scholarly journals Dynein-dependent Movements of the Mitotic Spindle inSaccharomyces cerevisiae Do Not Require Filamentous Actin

2000 ◽  
Vol 11 (3) ◽  
pp. 863-872 ◽  
Author(s):  
Richard A. Heil-Chapdelaine ◽  
Nguyen K. Tran ◽  
John A. Cooper
Keyword(s):  
Mitotic Spindle ◽  
Living Cell ◽  
Living Cells ◽  
Cell Populations ◽  
Cell Analysis ◽  
Mitotic Spindles ◽  
Filamentous Actin ◽  
Spindle Positioning ◽  
Latrunculin A ◽  
Two Phases

In budding yeast, the mitotic spindle is positioned in the neck between the mother and the bud so that both cells inherit one nucleus. The movement of the mitotic spindle into the neck can be divided into two phases: (1) Kip3p-dependent movement of the nucleus to the neck and alignment of the short spindle, followed by (2) dynein-dependent movement of the spindle into the neck and oscillation of the elongating spindle within the neck. Actin has been hypothesized to be involved in all these movements. To test this hypothesis, we disrupted the actin cytoskeleton with the use of mutations and latrunculin A (latrunculin). We assayed nuclear segregation in synchronized cell populations and observed spindle movements in individual living cells. In synchronized cell populations, no actin cytoskeletal mutant segregated nuclei as poorly as cells lacking dynein function. Furthermore, nuclei segregated efficiently in latrunculin-treated cells. Individual living cell analysis revealed that the preanaphase spindle was mispositioned and misaligned in latrunculin-treated cells and that astral microtubules were misoriented, confirming a role for filamentous actin in the early, Kip3p-dependent phase of spindle positioning. Surprisingly, mispositioned and misaligned mitotic spindles moved into the neck in the absence of filamentous actin, albeit less efficiently. Finally, dynein-dependent sliding of astral microtubules along the cortex and oscillation of the elongating mitotic spindle in the neck occurred in the absence of filamentous actin.

scholarly journals Adducin-1 is essential for mitotic spindle assembly through its interaction with myosin-X

2013 ◽  
Vol 204 (1) ◽  
pp. 19-28 ◽  
Author(s):  
Po-Chao Chan ◽  
Rosaline Y.C. Hsu ◽  
Chih-Wei Liu ◽  
Chien-Chen Lai ◽  
Hong-Chen Chen
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Actin Binding ◽  
Cyclin Dependent Kinase ◽  
Mitotic Spindles ◽  
Mitotic Progression ◽  
Filamentous Actin ◽  
Multipolar Spindles ◽  
Mitotic Spindle Assembly ◽  
Myosin X

Mitotic spindles are microtubule-based structures, but increasing evidence indicates that filamentous actin (F-actin) and F-actin–based motors are components of these structures. ADD1 (adducin-1) is an actin-binding protein that has been shown to play important roles in the stabilization of the membrane cortical cytoskeleton and cell–cell adhesions. In this study, we show that ADD1 associates with mitotic spindles and is crucial for proper spindle assembly and mitotic progression. Phosphorylation of ADD1 at Ser12 and Ser355 by cyclin-dependent kinase 1 enables ADD1 to bind to myosin-X (Myo10) and therefore to associate with mitotic spindles. ADD1 depletion resulted in distorted, elongated, and multipolar spindles, accompanied by aberrant chromosomal alignment. Remarkably, the mitotic defects caused by ADD1 depletion were rescued by reexpression of ADD1 but not of an ADD1 mutant defective in Myo10 binding. Together, our findings unveil a novel function for ADD1 in mitotic spindle assembly through its interaction with Myo10.

scholarly journals Long astral microtubules uncouple mitotic spindles from the cytokinetic furrow

2010 ◽  
Vol 190 (1) ◽  
pp. 35-43 ◽  
Author(s):  
Kathleen E. Rankin ◽  
Linda Wordeman
Keyword(s):  
Mitotic Spindle ◽  
Contractile Activity ◽  
Mitotic Spindles ◽  
Cortical Actin ◽  
Spindle Positioning ◽  
Actin Cortex ◽  
Nonmuscle Myosin Ii ◽  
Early Anaphase ◽  
Cortical Contractility ◽  
Spindle Position

