scholarly journals Ase1p Organizes Antiparallel Microtubule Arrays during Interphase and Mitosis in Fission Yeast

2005 ◽  
Vol 16 (4) ◽  
pp. 1756-1768 ◽  
Author(s):  
Isabelle Loïodice ◽  
Jayme Staub ◽  
Thanuja Gangi Setty ◽  
Nam-Phuong T. Nguyen ◽  
Anne Paoletti ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Mitotic Spindle ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Microtubule Organization ◽  
Cellular Processes ◽  
Spindle Midzone ◽  
Fluorescence Live Cell Imaging

Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Δ mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.

scholarly journals Feedback loops in the Plk4–STIL–HsSAS6 network coordinate site selection for procentriole formation

2019 ◽  
Author(s):  
Daisuke Takao ◽  
Koki Watanabe ◽  
Kanako Kuroki ◽  
Daiju Kitagawa
Keyword(s):  
Cell Cycle ◽  
Negative Feedback ◽  
Mitotic Spindle ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Centriole Duplication ◽  
Core Components ◽  
Entire Cell ◽  
Selection For ◽  
Assembly Site

SUMMARYCentrioles are duplicated once in every cell cycle, ensuring the bipolarity of the mitotic spindle. Although the core components have been identified, how they cooperate to achieve high fidelity in centriole duplication remains poorly understood. Here, we show that in centriole duplication the accumulation of STIL and HsSAS6, components of the cartwheel structure, provides negative feedback in the centriolar dynamics of Plk4. By live-cell imaging of endogenously tagged proteins in human cells throughout the entire cell cycle, we quantitatively tracked the dynamics of the critical duplication factors: Plk4, STIL, and HsSAS6. Centriolar Plk4 peaks and then starts decreasing during the late G1 phase, which coincides with the accumulation of STIL at centrioles. Shortly thereafter, the HsSAS6 level increases steeply at the procentriole assembly site. We also show that both STIL and HsSAS6 are necessary for attenuating Plk4 levels. Furthermore, our mathematical modeling and simulation convincingly reproduce the dynamics of these three proteins at centrioles, and suggest that the STIL–HsSAS6 complex in the cartwheel has a negative feedback effect on centriolar Plk4. Combined, these findings illustrate how the dynamic behavior of and interactions between the critical duplication factors coordinate the centriole-duplication process.

scholarly journals Transient Breakage of the Nucleocytoplasmic Barrier Controls Spore Maturation via Mobilizing the Proteasome Subunit Rpn11 in the Fission Yeast Schizosaccharomyces pombe

2020 ◽  
Vol 6 (4) ◽  
pp. 242
Author(s):  
Hui-Ju Yang ◽  
Haruhiko Asakawa ◽  
Chizuru Ohtsuki ◽  
Tokuko Haraguchi ◽  
Yasushi Hiraoka
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Nuclear Membrane ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Leading Edge ◽  
Imaging Analysis ◽  
Transmembrane Region ◽  
Nuclear Envelope Breakdown ◽  
Yeast Meiosis

Forespore membrane (FSM) closure is a process of specialized cytokinesis in yeast meiosis. FSM closure begins with the contraction of the FSM opening and finishes with the disassembly of the leading-edge proteins (LEPs) from the FSM opening. Here, we show that the FSM opening starts to contract when the event of virtual nuclear envelope breakdown (vNEBD) occurs in anaphase II of the fission yeast Schizosaccharomyces pombe. The occurrence of vNEBD controls the redistribution of the proteasomal subunit Rpn11 from the nucleus to the cytosol. To investigate the importance of Rpn11 re-localization during vNEBD, Rpn11 was sequestered at the inner nuclear membrane by fusion with the transmembrane region of Bqt4 (Rpn11-GFP-INM). Remarkably, in the absence of endogenous rpn11+, the cells carrying Rpn11-GFP-INM had abnormal or no spore formation. Live-cell imaging analysis further reveals that the FSM opening failed to contract when vNEBD occurred, and the LEP Meu14 was persistently present at the FSM in the rpn11-gfp-INM cells. The results suggest that the dynamic localization of Rpn11 during vNEBD is essential for spore development.

scholarly journals Live-cell imaging to detect phosphatidylserine externalization in brain endothelial cells exposed to ionizing radiation: implications for the treatment of brain arteriovenous malformations

