scholarly journals Astral microtubule crosslinking by Feo safeguards uniform nuclear distribution in the Drosophila syncytium

2019 ◽  
Author(s):  
Ojas Deshpande ◽  
Jorge de-Carvalho ◽  
Diana V. Vieira ◽  
Ivo A. Telley
Keyword(s):  
Internuclear Distance ◽  
Experimental Approach ◽  
Ex Vivo ◽  
Cell Cortex ◽  
Multinucleated Cell ◽  
Nuclear Distribution ◽  
Astral Microtubule ◽  
Mechanistic Insight ◽  
Microtubule Motors ◽  
Nuclear Separation

AbstractThe early insect embryo develops as a multinucleated cell distributing genome uniformly to the cell cortex. Mechanistic insight for nuclear positioning beyond cytoskeletal requirements is missing to date. Contemporary hypotheses propose actomyosin driven cytoplasmic movement transporting nuclei, or repulsion of neighbor nuclei driven by microtubule motors. Here, we show that microtubule crosslinking by Feo and Klp3A is essential for nuclear distribution and internuclear distance maintenance. Germline knockdown causes irregular, less dense nuclear delivery to the cell cortex and smaller distribution in ex vivo embryo explants. A minimal internuclear distance is maintained in explants from control embryos but not from Feo-depleted embryos, following micromanipulation assisted repositioning. A dimerization deficient Feo abolishes nuclear separation in embryo explants while the full-length protein rescues the genetic knockdown. We conclude that Feo and Klp3A crosslinking of antiparallel microtubule overlap generates a length-regulated mechanical link between neighboring microtubule asters. Enabled by a novel experimental approach, our study illuminates an essential process of embryonic multicellularity.

Astral microtubule cross-linking safeguards uniform nuclear distribution in the Drosophila syncytium

2021 ◽  
Vol 221 (1) ◽  
Author(s):  
Ojas Deshpande ◽  
Jorge de-Carvalho ◽  
Diana V. Vieira ◽  
Ivo A. Telley
Keyword(s):  
Internuclear Distance ◽  
Experimental Approach ◽  
Ex Vivo ◽  
Cell Cortex ◽  
Cross Linking ◽  
Nuclear Distribution ◽  
Astral Microtubule ◽  
Mechanistic Insight ◽  
Microtubule Motors ◽  
Nuclear Separation

The early insect embryo develops as a multinucleated cell distributing the genome uniformly to the cell cortex. Mechanistic insight for nuclear positioning beyond cytoskeletal requirements is missing. Contemporary hypotheses propose actomyosin-driven cytoplasmic movement transporting nuclei or repulsion of neighbor nuclei driven by microtubule motors. Here, we show that microtubule cross-linking by Feo and Klp3A is essential for nuclear distribution and internuclear distance maintenance in Drosophila. Germline knockdown causes irregular, less-dense nuclear delivery to the cell cortex and smaller distribution in ex vivo embryo explants. A minimal internuclear distance is maintained in explants from control embryos but not from Feo-inhibited embryos, following micromanipulation-assisted repositioning. A dimerization-deficient Feo abolishes nuclear separation in embryo explants, while the full-length protein rescues the genetic knockdown. We conclude that Feo and Klp3A cross-linking of antiparallel microtubule overlap generates a length-regulated mechanical link between neighboring microtubule asters. Enabled by a novel experimental approach, our study illuminates an essential process of embryonic multicellularity.

scholarly journals Disruption of Astral Microtubule Contact with the Cell Cortex Activates a Bub1, Bub3, and Mad3-dependent Checkpoint in Fission Yeast

2004 ◽  
Vol 15 (7) ◽  
pp. 3345-3356 ◽  
Author(s):  
Sylvie Tournier ◽  
Yannick Gachet ◽  
Vicky Buck ◽  
Jeremy S. Hyams ◽  
Jonathan B.A. Millar
Keyword(s):  
Actin Cytoskeleton ◽  
Fission Yeast ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Yeast Cells ◽  
Cell Cortex ◽  
Checkpoint Proteins ◽  
Sister Chromatids ◽  
Astral Microtubule ◽  
Spindle Rotation

In animal and yeast cells, the mitotic spindle is aligned perpendicularly to the axis of cell division. This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring. In fission yeast, spindle rotation is dependent upon the interaction of astral microtubules with the cortical actin cytoskeleton. In this article, we show that addition of Latrunculin A, which prevents spindle rotation, delays the separation of sister chromatids and anaphase promoting complex-mediated destruction of spindle-associated Securin and Cyclin B. Moreover, we find that whereas sister kinetochore pairs normally congress to the spindle midzone before anaphase onset, this congression is disrupted when astral microtubule contact with the actin cytoskeleton is disturbed. By analyzing the timing of kinetochore separation, we find that this anaphase delay requires the Bub3, Mad3, and Bub1 but not the Mad1 or Mad2 spindle assembly checkpoint proteins. In agreement with this, we find that Bub1 remains associated with kinetochores when spindles are mispositioned. These data indicate that, in fission yeast, astral microtubule contact with the medial cell cortex is monitored by a subset of spindle assembly checkpoint proteins. We propose that this checkpoint ensures spindles are properly oriented before anaphase takes place.

