Orientation of spindle axis and distribution of plasma membrane proteins during cell division in polarized MDCKII cells (original) (raw)
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Regulation of microtubule dynamics and nucleation during polarization in MDCK II cells
The Journal of Cell Biology, 1990
MDCK cells form a polarized epithelium when they reach confluence in tissue culture. We have previously shown that concomitantly with the establishment of intercellular junctions, centrioles separate and microtubules lose their radial organization (). In this work, we have examined the pattern of microtubule nucleation before and after the establishment of intercellular contacts. We analyzed the elongation rate and stability of microtubules in single and confluent cells. This was achieved by microinjection of Paramecium axonemal tubulin and detection of the newly incorporated subunits by an antibody directed specifically against the Paramecium axonemal tubulin. The determination of newly nucleated microtubule localization has been made possible by the use of advanced double-1. Abbreviations used in this paper: MAP, microtubule-associated protein; MCM, modular confocaI microscope.
Cytoskeleton, 2012
The mechanism by which spindle microtubules (MTs) determine the site of cell division in animal cells is still highly controversial. Putative cytokinesis ''signals'' have been proposed to be positioned by spindle MTs at equatorial cortical regions to increase cortical contractility and/or at polar regions to decrease contractility [Rappaport, 1986; von Dassow, 2009]. Given the relative paucity of MTs at the future division site, it has not been clear how MTs localize cytokinesis factors there. Here, we test cytokinesis models using computational and experimental approaches. We present a simple lattice-based model in which signal-kinesin complexes move by transient plus-end directed movements on MTs interspersed with occasions of uniform diffusion in the cytoplasm. In simulations, complexes distribute themselves initially at the spindle midzone and then move on astral MTs to accumulate with time at the equatorial cortex. Simulations accurately predict cleavage patterns of cells with different geometries and MT arrangements and elucidate several experimental observations that have defied easy explanation by previous models. We verify this model with experiments on indented sea urchin zygotes showing that cells often divide perpendicular to the spindle at sites distinct from the indentations. These studies support an equatorial stimulation model and provide a simple mechanism explaining how cytokinesis factors localize to the future division site. V
Cell Motility and The Cytoskeleton - CELL MOTIL CYTOSKELETON, 1983
The microtubule-containing structures that appear in eggs during fertilization and cell division in the sea urchins Lytechinus variegatus and Arbacia punctulata were detected by antitubulin immunofluorescence microscopy of detergent extracted cytoskeletal preparations. The extraction buffer, which is composed of 0.55 mM MgC12, 10 mM EGTA, 25 mM MES, 25% glycerol, 1% Nonidet P-40, and 25 pM PMSF, pH 6.7, allows for dramatically improved fluorescent images compared to those obtained using conventional staining procedures, with residual background staining being reduced to near zero.
Morphogenetic Properties of Microtubules and Mitotic Spindle Assembly
Cell, 1996
The second property is the polarity of microtubules: we discuss how interactions between polar microtubules Cell Biology Program European Molecular Biology Laboratoy and microtubule-based motors organize randomly polymerized microtubules into ordered arrays. Spindle as-Meyerhofstrasse 1 69012 Heidelberg sembly today is extensively studied in a number of different systems, and we make no attempt here to review Federal Republic of Germany the whole field. In particular, we do not address the problem of chromosome segregation, concentrating
The cytoskeleton in development of epithelial cell polarity
Journal of Cell Science, 1993
The polarization of intestinal epithelial cells and the stereotypic arrangement of their actin-based cytoskel eton have made these epithelia an excellent system to explore the organization and formation of a cortical actin-based cytoskeleton. Through a combined morpho logical and biochemical analysis, the molecular arrange ment of many of the components of the brush border has been elucidated. Study of brush border assembly in the Crypts of Lieberkiihn suggests that cytoskeletal mRNA and protein expression, as well as morphologi cal development, occur rapidly following cell differen tiation. Protein kinases appear to be important regula tors of intestinal cell growth, for differentiating cells in the crypts possess 15-fold higher levels of tyrosine phosphorylated proteins than differentiated cells of the villus. One of these kinases, pp60c'5Te, has a 4-to 7-fold higher activity in crypts and increased association with the cytoskeleton than it has in villus cells.
