whole-spindle" micromechanics assay. Microneedle-based micromanipulation can be a useful technique to examine cellular scale mechanics, but its use has been restricted by the difficulty in getting probes to penetrate the plasma membrane without disrupting cell physiology. As detailed here, the use of cell-free extracts prepared from metaphase-arrested Xenopus eggs can address this limitation. These micromanipulation studies also benefit from the use of frozen stocks of Xenopus egg extract. Together, these approaches can be used to decipher how micromechanics and biochemical activities ensure successful cell division.
Topics: Animals; Cell Division; Cell Membrane; Dyneins; Kinesins; Metaphase; Micromanipulation; Microtubules; Spindle Apparatus; Xenopus Proteins; Xenopus laevis
PubMed: 29957203
DOI: 10.1016/bs.mcb.2018.03.022 -
Nature Communications Sep 2019To facilitate proper mitotic cell partitioning, the Golgi disassembles by suppressing vesicle fusion. However, the underlying mechanism has not been characterized...
To facilitate proper mitotic cell partitioning, the Golgi disassembles by suppressing vesicle fusion. However, the underlying mechanism has not been characterized previously. Here, we report a Ran pathway-independent attenuation mechanism that allows Importin-α (a nuclear transport factor) to suppress the vesicle fusion mediated by p115 (a vesicular tethering factor) and is required for mitotic Golgi disassembly. We demonstrate that Importin-α directly competes with p115 for interaction with the Golgi protein GM130. This interaction, promoted by a phosphate moiety on GM130, is independent of Importin-β and Ran. A GM130 K34A mutant, in which the Importin-α-GM130 interaction is specifically disrupted, exhibited abundant Golgi puncta during metaphase. Importantly, a mutant showing enhanced p115-GM130 interaction presented proliferative defects and G2/M arrest, demonstrating that Importin-α-GM130 binding modulates the Golgi disassembly that governs mitotic progression. Our findings illuminate that the Ran and kinase-phosphatase pathways regulate multiple aspects of mitosis coordinated by Importin-α (e.g. spindle assembly, Golgi disassembly).
Topics: Autoantigens; Crystallography, X-Ray; G2 Phase Cell Cycle Checkpoints; Golgi Apparatus; Golgi Matrix Proteins; HEK293 Cells; Humans; Membrane Fusion; Membrane Proteins; Metaphase; Mitosis; Phosphorylation; Protein Binding; Vesicular Transport Proteins; alpha Karyopherins; beta Karyopherins; ran GTP-Binding Protein
PubMed: 31541088
DOI: 10.1038/s41467-019-12207-4 -
Cell Apr 1995We have isolated cDNAs and raised antibodies corresponding to the human homologs of the S. cerevisiae CDC27 and CDC16 proteins, which are tetratrico peptide repeat...
We have isolated cDNAs and raised antibodies corresponding to the human homologs of the S. cerevisiae CDC27 and CDC16 proteins, which are tetratrico peptide repeat (TPR)-containing proteins essential for mitosis in budding yeast. We find that the CDC27Hs and CDC16Hs proteins colocalize to the centrosome at all stages of the mammalian cell cycle, and to the mitotic spindle. Injection of affinity-purified anti-CDC27Hs antibodies into logarithmically growing HeLa cells causes a highly reproducible cell cycle arrest in metaphase with apparently normal spindle structure. We conclude that CDC27 and CDC16 are evolutionarily conserved components of the centrosome and mitotic spindle that control the onset of postmetaphase events during mitosis.
Topics: Anaphase; Antibodies; Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; Apc6 Subunit, Anaphase-Promoting Complex-Cyclosome; Cell Cycle; Cell Cycle Proteins; Centrosome; Cloning, Molecular; Fluorescent Antibody Technique; HeLa Cells; Humans; Immunoblotting; Metaphase; Microinjections; Molecular Sequence Data; Saccharomyces cerevisiae Proteins; Sequence Homology; Spindle Apparatus; Ubiquitin-Protein Ligases
PubMed: 7736578
DOI: 10.1016/0092-8674(95)90336-4 -
The Journal of Cell Biology Nov 1995We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase...
We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase mammalian cells by fluorescence photoactivation. Tubulin derivatized with photoactivatable fluorescein was microinjected into prometaphase LLC-PK and PtK1 cells and allowed to incorporate to steady-state. A fluorescent bar was generated across the MTs in a half-spindle of the mitotic cells using laser irradiation and the kinetics of fluorescence redistribution were determined in terms of a double exponential decay process. The movement of the activated zone was also measured along with chromosome movement and spindle elongation. To investigate the possible regulation of MT transport at the metaphase-anaphase transition, we performed double photoactivation analyses on the same spindles as the cell advanced from metaphase to anaphase. We determined values for the turnover of kMTs (t1/2 = 7.1 +/- 2.4 min at 30 degrees C) and demonstrated that the turnover of kMTs in metaphase is approximately an order of magnitude slower than that for non-kMTs. In anaphase, kMTs become dramatically more stable as evidenced by a fivefold increase in the fluorescence redistribution half-time (t1/2 = 37.5 +/- 8.5 min at 30 degrees C). Our results also indicate that MT transport slows abruptly at anaphase onset to one-half the metaphase value. In early anaphase, MT depolymerization at the kinetochore accounted, on average, for 84% of the rate of chromosome movement toward the pole whereas the relative contribution of MT transport and depolymerization at the pole contributed 16%. These properties reflect a dramatic shift in the dynamic behavior of kMTs at the metaphase-anaphase transition. A release-capture model is presented in which the stability of kMTs is increased at the onset of anaphase through a reduction in the probability of MT release from the kinetochore. The reduction in MT transport at the metaphase-anaphase transition suggests that motor activity and/or subunit dynamics at the centrosome are subject to modulation at this key cell cycle point.
Topics: Anaphase; Animals; Cell Cycle; Cell Line; Kidney Tubules, Proximal; Kinetochores; Metaphase; Microtubules; Photochemistry; Spindle Apparatus; Swine; Temperature; Time Factors; Tubulin
PubMed: 7593192
DOI: 10.1083/jcb.131.3.721 -
Cell Cycle (Georgetown, Tex.) 2014Kinesin-14 motor proteins play a variety of roles during metaphase and anaphase. However, it is not known whether members of this family of motors also participate in...
Kinesin-14 motor proteins play a variety of roles during metaphase and anaphase. However, it is not known whether members of this family of motors also participate in the dramatic changes in mitotic spindle organization during the transition from telophase to cytokinesis. We have identified the minus-end-directed motor, KIFC3, as an important contributor to central bridge morphology at this stage. KIFC3's unique motor-dependent localization at the central bridge allows it to congress microtubules, promoting efficient progress through cytokinesis. Conversely, when KIFC3 function is perturbed, abscission is delayed, and the central bridge is both widened and extended. Examination of KIFC3 on growing microtubules in interphase indicates that it caps microtubules released from the centrosome, both in the region of the centrosome and in the cell periphery. In line with other kinesin-14 family members, KIFC3 may guide free microtubules to their destination at the bridge and/or may slide and crosslink central bridge microtubules in order to stage the cells for abscission.
Topics: Anaphase; Animals; Centrosome; Cytokinesis; Dogs; HeLa Cells; Humans; Kinesins; Madin Darby Canine Kidney Cells; Metaphase; Microtubules; Spindle Apparatus
PubMed: 24275865
DOI: 10.4161/cc.27266