scholarly journals Evidence for dynein and astral microtubule–mediated cortical release and transport of Gαi/LGN/NuMA complex in mitotic cells

2013 ◽  
Vol 24 (7) ◽  
pp. 901-913 ◽  
Author(s):  
Zhen Zheng ◽  
Qingwen Wan ◽  
Jing Liu ◽  
Huabin Zhu ◽  
Xiaogang Chu ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescence Recovery After Photobleaching ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Mitotic Apparatus ◽  
Spindle Positioning ◽  
Spindle Poles ◽  
Astral Microtubule ◽  
Mammalian Homologue ◽  
Cortical Localization

Spindle positioning is believed to be governed by the interaction between astral microtubules and the cell cortex and involve cortically anchored motor protein dynein. How dynein is recruited to and regulated at the cell cortex to generate forces on astral microtubules is not clear. Here we show that mammalian homologue of Drosophila Pins (Partner of Inscuteable) (LGN), a Gαi-binding protein that is critical for spindle positioning in different systems, associates with cytoplasmic dynein heavy chain (DYNC1H1) in a Gαi-regulated manner. LGN is required for the mitotic cortical localization of DYNC1H1, which, in turn, also modulates the cortical accumulation of LGN. Using fluorescence recovery after photobleaching analysis, we show that cortical LGN is dynamic and the turnover of LGN relies, at least partially, on astral microtubules and DYNC1H1. We provide evidence for dynein- and astral microtubule–mediated transport of Gαi/LGN/nuclear mitotic apparatus (NuMA) complex from cell cortex to spindle poles and show that actin filaments counteract such transport by maintaining Gαi/LGN/NuMA and dynein at the cell cortex. Our results indicate that astral microtubules are required for establishing bipolar, symmetrical cortical LGN distribution during metaphase. We propose that regulated cortical release and transport of LGN complex along astral microtubules may contribute to spindle positioning in mammalian cells.

scholarly journals Cdk1-dependent destabilization of long astral microtubules is required for spindle orientation

2020 ◽  
Author(s):  
Divya Singh ◽  
Nadine Schmidt ◽  
Franziska Müller ◽  
Tanja Bange ◽  
Alexander W. Bird
Keyword(s):  
Mitotic Spindle ◽  
Cell Shape ◽  
Microtubule Dynamics ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Spindle Positioning ◽  
Tissue Organization ◽  
Microtubule Stability ◽  
Astral Microtubule ◽  
Early Mitosis

AbstractThe precise execution of mitotic spindle orientation in response to cell shape cues is important for tissue organization and development. The presence of astral microtubules extending from the centrosome towards the cell cortex is essential for this process, but little is understood about the contribution of astral microtubule dynamics to spindle positioning, or how astral microtubule dynamics are regulated spatiotemporally. The mitotic regulator Cdk1-CyclinB promotes destabilization of centrosomal microtubules and increased microtubule dynamics as cells transition from interphase to mitosis, but how Cdk1 activity specifically modulates astral microtubule stability, and whether it impacts spindle positioning, is unknown. Here we uncover a mechanism revealing that Cdk1 destabilizes astral microtubules to ensure spindle reorientation in response to cell shape. Phosphorylation of the EB1-dependent microtubule plus-end tracking protein GTSE1 by Cdk1 in early mitosis abolishes its interaction with EB1 and recruitment to microtubule plus-ends. Loss of Cdk1 activity, or mutation of phosphorylation sites in GTSE1, induces recruitment of GTSE1 to growing microtubule plus-ends in mitosis. This decreases the catastrophe frequency of astral microtubules, and causes an increase in the number of long astral microtubules reaching the cell cortex, which restrains the ability of cells to reorient spindles along the long cellular axis in early mitosis. Astral microtubules must thus not only be present, but also dynamic to allow the spindle to reorient in response to cell shape, a state achieved by selective destabilization of long astral microtubules via Cdk1.

scholarly journals Anillin promotes astral microtubule-directed cortical myosin polarization

2011 ◽  
Vol 22 (17) ◽  
pp. 3165-3175 ◽  
Author(s):  
Yu Chung Tse ◽  
Alisa Piekny ◽  
Michael Glotzer
Keyword(s):  
Myosin Ii ◽  
Cell Polarization ◽  
Cell Cortex ◽  
Polar Regions ◽  
Contractile Ring ◽  
Central Spindle ◽  
Astral Microtubule ◽  
Nonmuscle Myosin Ii ◽  
Evolutionarily Conserved ◽  
Cortical Asymmetry