Development, Growth and Differentiation, 1994
In order to study the dynamic behavior of the mitotic apparatus leading to unequal cleavage, we investigated the distribution of mitotic microtubules (MTs) during maturation division of starfish oocytes. When the mitotic apparatus attached to the cell surface at metaphase, in both the first and second meiotic division, it is revealed, by immunofluorescence, that the MT distribution in the spindle, as well as in the aster, became asymmetric. MTs in the peripheral half spindle increased in number compared with those in the inner half spindle. Furthermore, these results were confirmed in the living cell by polarization microscopy; shortly after the attachment, the birefringence retardation of the peripheral half spindle became greater than that of the inner one, and the difference increased with time during anaphase. By inhibiting the attachment of the mitotic apparatus by means of centrifugation, the MT distribution maintained a symmetrical pattern through mitosis. These results suggest that the attachment of the mitotic apparatus to the cell surface induces the asymmetrical distribution of MTs not only in the aster but also in the spindle. Such a rich distribution of MTs in the peripheral half spindle appears to ensure chromosome exclusion into the polar body by anchoring them firmly to the cell surface of the animal pole.
Effect of Cell Shape and Dimensionality on Spindle Orientation and Mitotic Timing
The formation and orientation of the mitotic spindle is a critical feature of mitosis. The morphology of the cell and the spatial distribution and composition of the cells' adhesive microenvironment all contribute to dictate the position of the spindle. However, the impact of the dimensionality of the cells' microenvironment has rarely been studied. In this study we present the use of a microwell platform, where the internal surfaces of the individual wells are coated with fibronectin, enabling the three-dimensional presentation of adhesive ligands to single cells cultured within the microwells. This platform was used to assess the effect of dimensionality and cell shape in a controlled microenvironment. Single HeLa cells cultured in circular microwells exhibited greater tilting of the mitotic spindle, in comparison to cells cultured in square microwells. This correlated with an increase in the time required to align the chromosomes at the metaphase plate due to prolonged activation of the spindle checkpoint in an actin dependent process. The comparison to 2D square patterns revealed that the dimensionality of cell adhesions alone affected both mitotic timings and spindle orientation; in particular the role of actin varied according to the dimensionality of the cells' microenvironment. Together, our data revealed that cell shape and the dimensionality of the cells' adhesive environment impacted on both the orientation of the mitotic spindle and progression through mitosis.
Girds ‘n’ cleeks o' cytokinesis: microtubule sticks and contractile hoops in cell division
Biochemical Society Transactions, 2008
Microtubules maintain an intimate relationship with the rings of anillin, septins and actomyosin filaments throughout cytokinesis. In Drosophila, peripheral microtubules emanating from the spindle poles contact the equatorial cell cortex to deliver the signal that initiates formation of the cytokinetic furrow. Mutations that affect microtubule stability lead to ectopic furrowing because peripheral microtubules contact inappropriate cortical sites. The PAV-KLP (Pavarotti-kinesin-like protein)/RacGAP50C (where GAP is GTPase-activating protein) centralspindlin complex moves towards the plus ends of microtubules to reach the cell equator. When RacGAP50C is tethered to the cell membrane, furrowing initiates at multiple non-equatorial sites, indicating that mis-localization of this single molecule is sufficient to promote furrowing. Furrow formation and ingression requires RhoA activation by the RhoGEF (guanine-nucleotide-exchange factor) Pebble, which interacts with RacGAP50C. RacGAP50C also binds anillin, which associates with actin, myosin and septins. Thus RacGAP50C plays a pivotal role during furrow formation by activating RhoA and linking the peripheral microtubules with the nascent rings through its interaction with anillin.