scholarly journals Augmin accumulation on long-lived microtubules drives amplification and kinetochore-directed growth

2019 ◽  
Vol 218 (7) ◽  
pp. 2150-2168 ◽  
Author(s):  
Ana F. David ◽  
Philippe Roudot ◽  
Wesley R. Legant ◽  
Eric Betzig ◽  
Gaudenz Danuser ◽  
...  
Keyword(s):  
Spindle Assembly ◽  
Light Sheet ◽  
Spindle Pole ◽  
Directional Bias ◽  
Fiber Assembly ◽  
Light Sheet Microscopy ◽  
Sister Chromatids ◽  
Spindle Dynamics ◽  
Dividing Cells ◽  
Microtubule Growth

Dividing cells reorganize their microtubule cytoskeleton into a bipolar spindle, which moves one set of sister chromatids to each nascent daughter cell. Early spindle assembly models postulated that spindle pole–derived microtubules search the cytoplasmic space until they randomly encounter a kinetochore to form a stable attachment. More recent work uncovered several additional, centrosome-independent microtubule generation pathways, but the contributions of each pathway to spindle assembly have remained unclear. Here, we combined live microscopy and mathematical modeling to show that most microtubules nucleate at noncentrosomal regions in dividing human cells. Using a live-cell probe that selectively labels aged microtubule lattices, we demonstrate that the distribution of growing microtubule plus ends can be almost entirely explained by Augmin-dependent amplification of long-lived microtubule lattices. By ultrafast 3D lattice light-sheet microscopy, we observed that this mechanism results in a strong directional bias of microtubule growth toward individual kinetochores. Our systematic quantification of spindle dynamics reveals highly coordinated microtubule growth during kinetochore fiber assembly.

scholarly journals Augmin-mediated amplification of long-lived spindle microtubules directs plus-ends to kinetochores

2018 ◽  
Author(s):  
Ana F. David ◽  
Philippe Roudot ◽  
Wesley R. Legant ◽  
Eric Betzig ◽  
Gaudenz Danuser ◽  
...  
Keyword(s):  
Spindle Assembly ◽  
Light Sheet ◽  
Spindle Pole ◽  
Directional Bias ◽  
Fiber Assembly ◽  
Light Sheet Microscopy ◽  
Sister Chromatids ◽  
Spindle Dynamics ◽  
Dividing Cells ◽  
Microtubule Growth

AbstractDividing cells reorganize their microtubule cytoskeleton into a bipolar spindle, which moves one set of sister chromatids to each nascent daughter cell. Early spindle assembly models postulated that spindle-pole-derived microtubules search the cytoplasmic space until they randomly encounter a kinetochore to form a stable attachment. More recent work uncovered several additional, centrosome-independent microtubule generation pathways, but the contributions of each pathway to spindle assembly have remained unclear. Here, we combined live microscopy and mathematical modeling to show that most microtubules nucleate at non-centrosomal regions in dividing human cells. Using a live-cell probe that selectively labels aged microtubule lattices, we demonstrate that the distribution of growing microtubule plus-ends can be almost entirely explained by Augmin-dependent amplification of long-lived microtubules. By ultra-fast 3D lattice light-sheet microscopy, we observed that this mechanism results in a strong directional bias of microtubule growth towards individual kinetochores. Our systematic quantification of spindle dynamics reveals highly coordinated microtubule growth during kinetochore-fiber assembly.

scholarly journals Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells

2014 ◽  
Vol 204 (7) ◽  
pp. 1111-1121 ◽  
Author(s):  
Emmanuel Gallaud ◽  
Renaud Caous ◽  
Aude Pascal ◽  
Franck Bazile ◽  
Jean-Philippe Gagné ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Microtubule Associated Proteins ◽  
Microtubule Polymerization ◽  
Sister Chromatids ◽  
Centrosome Separation ◽  
Associated Proteins ◽  
Microtubule Growth ◽  
Mitotic Spindle Assembly

The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.

scholarly journals Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy

2016 ◽  
Vol 27 (22) ◽  
pp. 3418-3435 ◽  
Author(s):  
François Aguet ◽  
Srigokul Upadhyayula ◽  
Raphaël Gaudin ◽  
Yi-ying Chou ◽  
Emanuele Cocucci ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Imaging ◽  
Single Cells ◽  
Light Sheet ◽  
Membrane Dynamics ◽  
Bottom Surface ◽  
Light Sheet Microscopy ◽  
Dividing Cells ◽  
Entire Cell ◽  
Membrane Bound