Assembly of a cytokinetic contractile ring is a form of cell polarization in which the equatorial cell cortex becomes differentiated from the polar regions. Microtubules direct cytokinetic polarization via the central spindle and astral microtubules. The mechanism of central spindle–directed furrow formation is reasonably well understood, but the aster-directed pathway is not. In aster-directed furrowing, cytoskeletal factors accumulate to high levels at sites distal to the asters and at reduced levels at cortical sites near the asters. In this paper, we demonstrate that the cytoskeletal organizing protein anillin (ANI-1) promotes the formation of an aster-directed furrow in Caenorhabditis elegans embryos. Microtubule-directed nonmuscle myosin II polarization is aberrant in embryos depleted of ANI-1. In contrast, microtubule-directed polarized ANI-1 localization is largely unaffected by myosin II depletion. Consistent with a role in the induction of cortical asymmetry, ANI-1 also contributes to the polarization of arrested oocytes. Anillin has an evolutionarily conserved capacity to associate with microtubules, possibly providing an inhibitory mechanism to promote polarization of the cell cortex.

Fourier Transform Infrared Spectroscopy of Protein Adsorption from Whole Blood: Ex Vivo Dog Studies

1981 ◽  
Vol 35 (4) ◽  
pp. 353-357 ◽  
Author(s):  
R. M. Gendreau ◽  
S. Winters ◽  
R. I. Leininger ◽  
D. Fink ◽  
C. R. Hassler ◽  
...  
Keyword(s):  
Fourier Transform ◽  
Protein Adsorption ◽  
Whole Blood ◽  
Experimental Approach ◽  
Ex Vivo ◽  
Fourier Transform Infrared ◽  
Ex Vivo Model ◽  
Flowing Blood ◽  
The Relationship ◽  
Exact Relationship

A Fourier transform infrared/attenuated total reflectance technique has been developed to study protein adsorption onto surfaces. The application of this technique to an ex vivo model using a beagle dog as the source of whole, flowing blood is described (currently, high-quality infrared spectra are being collected at 5-s intervals of protein adsorption). This approach has enabled the authors to identify albumin and glycoproteins as the initially adsorbing species, with the subsequent competitive replacement of part of this protein layer with fibrinogen and other proteins. The exact relationship between the pattern of protein adsorption from whole blood and the generation of a thrombus (clot) is not yet clear, but it is hoped that this type of experimental approach will help clarify the relationship.

scholarly journals Bridging Organ- and Cellular-Level Behavior in Ex Vivo Experimental Platforms Using Populations of Models of Cardiac Electrophysiology

2018 ◽  
Vol 1 (4) ◽  
Author(s):  
Carlos A. Ledezma ◽  
Benjamin Kappler ◽  
Veronique Meijborg ◽  
Bas Boukens ◽  
Marco Stijnen ◽  
...  
Keyword(s):  
Cardiac Electrophysiology ◽  
Experimental Approach ◽  
Potassium Concentration ◽  
Ex Vivo ◽  
Cellular Level ◽  
Proof Of Concept ◽  
Physiological Variability ◽  
Novel Method ◽  
Organ Level ◽  
Pathological Behavior

The inability to discern between pathology and physiological variability is a key issue in cardiac electrophysiology since this prevents the use of minimally invasive acquisitions to predict early pathological behavior. The goal of this work is to demonstrate how experimentally calibrated populations of models (ePoM) may be employed to inform which cellular-level pathologies are responsible for abnormalities observed in organ-level acquisitions while accounting for intersubject variability; this will be done through an exemplary computational and experimental approach. Unipolar epicardial electrograms (EGM) were acquired during an ex vivo porcine heart experiment. A population of the Ten Tusscher 2006 model was calibrated to activation–recovery intervals (ARI), measured from the electrograms, at three representative times. The distributions of the parameters from the resulting calibrated populations were compared to reveal statistically significant pathological variations. Activation–recovery interval reduction was observed in the experiments, and the comparison of the calibrated populations of models suggested a reduced L-type calcium conductance and a high extra-cellular potassium concentration as the most probable causes for the abnormal electrograms. This behavior was consistent with a reduction in the cardiac output (CO) and was confirmed by other experimental measurements. A proof of concept method to infer cellular pathologies by means of organ-level acquisitions is presented, allowing for an earlier detection of pathology than would be possible with current methods. This novel method that uses mathematical models as a tool for formulating hypotheses regarding the cellular causes of observed organ-level behaviors, while accounting for physiological variability has been unexplored.

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