Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

10.3791/60255 ◽  
2019 ◽  
Author(s):  
Dayna L. Mercadante ◽  
Elizabeth A. Crowley ◽  
Amity L. Manning
Keyword(s):  
Cell Fate ◽  
Mitotic Spindle ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell

O-154 First mitotic spindle formation led by sperm centrosome-dependent microtubule organising centres may cause high incidence of zygotic division errors in humans

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Y Kai ◽  
H Kawano ◽  
N Yamashita
Keyword(s):  
Mitotic Spindle ◽  
High Frequency ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Immunofluorescence Assay ◽  
Live Cell ◽  
Microtubule Assembly ◽  
Cell Stage ◽  
Spindle Formation

Abstract Study question Why do multinucleated blastomeres appear at high frequency in two-cell-stage embryos in humans? Summary answer Failure in microtubule assembly during the first mitotic spindle body formation by sperm centrosome-dependent microtubule organising centres (MTOCs) may lead to chromosomal instability. What is known already Unlike that in mice, multinucleated blastomeres appear at high frequency in two-cell-stage embryos in humans. However, the underlying mechanism remains elusive. In mice, multiple acentriolar MTOCs appear around the male and female pronuclei after pronuclear disappearance and contribute to dual-spindle formation, engulfing each parental chromosome. This spindle formation may ensure an error-free division, keeping the chromosomes stable during the first cleavage, as observed in mice, but it is unclear whether a similar mechanism exists in humans. Study design, size, duration To examine how sperm centrosomes contribute to MTOC formation in humans, two types of 3PN zygotes derived fromeither conventional in vitro fertilization (c-IVF, n = 30) or intracytoplasmic sperm injection (ICSI, n = 10) were used. The zygotes were collected from October 2018 to January 2020. MTOC and mitotic spindle formation at consecutive stages of development during the first cleavage were analysed under static and dynamic conditions using immunofluorescence assay and fluorescent live-cell imaging. Participants/materials, setting, methods Under ethics approval, 3PN zygotes were donated by infertile couples undergoing c-IVF or ICSI cycles at the Yamashita Shonan Yume Clinic in Japan. All participants provided informed consent. Immunofluorescence assay was performed using antibodies against α-tubulin, pericentrin, and H3K9me3 after fixation with MTSB-XF solution. Fluorescent live-cell imaging was performed using TagGFP2-H2B mRNA (chromosome marker) and FusionRed-MAP4 mRNA (microtubule marker). Main results and the role of chance Immunofluorescence revealed that while 3PN zygotes derived from c-IVF showed four pericentrin dots, those derived from ICSI exhibited two pericentrin dots. In pro-metaphase, an independent group of chromosomes derived from each pronucleus and MTOCs were formed by the sperm centrosome at the core. Microtubules from each MTOC extended toward the chromosomes in the early metaphase; a quadrupolar spindle was formed in the c-IVF-derived zygotes, and a bipolar spindle was formed in the ICSI-derived zygotes by the MTOCs at the zygote apex after chromosome alignment. In pro-metaphase, the microtubules extended from the MTOCs to the nearest chromosome. Since microtubule assembly was found on oocyte-derived chromosomes, we hypothesised that whether a chromosome is surrounded by microtubules depends on the location of the MTOCs, irrespective of its origin. Live-cell imaging of histone H2B and MAP4 revealed that four MTOCs appeared around the three pronuclei just before the disappearance of the pronuclear membrane; microtubules then extended from the MTOCs toward the chromosomes, beginning to form a mitotic spindle as the chromosomes moved to the centre of the oocyte. Interestingly, one of the three assembled chromosome groups showed no microtubule assembly in the pro-metaphase. Similar results were obtained in all six 3PN zygotes subjected. Limitations, reasons for caution We demonstrated the high risk of developing bare chromosomes not surrounded by microtubules during the formation of the first mitotic spindle, using human tripronuclear zygotes. However, owing to unavailability of normal fertilized oocytes for this study because of the clinical use, we were unable to confirm this in normal zygotes. Wider implications of the findings Although two sperm centrosome-dependent MTOCs are expected to be formed in normal fertilized oocytes, these MTOCs are not sufficient to completely enclose physically separated female and male chromosomes with the microtubules. This explains the high frequency of zygotic division errors that lead to unstable human chromosomes. Trial registration number not applicable

scholarly journals Kinesin-5 regulates the growth of the axon by acting as a brake on its microtubule array