Astral microtubules (MTs) are known to be important for cleavage furrow induction and spindle positioning, and loss of astral MTs has been reported to increase cortical contractility. To investigate the effect of excess astral MT activity, we depleted the MT depolymerizer mitotic centromere-associated kinesin (MCAK) from HeLa cells to produce ultra-long, astral MTs during mitosis. MCAK depletion promoted dramatic spindle rocking in early anaphase, wherein the entire mitotic spindle oscillated along the spindle axis from one proto-daughter cell to the other, driven by oscillations of cortical nonmuscle myosin II. The effect was phenocopied by taxol treatment. Live imaging revealed that cortical actin partially vacates the polar cortex in favor of the equatorial cortex during anaphase. We propose that this renders the polar actin cortex vulnerable to rupture during normal contractile activity and that long astral MTs enlarge the blebs. Excessively large blebs displace mitotic spindle position by cytoplasmic flow, triggering the oscillations as the blebs resolve.

scholarly journals Dynein is regulated by the stability of its microtubule track

2017 ◽  
Vol 216 (7) ◽  
pp. 2047-2058 ◽  
Author(s):  
Cassi Estrem ◽  
Colby P. Fees ◽  
Jeffrey K. Moore
Keyword(s):  
Mitotic Spindle ◽  
Living Cells ◽  
High Resolution Imaging ◽  
Primary Role ◽  
Spindle Positioning ◽  
Rich Domain ◽  
Positioning Errors ◽  
Resolution Imaging ◽  
The Stability ◽  
Dynactin Complex

How dynein motors accurately move cargoes is an important question. In budding yeast, dynein moves the mitotic spindle to the predetermined site of cytokinesis by pulling on astral microtubules. In this study, using high-resolution imaging in living cells, we discover that spindle movement is regulated by changes in microtubule plus-end dynamics that occur when dynein generates force. Mutants that increase plus-end stability increase the frequency and duration of spindle movements, causing positioning errors. We find that dynein plays a primary role in regulating microtubule dynamics by destabilizing microtubules. In contrast, the dynactin complex counteracts dynein and stabilizes microtubules through a mechanism involving the shoulder subcomplex and the cytoskeletal-associated protein glycine-rich domain of Nip100/p150glued. Our results support a model in which dynein destabilizes its microtubule substrate by using its motility to deplete dynactin from the plus end. We propose that interplay among dynein, dynactin, and the stability of the microtubule substrate creates a mechanism that regulates accurate spindle positioning.

scholarly journals Microtubule-based mitotic spindles contain a micron-sized mixed-nucleotide zone

2021 ◽  
Author(s):  
Cedric Castrogiovanni ◽  
Alessio V. Inchingolo ◽  
Jonathan U. Harrison ◽  
Damian Dudka ◽  
Onur Sen ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Computational Simulation ◽  
Living Cells ◽  
Mitotic Spindles ◽  
In Cells

Current models infer that the microtubule-based mitotic spindle is built from GDP-tubulin with small GTP caps at microtubule plus-ends, including those that attach to kinetochores (K-fibres). Here we reveal that K-fibres additionally contain a dynamic mixed-nucleotide zone that reaches several microns in length. This zone becomes visible in cells expressing fluorescently labelled EBs, a known marker for GTP-tubulin, and endogenously-labelled HURP - a protein which we show to preferentially bind the GDP microtubule lattice in vitro. In living cells HURP accumulates on the ends of depolymerising K-fibres, whilst avoiding recruitment to nascent polymerising K-fibres. This gives rise to a growing 'HURP-gap' which we can recapitulate in a minimal computational simulation. We therefore postulate that the K-fibre lattice contains a dynamic, micron-sized mixed-nucleotide zone.

Modulated polarization microscopy displays the dynamics of cytoskeletal structures in living, motile cells

1995 ◽  
Vol 53 ◽  
pp. 902-903
Author(s):  
J. R. Kuhn ◽  
M. Poenie
Keyword(s):  
Mitotic Spindle ◽  
Actin Filaments ◽  
Cell Shape ◽  
Dynamic Structure ◽  
Living Cells ◽  
Cytoskeletal Proteins ◽  
Polarization Microscopy ◽  
Video Enhancement ◽  
Ultrastructural Studies ◽  
Motile Cells

Cell shape and movement are controlled by elements of the cytoskeleton including actin filaments an microtubules. Unfortunately, it is difficult to visualize the cytoskeleton in living cells and hence follow it dynamics. Immunofluorescence and ultrastructural studies of fixed cells while providing clear images of the cytoskeleton, give only a static picture of this dynamic structure. Microinjection of fluorescently Is beled cytoskeletal proteins has proved useful as a way to follow some cytoskeletal events, but long terry studies are generally limited by the bleaching of fluorophores and presence of unassembled monomers.Polarization microscopy has the potential for visualizing the cytoskeleton. Although at present, it ha mainly been used for visualizing the mitotic spindle. Polarization microscopy is attractive in that it pro vides a way to selectively image structures such as cytoskeletal filaments that are birefringent. By combing ing standard polarization microscopy with video enhancement techniques it has been possible to image single filaments. In this case, however, filament intensity depends on the orientation of the polarizer and analyzer with respect to the specimen.

