Formation and functions of asymmetric microtubule organization in polarized cells

Although microtubules are known to be essential for chromosome segregation during cell division, they also play important roles in the regulation and function of cell polarity. Cell polarization is fundamental to appropriate tissue patterning and the regulation of cellular diversity during animal development. In polarized cells, microtubules are often organized asymmetrically along the polarity axis. Recent studies show that such asymmetry in microtubule organization is important to connect a cell's polarization with its polarized functions. In some cases, asymmetrically organized microtubule arrays themselves induce cell polarity. Here we present an overview of the mechanisms and functions of asymmetric microtubule organization and discuss the possible role of microtubule asymmetry in the symmetry-breaking that leads to cell polarization.     

Regulation of a formin complex by the microtubule plus end protein tea1p

The plus ends of microtubules have been speculated to regulate the actin cytoskeleton for the proper positioning of sites of cell polarization and cytokinesis. In the fission yeast Schizosaccharomyces pombe, interphase microtubules and the kelch repeat protein tea1p regulate polarized cell growth. Here, we show that tea1p is directly deposited at cell tips by microtubule plus ends. Tea1p associates in large "polarisome" complexes with bud6p and for3p, a formin that assembles actin cables. Tea1p also interacts in a separate complex with the CLIP-170 protein tip1p, a microtubule plus end-binding protein that anchors tea1p to the microtubule plus end. Localization experiments suggest that tea1p and bud6p regulate formin distribution and actin cable assembly. Although single mutants still polarize, for3Deltabud6Deltatea1Delta triple-mutant cells lack polarity, indicating that these proteins contribute overlapping functions in cell polarization. Thus, these experiments begin to elucidate how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth.     

Shaping fission yeast cells by rerouting actin-based transport on microtubules

Kinesins and myosins transport cargos to specific locations along microtubules and actin filaments, respectively. The relative contribution of the two transport systems for cell polarization varies extensively in different cell types, with some cells relying exclusively on actin-based transport while others mainly use microtubules. Using fission yeast, we asked whether one transport system can substitute for the other. In this organism, microtubules and actin cables both contribute to polarized growth by transporting cargos to cell poles, but with distinct roles: microtubules transport landmarks to label cell poles for growth and actin assembly but do not directly contribute to the growth process [1]. Actin cables serve as tracks for myosin V delivery of growth vesicles to cell poles [ [2] , [3] and [4] ]. We engineered a chimera between the motor domain of the kinesin 7 Tea2 and the globular tail of the myosin V Myo52, which we show transports Ypt3, a myosin cargo receptor, to cell poles along microtubules. Remarkably, this chimera restores polarized growth and viability to cells lacking actin cables. It also bypasses the normal microtubule-dependent marking of cell poles for polarized growth, but not for other functions. Thus, a synthetic motor protein successfully redirects cargos along a distinct cytoskeletal route.

Video Abstract

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4.

Structural change in the cell center during fibroblast polarization and movement     

G O Gudima  I A Vorob'ev  Iu S Chentsov 《Tsitologiia》1983,25(8):883-888

In polarizing and migrating 3T3/Balb mouse fibroblasts, the centrioles are located between the nucleus and the leading edge of the cell. In cells within the monolayer and in migrating cells, the centrioles have a random orientation towards the substrate. In polarized cells, that still remain in the monolayer, one centriole may be perpendicular to the substrate plane in 70% of cases. Upon polarization and migration of fibroblasts, the number of microtubules, which radiate from the centriolar region, increases. These data support a hypothesis that the number of microtubules in the cell centre characterizes the rate of their renovation in the cytoplasm. It is concluded that the cell centre is strongly involved in polarization and migration of fibroblasts.     

5.