2007 ◽  
Vol 178 (6) ◽  
pp. 1081-1091 ◽  
Author(s):  
Kenneth A. Myers ◽  
Peter W. Baas
Keyword(s):  
Growth Rates ◽  
Mitotic Spindle ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Motor Protein ◽  
Live Cell ◽  
Cultured Neurons ◽  
Wild Type ◽  
Microtubule Array ◽  
Inverse Effect

Kinesin-5 is a homotetrameric motor protein that interacts with adjacent microtubules in the mitotic spindle. Kinesin-5 is also highly expressed in developing postmitotic neurons. Axons of cultured neurons experimentally depleted of kinesin-5 grow up to five times longer than controls and display more branches. The faster growth rates are accompanied by a doubling of the frequency of transport of short microtubules, suggesting a major role for kinesin-5 in the balance of motor-driven forces on the axonal microtubule array. Live-cell imaging reveals that the effects on axonal length of kinesin-5 depletion are caused partly by a lower propensity of the axon and newly forming branches to undergo bouts of retraction. Overexpression of wild-type kinesin-5, but not a rigor mutant of kinesin-5, has the inverse effect on axonal length. These results indicate that kinesin-5 imposes restrictions on the growth of the axon and does so at least in part by generating forces on the axonal microtubule array.

scholarly journals Factor graph analysis of live cell–imaging data reveals mechanisms of cell fate decisions

2015 ◽  
Vol 31 (11) ◽  
pp. 1816-1823 ◽  
Author(s):  
Theresa Niederberger ◽  
Henrik Failmezger ◽  
Diana Uskat ◽  
Don Poron ◽  
Ingmar Glauche ◽  
...  
Keyword(s):  
Cell Fate ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Factor Graph ◽  
Graph Analysis ◽  
Imaging Data ◽  
Cell Fate Decisions

scholarly journals The Fusome Mediates Intercellular Endoplasmic Reticulum Connectivity in Drosophila Ovarian Cysts

2004 ◽  
Vol 15 (10) ◽  
pp. 4512-4521 ◽  
Author(s):  
Erik L. Snapp ◽  
Takako Iida ◽  
David Frescas ◽  
Jennifer Lippincott-Schwartz ◽  
Mary A. Lilly
Keyword(s):  
Endoplasmic Reticulum ◽  
Mitotic Spindle ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Ovarian Cysts ◽  
Cleavage Furrow ◽  
Cytoskeletal Component ◽  
The Common ◽  
Oocyte Differentiation

Drosophila ovarian cysts arise through a series of four synchronous incomplete mitotic divisions. After each round of mitosis, a membranous organelle, the fusome, grows along the cleavage furrow and the remnants of the mitotic spindle to connect all cystocytes in a cyst. The fusome is essential for the pattern and synchrony of the mitotic cyst divisions as well as oocyte differentiation. Using live cell imaging, greenfluorescent protein–tagged proteins, and photobleaching techniques, we demonstrate that fusomal endomembranes are part of a single continuous endoplasmic reticulum (ER) that is shared by all cystocytes in dividing ovarian cysts. Membrane and lumenal proteins of the common ER freely and rapidly diffuse between cystocytes. The fusomal ER mediates intercellular ER connectivity by linking the cytoplasmic ER membranes of all cystocytes within a cyst. Before entry into meiosis and onset of oocyte differentiation (between region 1 and region 2A), ER continuity between cystocytes is lost. Furthermore, analyses of hts and Dhc64c mutants indicate that intercellular ER continuity within dividing ovarian cysts requires the fusome cytoskeletal component and suggest a possible role for the common ER in synchronizing mitotic cyst divisions.

The Spindle Checkpoint Protein BubR1 Is Differentially Expressed in the Lymphatic and Myeloid Lineage and Is a Determinator of Response to Spindle Poisons

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1407-1407
Author(s):  
Dominik Schnerch ◽  
Marie Follo ◽  
Andrea Schmidts ◽  
Julika Krohs ◽  
Julia Felthaus ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mitotic Spindle ◽  
Cancer Biology ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cpg Island ◽  
Mitotic Checkpoint ◽  
Live Cell ◽  
Cyclin B ◽  
Upstream Region