2016 ◽  
Vol 124 (6) ◽  
pp. 1780-1787 ◽  
Author(s):  
Zhenjun Zhao ◽  
Michael S. Johnson ◽  
Biyi Chen ◽  
Michael Grace ◽  
Jaysree Ukath ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Endothelial Cells ◽  
Ionizing Radiation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Vascular Targeting ◽  
Radiation Doses ◽  
Radiation Induced ◽  
Polarity Sensitive

OBJECT Stereotactic radiosurgery (SRS) is an established intervention for brain arteriovenous malformations (AVMs). The processes of AVM vessel occlusion after SRS are poorly understood. To improve SRS efficacy, it is important to understand the cellular response of blood vessels to radiation. The molecular changes on the surface of AVM endothelial cells after irradiation may also be used for vascular targeting. This study investigates radiation-induced externalization of phosphatidylserine (PS) on endothelial cells using live-cell imaging. METHODS An immortalized cell line generated from mouse brain endothelium, bEnd.3 cells, was cultured and irradiated at different radiation doses using a linear accelerator. PS externalization in the cells was subsequently visualized using polarity-sensitive indicator of viability and apoptosis (pSIVA)-IANBD, a polarity-sensitive probe. Live-cell imaging was used to monitor PS externalization in real time. The effects of radiation on the cell cycle of bEnd.3 cells were also examined by flow cytometry. RESULTS Ionizing radiation effects are dose dependent. Reduction in the cell proliferation rate was observed after exposure to 5 Gy radiation, whereas higher radiation doses (15 Gy and 25 Gy) totally inhibited proliferation. In comparison with cells treated with sham radiation, the irradiated cells showed distinct pseudopodial elongation with little or no spreading of the cell body. The percentages of pSIVA-positive cells were significantly higher (p = 0.04) 24 hours after treatment in the cultures that received 25- and 15-Gy doses of radiation. This effect was sustained until the end of the experiment (3 days). Radiation at 5 Gy did not induce significant PS externalization compared with the sham-radiation controls at any time points (p > 0.15). Flow cytometric analysis data indicate that irradiation induced growth arrest of bEnd.3 cells, with cells accumulating in the G2 phase of the cell cycle. CONCLUSIONS Ionizing radiation causes remarkable cellular changes in endothelial cells. Significant PS externalization is induced by radiation at doses of 15 Gy or higher, concomitant with a block in the cell cycle. Radiation-induced markers/targets may have high discriminating power to be harnessed in vascular targeting for AVM treatment.

Overexpression Phenotypes of Plk1 and Ndrg2 in Schizosaccharomyces Pombe: Fission Yeast System for Mammalian Gene Study

2005 ◽  
Vol 277-279 ◽  
pp. 1-6 ◽  
Author(s):  
Young Joo Jang ◽  
Young Sook Kil ◽  
Jee Hee Ahn ◽  
Jae Hoon Ji ◽  
Jong Seok Lim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Rna Splicing ◽  
Mammalian Cells ◽  
Protein Modification ◽  
Genetic System ◽  
Functional Study ◽  
Mammalian Genes

The fission yeast, Schizosaccharomyces pombe is a single-celled free-living fungus that shares many features with cells of more complicated eukaryotes. Many of the genes required for the cell-cycle control, proteolysis, protein modification, and RNA splicing are highly conserved with those of higher eukaryotes. Moreover, fission yeast has the merit of genetics and its genetic system is already well characterized. As such, the current study evaluated the use of a fission yeast system as a tool for the functional study of mammalian genes and attempted to set up an assay system for novel genes. Since the phenotypes of a deletion mutant and the overexpression of a gene are generally analyzed for a functional study of specific genes in yeast, the present study used overexpression phenotypes to study the functions of mammalian genes. Therefore, based on using a thiamine-repressive promoter, two mammalian genes were expressed in fission yeast, and their overexpressed phenotypes compared with those in mammalian cells. The phenotypes resulting from overexpression were analyzed using a FACS, which analyzes the DNA contents, and a microscope. One of the selected genes was the mammalian Polo-like kinase 1 (Plk1), which is activated and plays a role in the mitotic phase of the cell division cycle. The overexpression of various constructs of Plk1 in the HeLa cells caused cell cycle defects, suggesting that the ectopic Plk1s blocked the endogenous Plk1 in the cells. As expected, when the constructs were overexpressed in the fission yeast system, the cells were arrested in mitosis and defected at the end of mitosis. As such, this data suggests that the Plk1-overexpressed phenotypes were similar in the mammalian cells and the fission yeast, thereby enabling the mammalian Plk1 functions to be approximated in the fission yeast. The other selected gene was the N-Myc downstream-regulated gene 2 (ndrg2), which is upregulated during cell differentiation, yet still not well characterized. When the ndrg2 gene was overexpressed in the fission yeast, the cells contained multi-septa. The septa were positioned well, yet their number increased per cell. Therefore, this gene was speculated to block cell division in the last stage of the cell cycle, making the phenotype potentially useful for explaining cell growth and differentiation in mammalian cells. Accordingly, fission yeast is demonstrated to be an appropriate species for the functional study of mammalian genes.