Cell biology of planar polarity transmission in the Drosophila wing     

Eaton S 《Mechanisms of development》2003,120(11):1257-1264

The coordination of epithelial planar polarization is a critical step in the formation of well-ordered tissues. The process has been extensively studied in Drosophila, where genetic analysis has identified a set of "tissue polarity" genes that serve to coordinate planar polarity of cells in the developing wings, bristles and eyes. In the last several years, it has emerged that six of these genes encode junctional proteins. In the wing epithelium, these proteins undergo a polarized redistribution, forming separate proximal and distal cortical domains within each cell. The mechanisms that mediate cortical polarization and cue its direction have been the subject of intense investigation. Cuing the orientation of cortical polarization appears to depend on the atypical Cadherins Fat and Dachsous, although these proteins do not become polarized themselves, nor do they colocalize with components of polarized cortical domains. Interestingly, these Cadherins also act at earlier developmental stages to polarize tissue growth along the proximal-distal axis and it will be interesting to see whether these processes are mechanistically related. Once the axis of polarization is determined, cortical polarity seems to be propagated, at least locally, by a cascade of direct cell-cell interactions mediated by the proximal and distal domains. The cell biological mechanisms leading to polarization are still unclear, but the process depends on the control of Protein Phosphatase 2A activity by its regulatory subunit, Widerborst. Interestingly, Widerborst is found on a planar web of microtubules with connections to apical junctions, suggesting that these microtubules may have an important function in polarizing the cortex.     

6.

Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170   总被引：27，自引：0，他引：27  

Fukata M  Watanabe T  Noritake J  Nakagawa M  Yamaga M  Kuroda S  Matsuura Y  Iwamatsu A  Perez F  Kaibuchi K 《Cell》2002,109(7):873-885

Linkage of microtubules to special cortical regions is essential for cell polarization. CLIP-170 binds to the growing ends of microtubules and plays pivotal roles in orientation. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts with CLIP-170. In Vero fibroblasts, IQGAP1 localizes at the polarized leading edge. Expression of carboxy-terminal fragment of IQGAP1, which includes the CLIP-170 binding region, delocalizes GFP-CLIP-170 from the tips of microtubules and alters the microtubule array. Activated Rac1/Cdc42, IQGAP1, and CLIP-170 form a tripartite complex. Furthermore, expression of an IQGAP1 mutant defective in Rac1/Cdc42 binding induces multiple leading edges. These results indicate that Rac1/Cdc42 marks special cortical spots where the IQGAP1 and CLIP-170 complex is targeted, leading to a polarized microtubule array and cell polarization.     

7.

Interplay between microtubule dynamics and intracellular organization     

de Forges H  Bouissou A  Perez F 《The international journal of biochemistry & cell biology》2012,44(2):266-274

Microtubules are hollow tubes essential for many cellular functions such as cell polarization and migration, intracellular trafficking and cell division. They are polarized polymers composed of α and β tubulin that are, in most cells, nucleated at the centrosome at the center of the cell. Microtubule plus-ends are oriented towards the periphery of the cell and explore the cytoplasm in a very dynamic manner. Microtubule alternate between phases of growth and shrinkage in a manner described as dynamic instability. Their dynamics is highly regulated by multiple factors: tubulin post-translational modifications such as detyrosination or acetylation, and microtubule-associated proteins, among them the plus-tip tracking proteins. This regulation is necessary for microtubule functions in the cell. In this review, we will focus on the role of microtubules in intracellular organization. After an overview of the mechanisms responsible for the regulation of microtubule dynamics, the major roles of microtubules dynamics in organelle positioning and organization in interphase cells will be discussed. Conversely, the role of certain organelles, like the nucleus and the Golgi apparatus as microtubule organizing centers will be reviewed. We will then consider the role of microtubules in the establishment and maintenance of cell polarity using few examples of cell polarization: epithelial cells, neurons and migrating cells. In these cells, the microtubule network is reorganized and undergoes specific and local regulation events; microtubules also participate in the intracellular reorganization of different organelles to ensure proper cell differentiation.     

8.

Transmembrane control of laminin, lectin receptors and surface villi in teratocarcinoma-derived endodermal cells     

H Hamada  M Sato  M Ozawa  Y Ohi  T Muramatsu 《Experimental cell research》1984,154(1):299-303

Distribution of laminin on the surface of teratocarcinoma-derived parietal endoderm cells was studied by immuno-histochemical staining of the fixed specimen using affinity-purified anti-laminin antibody. Laminin was distributed on the basal surface of the cells, while treatment either with colchicine or with cytochalasin D (CD) resulted in a severely polarized distribution; laminin was seen only at one end of the cell. Treatment with both the reagents did not cause the severe polarization. Receptors for lectins and cell surface villi were polarized by treatment with CD but not by treatment with colchicine. These results suggest that laminin--or its cell surface receptor--is linked to both microfilament and microtubules and that the mode of transmembrane control for laminin is different from certain other cell surface components of the cells.     

9.