Abstract Abstract 1407 Introduction: Spindle poisons disrupt the mitotic spindle thus leading to activation of the spindle assembly checkpoint (SAC). The SAC is a mitotic surveillance mechanism that can interfere with anaphase-promoting complex/cyclosome- (APC/C-) dependent proteolysis of key cell cycle regulators, such as securin and cyclin B, to delay cells at the metaphase to anaphase transition. The SAC protein BubR1 is a tumor suppressor with critical functions during mitosis and has also been shown to be important for the stablilization of cyclin B during interphase. We found this critical player to be downregulated in AML cell lines, primary AML blast populations and myeloblast-like murine 32D cells when compared to ALL cell lines and lymphoblast-like murine BaF3 cells. While myeloblast-like 32D cells are untransformed cells but also exhibit BubR1 repression we speculate that there might be a physiological role for a reduced BubR1 expression level in healthy myelopoiesis. However, in highly proliferative AML cells BubR1 repression might be a source of genetic instability due to a less efficient SAC-mediated interference with APC/C-dependent proteolysis in the presence of inaccuracies during mitosis. Methods and results: Since repression of BubR1 is known to shorten the metaphase duration, we performed live-cell imaging of leukemia cells and found myeloblastic Kasumi-1 cells, expressing histone H2-GFP, to proceed faster through mitosis as compared to lymphoblastic DG-75 cells. While DG-75 cells exhibited a stable metaphase arrest upon spindle disruption using the spindle poison nocodazole, Kasumi-1 cells showed only a transient arrest, degraded securin and cyclin B and underwent sister-chromatid separation in the absence of a functional mitotic spindle. These findings suggest that the mitotic checkpoint is unable to properly interfere with APC/C-dependent proteolysis to prevent mitotic progression in myeloblastic leukemia upon treatment with spindle poisons. By using inducible retroviral reexpression of BubR1 and its downstream effector cyclin B we could enhance the ability of Kasumi-1 cells to accumulate in mitosis upon spindle disruption. Moreover, restoration of BubR1 led to higher cyclin B levels. Live-cell imaging analyses of Kasumi-1 cells, which expressed doxycyclin-inducible BubR1, revealed a prolonged metaphase, suggesting a more stringent control by the mitotic checkpoint when BubR1 expression is restored. Prolonged metaphase delays were also detected after reexpression of BubR1 when we challenged the cells with lower doses of spindle poisons suggesting that BubR1 is an important sensitizer for antimitotic therapies. Therefore, our finding of low BubR1 expression in AML provides an explanation for the poor response of myeloid leukemia to spindle poisons as compared to lymphoblastic leukemia. BubR1 has also been reported to be inactivated through promoter hypermethylation in various malignancies. The existence of a CpG island in the upstream region of the Bub1b locus (BubR1 coding sequence) tempted us to treat Kasumi-1 cells with the demethylating agent decitabine. Promotor demethylation led to an upregulation of BubR1 in mitotic AML cells providing evidence that BubR1 is a druggable target to enhance mitotic surveillance in AML cells. Conclusions: Because mitotic therapies are widely used in the treatment of different malignancies, a further understanding of these processes might lead to a better understanding of cancer biology and improved therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Single live-cell imaging for systems biology 9

2008 ◽  
Vol 45 ◽  
pp. 121-134 ◽  
Author(s):  
Dhanya Mullassery ◽  
Caroline A. Horton ◽  
Christopher D. Wood ◽  
Michael R.H. White
Keyword(s):  
Gene Expression ◽  
Systems Biology ◽  
Cell Fate ◽  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Protein Localization ◽  
Cell Signalling ◽  
Computational Modelling ◽  
Live Cell

Understanding how mammalian cells function requires a dynamic perspective. However, owing to the complexity of signalling networks, these non-linear systems can easily elude human intuition. The central aim of systems biology is to improve our understanding of the temporal complexity of cell signalling pathways, using a combination of experimental and computational approaches. Live-cell imaging and computational modelling are compatible techniques which allow quantitative analysis of cell signalling pathway dynamics. Non-invasive imaging techniques, based on the use of various luciferases and fluorescent proteins, trace cellular events such as gene expression, protein–protein interactions and protein localization in cells. By employing a number of markers in a single assay, multiple parameters can be measured simultaneously in the same cell. Following acquisition using specialized microscopy, analysis of multi-parameter time-lapse images facilitates the identification of important qualitative and quantitative relationships–linking intracellular signalling, gene expression and cell fate.

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