Membrane remodeling is an essential part of transferring components to and from the cell surface and membrane-bound organelles and for changes in cell shape, which are particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging and mostly restricted by technical necessity to the attached bottom surface, showed persistent formation of endocytic clathrin pits and vesicles during mitosis. Taking advantage of the resolution, speed, and noninvasive illumination of the newly developed lattice light-sheet fluorescence microscope, we reexamined their assembly dynamics over the entire cell surface and found that clathrin pits form at a lower rate during late mitosis. Full-cell imaging measurements of cell surface area and volume throughout the cell cycle of single cells in culture and in zebrafish embryos showed that the total surface increased rapidly during the transition from telophase to cytokinesis, whereas cell volume increased slightly in metaphase and was relatively constant during cytokinesis. These applications demonstrate the advantage of lattice light-sheet microscopy and enable a new standard for imaging membrane dynamics in single cells and multicellular assemblies.

scholarly journals Three-dimensional tracking of plus-tips by lattice light-sheet microscopy permits the quantification of microtubule growth trajectories within the mitotic apparatus

2015 ◽  
Vol 20 (10) ◽  
pp. 101206 ◽  
Author(s):  
Norio Yamashita ◽  
Masahiko Morita ◽  
Wesley R. Legant ◽  
Bi-Chang Chen ◽  
Eric Betzig ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Light Sheet ◽  
Growth Trajectories ◽  
Mitotic Apparatus ◽  
Light Sheet Microscopy ◽  
Microtubule Growth

scholarly journals Non-rodent mammalian zygotes assemble dual spindles despite the presence of paternal centrosomes

2020 ◽  
Author(s):  
Isabell Schneider ◽  
Marta de Ruijter-Villani ◽  
M. Julius Hossain ◽  
Tom A. E. Stout ◽  
Jan Ellenberg
Keyword(s):  
Self Assembly ◽  
Spindle Assembly ◽  
Light Sheet ◽  
Self Organization ◽  
Parental Genome ◽  
Mammalian Embryo ◽  
Light Sheet Microscopy ◽  
Assembly Pathway ◽  
Spindle Structure ◽  
Human Ivf

AbstractThe first mitosis of the mammalian embryo must partition the parental genomes contained in two pronuclei. In rodent zygotes, sperm centrosomes are degraded and, instead, acentriolar microtubule organizing centers and microtubule self-organization guide the assembly of two separate spindles around the genomes. In non-rodent mammals, including human or bovine, centrosomes are inherited from the sperm and have been widely assumed to be active. Whether non-rodent zygotes assemble a single centrosomal spindle around both genomes, or follow the dual spindle self-assembly pathway is unclear. To address this, we investigated spindle assembly in bovine zygotes by systematic immunofluorescence and real-time light-sheet microscopy. We show that two independent spindles form around the parental genomes despite the presence of centrosomes, which had little effect on spindle structure and were only loosely connected to the two spindles. We conclude that the dual spindle assembly pathway is conserved in non-rodent mammals. This could explain whole parental genome loss frequently observed in blastomeres of human IVF embryos.SummaryThis study investigates spindle assembly during the first embryonic division in bovine zygotes that, like human, inherit centrosomes from the sperm. It shows that two independent microtubule arrays form by self-organization around parental genomes with only loosely connected centrosomes.

Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells

2001 ◽  
Vol 114 (24) ◽  
pp. 4385-4395 ◽  
Author(s):  
Stephen S. Taylor ◽  
Deema Hussein ◽  
Yunmei Wang ◽  
Sarah Elderkin ◽  
Christopher J. Morrow
Keyword(s):  
Cell Cycle ◽  
Protein Kinases ◽  
Budding Yeast ◽  
Spindle Assembly ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Cell Cycle Regulators ◽  
Yeast Gene ◽  
Microtubule Attachment ◽  
Spindle Dynamics