Microtubule targeting of substrate contacts promotes their relaxation and dissociation.   总被引：13，自引：0，他引：13  

I Kaverina  O Krylyshkina  J V Small 《The Journal of cell biology》1999,146(5):1033-1044

We recently showed that substrate contact sites in living fibroblasts are specifically targeted by microtubules (Kaverina, I., K. Rottner, and J.V. Small. 1998. J. Cell Biol. 142:181-190). Evidence is now provided that microtubule contact targeting plays a role in the modulation of substrate contact dynamics. The results are derived from spreading and polarized goldfish fibroblasts in which microtubules and contact sites were simultaneously visualized using proteins conjugated with Cy-3, rhodamine, or green fluorescent protein.For cells allowed to spread in the presence of nocodazole the turnover of contacts was retarded, as compared with controls and adhesions that were retained under the cell body were dissociated after microtubule reassembly. In polarized cells, small focal complexes were found at the protruding cell front and larger adhesions, corresponding to focal adhesions, at the retracting flanks and rear. At retracting edges, multiple microtubule contact targeting preceded contact release and cell edge retraction. The same effect could be observed in spread cells, in which microtubules were allowed to reassemble after local disassembly by the application of nocodazole to one cell edge. At the protruding front of polarized cells, focal complexes were also targeted and as a result remained either unchanged in size or, more rarely, were disassembled. Conversely, when contact targeting at the cell front was prevented by freezing microtubule growth with 20 nM taxol and protrusion stimulated by the injection of constitutively active Rac, peripheral focal complexes became abnormally enlarged. We further found that the local application of inhibitors of myosin contractility to cell edges bearing focal adhesions induced the same contact dissociation and edge retraction as observed after microtubule targeting.Our data are consistent with a mechanism whereby microtubules deliver localized doses of relaxing signals to contact sites to retard or reverse their development. We propose that it is via this route that microtubules exert their well-established control on cell polarity.     

10.

Alternative routes for the establishment of surface polarity during compaction of the mouse embryo     

E Houliston  S J Pickering  B Maro 《Developmental biology》1989,134(2):342-350

During the process of compaction, mouse 8-cell blastomeres flatten upon each other and polarize along an axis perpendicular to cell contacts. If the process of flattening is prevented, polarization can still occur, but does so in a lower proportion of cells than for control populations, and without the normal contact-directed orientation. We compared contact-directed and noncontact-directed processes to see if they involve common mechanisms. In nonflattened cells, surface polarization was favored in cells with nuclei located close to the cell surface, and the positions of surface poles and of nuclei tended to coincide. We present evidence that microtubules are involved in the development of microvillous poles associated with nuclei. In contrast it is known that polarization of microvilli occurs in the absence of microtubules if blastomeres are allowed to flatten. We conclude that surface polarization of mouse blastomeres can be accomplished by at least two alternative routes. One requires flattening but is independent of microtubules, and another can occur without flattening but involves a microtubule-mediated interaction between the nucleus and the cell cortex. It seems that both these pathways operate in the undisturbed embryo.     

11.

Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid     

Yap AS  Manley SW 《Cell motility and the cytoskeleton》2001,48(3):201-212

In this study, we examined the contribution of microtubules to epithelial morphogenesis in primary thyroid cell cultures. Thyroid follicles consist of a single layer of polarized epithelial cells surrounding a closed compartment, the follicular lumen. Freshly isolated porcine thyroid cells aggregate and reorganize to form follicles when grown in primary cultures. Follicular reorganization is principally a morphogenetic process that entails the assembly of biochemically distinct apical and basolateral membrane domains, delimited by tight junctions. The establishment of cell surface polarity during folliculogenesis coincided with the polarized redistribution of microtubules, predominantly in the developing apical poles of cells. Disruption of microtubule integrity using either colchicine or nocodazole caused loss of defined apical membrane domains, tight junctions and follicular lumina. Apical membrane and tight junction markers became randomly distributed at the outer surfaces of aggregates. In contrast, the basolateral surface markers, E-cadherin and Na(+),K(+)-ATPase, remained correctly localized at sites of cell-cell contact and at the free surfaces of cell aggregates. These findings demonstrate that microtubules play a necessary role in thyroid epithelial morphogenesis. Specifically, microtubules are essential to preserve the correct localization of apical membrane components within enclosed cellular aggregates, a situation that is also likely to pertain where lumina must be formed from solid aggregates of epithelial precursors.     

12.