scholarly journals “Probe, Sample, and Instrument (PSI)”: The Hat-Trick for Fluorescence Live Cell Imaging

Chemosensors ◽  
2018 ◽  
Vol 6 (3) ◽  
pp. 40 ◽  
Author(s):  
Ludovic Galas ◽  
Thibault Gallavardin ◽  
Magalie Bénard ◽  
Arnaud Lehner ◽  
Damien Schapman ◽  
...  
Keyword(s):  
Organic Synthesis ◽  
Fluorescent Probes ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Cell Labeling ◽  
Key Points ◽  
Research Infrastructures ◽  
New Strategies ◽  
Fluorescence Live Cell Imaging

Cell Imaging Platforms (CIPs) are research infrastructures offering support to a number of scientific projects including the choice of adapted fluorescent probes for live cell imaging. What to detect in what type of sample and for how long is a major issue with fluorescent probes and, for this, the “hat-trick” “Probe–Sample–Instrument” (PSI) has to be considered. We propose here to deal with key points usually discussed in CIPs including the properties of fluorescent organic probes, the modality of cell labeling, and the best equipment to obtain appropriate spectral, spatial, and temporal resolution. New strategies in organic synthesis and click chemistry for accessing probes with enhanced photophysical characteristics and targeting abilities will also be addressed. Finally, methods for image processing will be described to optimize exploitation of fluorescence signals.

scholarly journals Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia

2007 ◽  
Vol 204 (9) ◽  
pp. 2213-2224 ◽  
Author(s):  
Dale A. Moulding ◽  
Michael P. Blundell ◽  
David G. Spiller ◽  
Michael R.H. White ◽  
Giles O. Cory ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Actin Polymerization ◽  
Cell Imaging ◽  
Human Gene ◽  
Filamentous Actin ◽  
Gene Encoding ◽  
Activating Mutations ◽  
Spindle Midzone ◽  
Underlying Mechanisms ◽  
Syndrome Protein

Specific mutations in the human gene encoding the Wiskott-Aldrich syndrome protein (WASp) that compromise normal auto-inhibition of WASp result in unregulated activation of the actin-related protein 2/3 complex and increased actin polymerizing activity. These activating mutations are associated with an X-linked form of neutropenia with an intrinsic failure of myelopoiesis and an increase in the incidence of cytogenetic abnormalities. To study the underlying mechanisms, active mutant WASpI294T was expressed by gene transfer. This caused enhanced and delocalized actin polymerization throughout the cell, decreased proliferation, and increased apoptosis. Cells became binucleated, suggesting a failure of cytokinesis, and micronuclei were formed, indicative of genomic instability. Live cell imaging demonstrated a delay in mitosis from prometaphase to anaphase and confirmed that multinucleation was a result of aborted cytokinesis. During mitosis, filamentous actin was abnormally localized around the spindle and chromosomes throughout their alignment and separation, and it accumulated within the cleavage furrow around the spindle midzone. These findings reveal a novel mechanism for inhibition of myelopoiesis through defective mitosis and cytokinesis due to hyperactivation and mislocalization of actin polymerization.

The cell cycle genes cdc22 + and suc22 + of the fission yeast Schizosaccharomyces pombe encode the large and small subunits of ribonucleotide reductase

1993 ◽  
Vol 238-238 (1-2) ◽  
pp. 241-251 ◽  
Author(s):  
Maria-Jose Fernandez Sarabia ◽  
Christopher McInerny ◽  
Pamela Harris ◽  
Colin Gordon ◽  
Peter Fantes
Keyword(s):  
Cell Cycle ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Ribonucleotide Reductase ◽  
Cell Cycle Genes
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