BUB1 is a budding yeast gene required to ensure that progression through mitosis is coupled to correct spindle assembly. Two related human protein kinases, Bub1 and BubR1, both localise to kinetochores during mitosis, suggesting that they play a role in delaying anaphase until all chromosomes achieve correct, bipolar attachment to the spindle. However, how the activities of Bub1 and BubR1 are regulated by spindle events and how their activities regulate downstream cell cycle events is not known.To investigate how spindle events regulate Bub1 and BubR1, we characterised their relative localisations during mitosis in the presence and absence of microtubule toxins. In prometaphase cells, both kinases colocalise to the same domain of the kinetochore. However, whereas the localisation of BubR1 at sister kinetochores is symmetrical, localisation of Bub1 is often asymmetrical. This asymmetry is dependent on microtubule attachment, and the kinetochore exhibiting weaker Bub1 staining is typically closer to the nearest spindle pole. In addition, a 30 minute nocodazole treatment dramatically increases the amount of Bub1 localising to kinetochores but has little effect on BubR1. Furthermore, Bub1 levels increase at metaphase kinetochores following loss of tension caused by taxol treatment. Thus, these observations suggest that Bub1 localisation is sensitive to changes in both tension and microtubule attachment.Consistent with this, we also show that Bub1 is rapidly phosphorylated following brief treatments with nocodazole or taxol. In contrast, BubR1 is phosphorylated in the absence of microtubule toxins, and spindle damage has little additional effect. Although these observations indicate that Bub1 and BubR1 respond differently to spindle dynamics, they are part of a common complex during mitosis. We suggest therefore that Bub1 and BubR1 may integrate different ‘spindle assembly signals’ into a single signal which can then be interpreted by downstream cell cycle regulators.

scholarly journals Dual spindle formation in zygotes keeps parental genomes apart in early mammalian embryos

2017 ◽  
Author(s):  
Judith Reichmann ◽  
Bianca Nijmeijer ◽  
M. Julius Hossain ◽  
Manuel Eguren ◽  
Isabell Schneider ◽  
...  
Keyword(s):  
Genetic Material ◽  
Spindle Assembly ◽  
Light Sheet ◽  
Light Sheet Microscopy ◽  
Nuclear Compartments ◽  
Assembly Mechanism ◽  
Mammalian Embryos ◽  
Cell Embryo ◽  
Bipolar Spindles ◽  
Fertilized Egg

AbstractAt the beginning of mammalian life the genetic material from each parent meets when the fertilized egg divides. It was previously thought that a single microtubule spindle is responsible to spatially combine the two genomes and then segregate them to create the two-cell embryo. Utilizing light-sheet microscopy, we showed that two bipolar spindles form in the zygote, that independently congress the maternal and paternal genomes. These two spindles aligned their poles prior to anaphase but kept the parental genomes apart during the first cleavage. This spindle assembly mechanism provides a rationale for erroneous divisions into more than two blastomeric nuclei observed in mammalian zygotes and reveals the mechanism behind the observation that parental genomes occupy separate nuclear compartments in the two-cell embryo.One Sentence Summary: After fertilization, two spindles form around pro-nuclei in mammalian zygotes and keep the parental genomes apart during the first division.

scholarly journals Dual spindles assemble in bovine zygotes despite the presence of paternal centrosomes

2021 ◽  
Vol 220 (11) ◽  
Author(s):  
Isabell Schneider ◽  
Marta de Ruijter-Villani ◽  
M. Julius Hossain ◽  
Tom A.E. Stout ◽  
Jan Ellenberg
Keyword(s):  
Self Assembly ◽  
Spindle Assembly ◽  
Light Sheet ◽  
Self Organization ◽  
Parental Genome ◽  
Mammalian Embryo ◽  
Light Sheet Microscopy ◽  
Assembly Pathway ◽  
Spindle Structure ◽  
Human Ivf

The first mitosis of the mammalian embryo must partition the parental genomes contained in two pronuclei. In rodent zygotes, sperm centrosomes are degraded, and instead, acentriolar microtubule organizing centers and microtubule self-organization guide the assembly of two separate spindles around the genomes. In nonrodent mammals, including human or bovine, centrosomes are inherited from the sperm and have been widely assumed to be active. Whether nonrodent zygotes assemble a single centrosomal spindle around both genomes or follow the dual spindle self-assembly pathway is unclear. To address this, we investigated spindle assembly in bovine zygotes by systematic immunofluorescence and real-time light-sheet microscopy. We show that two independent spindles form despite the presence of centrosomes, which had little effect on spindle structure and were only loosely connected to the two spindles. We conclude that the dual spindle assembly pathway is conserved in nonrodent mammals. This could explain whole parental genome loss frequently observed in blastomeres of human IVF embryos.