3-D reconstruction and analysis of mammalian mitotic spindle structure

1992 ◽  
Vol 50 (1) ◽  
pp. 578-579
Author(s):  
Kent McDonald ◽  
David Mastronarde ◽  
Rubai Ding ◽  
Eileen O'Toole ◽  
J. Richard McIntosh
Keyword(s):  
Cross Sections ◽  
Mitotic Spindle ◽  
Experimental Studies ◽  
Video Camera ◽  
Three Dimensions ◽  
Mitotic Spindles ◽  
Black And White ◽  
Reconstruction Methods ◽  
Spindle Structure ◽  
Tissue Culture Line

Mammalian spindles are generally large and may contain over a thousand microtubules (MTs). For this reason they are difficult to reconstruct in three dimensions and many researchers have chosen to study the smaller and simpler spindles of lower eukaryotes. Nevertheless, the mammalian spindle is used for many experimental studies and it would be useful to know its detailed structure.We have been using serial cross sections and computer reconstruction methods to analyze MT distributions in mitotic spindles of PtK cells, a mammalian tissue culture line. Images from EM negatives are digtized on a light box by a Dage MTI video camera containing a black and white Saticon tube. The signal is digitized by a Parallax 1280 graphics device in a MicroVax III computer. Microtubules are digitized at a magnification such that each is 10-12 pixels in diameter.

Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

1990 ◽  
Vol 48 (3) ◽  
pp. 166-167
Author(s):  
J. Holy ◽  
G. Schatten
Keyword(s):  
Confocal Microscopy ◽  
Mitotic Spindle ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Two Dimensions ◽  
Embryonic Cells ◽  
Mitotic Spindles ◽  
Cleavage Division ◽  
Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

scholarly journals Nanoarchitectonics on living cells

RSC Advances ◽  
2021 ◽  
Vol 11 (31) ◽  
pp. 18898-18914
Author(s):  
Katsuhiko Ariga ◽  
Rawil Fakhrullin
Keyword(s):  
Living Cell ◽  
Living Cells ◽  
Material Surfaces ◽  
As If

We can introduce functional structures with various components on a living cell as if architectures were constructed on material surfaces.

scholarly journals NuMA-microtubule interactions are critical for spindle orientation and the morphogenesis of diverse epidermal structures

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Lindsey Seldin ◽  
Andrew Muroyama ◽  
Terry Lechler
Keyword(s):  
Cell Fate ◽  
Mitotic Spindle ◽  
Cell Types ◽  
Epidermal Differentiation ◽  
Spindle Orientation ◽  
Direct Function ◽  
Spindle Positioning ◽  
Adult Mice ◽  
Neonatal Lethality ◽  
The Matrix

Mitotic spindle orientation is used to generate cell fate diversity and drive proper tissue morphogenesis. A complex of NuMA and dynein/dynactin is required for robust spindle orientation in a number of cell types. Previous research proposed that cortical dynein/dynactin was sufficient to generate forces on astral microtubules (MTs) to orient the spindle, with NuMA acting as a passive tether. In this study, we demonstrate that dynein/dynactin is insufficient for spindle orientation establishment in keratinocytes and that NuMA’s MT-binding domain, which targets MT tips, is also required. Loss of NuMA-MT interactions in skin caused defects in spindle orientation and epidermal differentiation, leading to neonatal lethality. In addition, we show that NuMA-MT interactions are also required in adult mice for hair follicle morphogenesis and spindle orientation within the transit-amplifying cells of the matrix. Loss of spindle orientation in matrix cells results in defective differentiation of matrix-derived lineages. Our results reveal an additional and direct function of NuMA during mitotic spindle positioning, as well as a reiterative use of spindle orientation in the skin to build diverse structures.

scholarly journals Unmasking Chaotic Attributes in Time Series of Living Cell Populations

PLoS ONE ◽  
2010 ◽  
Vol 5 (2) ◽  
pp. e9346 ◽  
Author(s):  
Michel Laurent ◽  
Jean Deschatrette ◽  
Claire M. Wolfrom
Keyword(s):  
Time Series ◽  
Living Cell ◽  
Cell Populations
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