The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium   总被引：18，自引：11，他引：7       下载免费PDF全文

《The Journal of cell biology》1989,109(6):2817-2832

Studies of the developing trophectoderm in the mouse embryo have shown that extensive cellular remodeling occurs during epithelial formation. In this investigation, confocal immunofluorescence microscopy is used to examine the three-dimensional changes in cellular architecture that take place during the polarization of a terminally differentiated epithelial cell line. Madin-Darby canine kidney cells were plated at a low density on permeable filter supports. Antibodies that specifically recognize components of the tight junction, adherens junction, microtubules, centrosomes, and the Golgi complex were used to study the spatial remodeling of the cytoarchitecture during the formation of the polarized cell layer. The immunofluorescence data were correlated with establishment of functional tight junctions as measured by transepithelial resistance and back-exchange of the cell surface, labeled with metabolites of the fluorescent lipid analogue N-(7-[4- nitrobenzo-2-oxa-1,3-diazole]) aminocaproyl sphingosine. 1 d after plating, single cells had microtubules, radiating from a broad region, that contained the centrosomes and the Golgi complex. 2 d after plating, the cells had grown to confluence and had formed functional tight junctions close to the substratum. The centrioles had split and no longer organized the microtubules which were running above and below the nucleus. The Golgi complex had spread around the nucleus. By the fifth day after plating, the final polarized state had been achieved. The junctional complex had moved greater than 10 microns upward from its basal location. The centrioles were together below the apical membrane, and the Golgi complex formed a ribbon-like convoluted structure located in the apical region above the nucleus. The microtubules were organized in an apical web and in longitudinal microtubule bundles in the apical-basal axis of the columnar cell. The longitudinal microtubules were arranged with their minus ends spread over the apical region of the cell and their plus ends toward the basal region. These findings show that there is an extensive remodeling of epithelial cytoarchitecture after formation of cell-cell contacts. Reorganization of the microtubule network results in functional polarization of the cytoplasm.     

13.

The Rho-mDia1 pathway regulates cell polarity and focal adhesion turnover in migrating cells through mobilizing Apc and c-Src          下载免费PDF全文

Yamana N  Arakawa Y  Nishino T  Kurokawa K  Tanji M  Itoh RE  Monypenny J  Ishizaki T  Bito H  Nozaki K  Hashimoto N  Matsuda M  Narumiya S 《Molecular and cellular biology》2006,26(18):6844-6858

Directed cell migration requires cell polarization and adhesion turnover, in which the actin cytoskeleton and microtubules work critically. The Rho GTPases induce specific types of actin cytoskeleton and regulate microtubule dynamics. In migrating cells, Cdc42 regulates cell polarity and Rac works in membrane protrusion. However, the role of Rho in migration is little known. Rho acts on two major effectors, ROCK and mDia1, among which mDia1 produces straight actin filaments and aligns microtubules. Here we depleted mDia1 by RNA interference and found that mDia1 depletion impaired directed migration of rat C6 glioma cells by inhibiting both cell polarization and adhesion turnover. Apc and active Cdc42, which work together for cell polarization, localized in the front of migrating cells, while active c-Src, which regulates adhesion turnover, localized in focal adhesions. mDia1 depletion impaired localization of these molecules at their respective sites. Conversely, expression of active mDia1 facilitated microtubule-dependent accumulation of Apc and active Cdc42 in the polar ends of the cells and actin-dependent recruitment of c-Src in adhesions. Thus, the Rho-mDia1 pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and delivering Apc/Cdc42 and c-Src to their respective sites of action.     

14.

Regulation of cell migration by dynamic microtubules     

Kaverina I  Straube A 《Seminars in cell & developmental biology》2011,(9):968-974

Microtubules define the architecture and internal organization of cells by positioning organelles and activities, as well as by supporting cell shape and mechanics. One of the major functions of microtubules is the control of polarized cell motility. In order to support the asymmetry of polarized cells, microtubules have to be organized asymmetrically themselves. Asymmetry in microtubule distribution and stability is regulated by multiple molecular factors, most of which are microtubule-associated proteins that locally control microtubule nucleation and dynamics. At the same time, the dynamic state of microtubules is key to the regulatory mechanisms by which microtubules regulate cell polarity, modulate cell adhesion and control force-production by the actin cytoskeleton. Here, we propose that even small alterations in microtubule dynamics can influence cell migration via several different microtubule-dependent pathways. We discuss regulatory factors, potential feedback mechanisms due to functional microtubule-actin crosstalk and implications for cancer cell motility.     

15.