scholarly journals Unrestrained Spindle Elongation during Recovery from Spindle Checkpoint Activation in cdc15-2 Cells Results in Mis-Segregation of Chromosomes

2010 ◽  
Vol 21 (14) ◽  
pp. 2384-2398 ◽  
Author(s):  
Chuan Chung Chai ◽  
Ee Mei Teh ◽  
Foong May Yeong
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Spindle Checkpoint ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Checkpoint Activation ◽  
Sister Chromatids ◽  
Spindle Pole Bodies ◽  
Balance Of Forces ◽  
Spindle Elongation ◽  
Pulling Force

During normal metaphase in Saccharomyces cerevisiae, chromosomes are captured at the kinetochores by microtubules emanating from the spindle pole bodies at opposite poles of the dividing cell. The balance of forces between the cohesins holding the replicated chromosomes together and the pulling force from the microtubules at the kinetochores result in the biorientation of the sister chromatids before chromosome segregation. The absence of kinetochore–microtubule interactions or loss of cohesion between the sister chromatids triggers the spindle checkpoint which arrests cells in metaphase. We report here that an MEN mutant, cdc15-2, though competent in activating the spindle assembly checkpoint when exposed to Noc, mis-segregated chromosomes during recovery from spindle checkpoint activation. cdc15-2 cells arrested in Noc, although their Pds1p levels did not accumulate as well as in wild-type cells. Genetic analysis indicated that Pds1p levels are lower in a mad2Δ cdc15-2 and bub2Δ cdc15-2 double mutants compared with the single mutants. Chromosome mis-segregation in the mutant was due to premature spindle elongation in the presence of unattached chromosomes, likely through loss of proper control on spindle midzone protein Slk19p and kinesin protein, Cin8p. Our data indicate that a slower rate of transition through the cell division cycle can result in an inadequate level of Pds1p accumulation that can compromise recovery from spindle assembly checkpoint activation.

scholarly journals Hec1 Contributes to Mitotic Centrosomal Microtubule Growth for Proper Spindle Assembly through Interaction with Hice1

2009 ◽  
Vol 20 (22) ◽  
pp. 4686-4695 ◽  
Author(s):  
Guikai Wu ◽  
Randy Wei ◽  
Eric Cheng ◽  
Bryan Ngo ◽  
Wen-Hwa Lee
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
De Novo ◽  
Coiled Coil ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Microtubule Growth ◽  
Microtubule Aster ◽  
Interfering Rna

Previous studies have stipulated Hec1 as a conserved kinetochore component critical for mitotic control in part by directly binding to kinetochore fibers of the mitotic spindle and by recruiting spindle assembly checkpoint proteins Mad1 and Mad2. Hec1 has also been reported to localize to centrosomes, but its function there has yet to be elucidated. Here, we show that Hec1 specifically colocalizes with Hice1, a previously characterized centrosomal microtubule-binding protein, at the spindle pole region during mitosis. In addition, the C-terminal region of Hec1 directly binds to the coiled-coil domain 1 of Hice1. Depletion of Hice1 by small interfering RNA (siRNA) reduced levels of Hec1 in the cell, preferentially at centrosomes and spindle pole vicinity. Reduction of de novo microtubule nucleation from mitotic centrosomes can be observed in cells treated with Hec1 or Hice1 siRNA. Consistently, neutralization of Hec1 or Hice1 by specific antibodies impaired microtubule aster formation from purified mitotic centrosomes in vitro. Last, disruption of the Hec1/Hice1 interaction by overexpressing Hice1ΔCoil1, a mutant defective in Hec1 interaction, elicited abnormal spindle morphology often detected in Hec1 and Hice1 deficient cells. Together, the results suggest that Hec1, through cooperation with Hice1, contributes to centrosome-directed microtubule growth to facilitate establishing a proper mitotic spindle.

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