Analogous ultrastructure and surface properties during capping and phagocytosis in leukocytes   总被引：27，自引：12，他引：15       下载免费PDF全文

R D Berlin  J M Oliver 《The Journal of cell biology》1978,77(3):789-804

Ultrastructural analyses have revealed striking similarities between Concanavalin A capping and phagocytosis in leukocytes. Both processes involve extensive membrane movement to form a protuberance or pseudopods; a dense network of microfilaments is recruited into both the protuberance and the pseudopods; microtubules are disassembled either generally (capping) or in the local region of the pseudopods (phagocytosis); and cells generally depleted of microtubules by colchicine show polarized phagocytosis via the microfilament-rich protuberance rather than uniform peripheral ingestion of particles via individual pseudopods. Cap formation can thus be viewed as occurring as an exaggeration of the same ultrastructural events that mediate phagocytosis. Similar changes in cell surface topography also accompany capping and phagocytosis. Thus, in nonfixed cells, Concanavalin A-receptor complexes aggregate into the region of the protuberance in colchicine-treated leukocytes (conventional capping) or into the region of pseudopod formation in phagocytizing leukocytes. In the latter case, the movement of lectin-receptor complexes occurs from membrane overlying peripheral microtubules into filament-rich pseudopods that exclude microtubules. These data provide evidence against a role for microtubules as "anchors" for lectin receptors. Rather, they indicate a preferential movement of cell surface Concanavalin A-receptor complexes towards areas of extensive (the protuberance) or localized (pseudopods) microfilament concentration. In conventional capping, Concanavalin A must be added to the colchicine-treated cells before fixation in order to demonstrate movement of receptors from a diffuse distribution into the protuberance. However, Convanavalin A receptors are enriched in the membrane associated with phagocytic particles as compared to the remaining membrane. This particle-induced redistribution of receptors is particularly prominent in colchicine-treated cells that phagocytize and are then fixed and Concanavalin A labeled; both lectin receptors and beads are concentrated over the protuberance. Thus, the final analogy between conventionally capped and phagocytic cells is that in both cases the properties of the plasma membrane in regions of microfilament concentration are modified by Concanavalin A itself (capping) or by the phagocytized particle, to limit locally the diffusion of Concanavalin A receptors.     

16.

The posttranslational modification of tubulin undergoes a switch from detyrosination to acetylation as epithelial cells become polarized     

Quinones GB  Danowski BA  Devaraj A  Singh V  Ligon LA 《Molecular biology of the cell》2011,22(7):1045-1057

Tubulin posttranslational modifications (PTMs) have been suggested to provide navigational cues for molecular motors to deliver cargo to spatially segregated subcellular domains, but the molecular details of this process remain unclear. Here we show that in Madin-Darby Canine Kidney (MDCK) epithelial cells, microtubules express several tubulin PTMs. These modifications, however, are not coordinated, and cells have multiple subpopulations of microtubules that are marked by different combinations of PTMs. Furthermore these subpopulations show differential sensitivity to both drug- and cold-induced depolymerization, suggesting that they are functionally different as well. The composition and distribution of modified microtubules change as cells undergo the morphogenesis associated with polarization. Two-dimensionally polarized spreading cells have more detyrosinated microtubules that are oriented toward the leading edge, but three-dimensionally polarized cells have more acetylated microtubules that are oriented toward the apical domain. These data suggest that the transition from 2D polarity to 3D polarity involves both a reorganization of the microtubule cytoskeleton and a change in tubulin PTMs. However, in both 2D polarized and 3D polarized cells, the modified microtubules are oriented to support vectorial cargo transport to areas of high need.     

17.

Regulation of microtubules in cell migration     

Watanabe T  Noritake J  Kaibuchi K 《Trends in cell biology》2005,15(2):76-83

Directional cell migration is a fundamental process in all organisms that is stringently regulated during tissue development, chemotaxis and wound healing. Migrating cells have a polarized morphology with an asymmetrical distribution of signaling molecules and the cytoskeleton. Microtubules are indispensable for the directional migration of certain cells. Recent studies have shown that Rho family GTPases, which are key regulators of cell migration, affect microtubules, in addition to the actin cytoskeleton and adhesion. Rho family GTPases capture and stabilize microtubules through their effectors at the cell cortex, leading to a polarized microtubule array; in turn, microtubules modulate the activities of Rho family GTPases. In this article, we discuss how a polarized microtubule array is established and how microtubules facilitate cell migration.     

18.

Shaping Fission Yeast with Microtubules     

Fred Chang  Sophie G. Martin 《Cold Spring Harbor perspectives in biology》2009,1(1)

For cell morphogenesis, the cell must establish distinct spatial domains at specified locations at the cell surface. Here, we review the molecular mechanisms of cell polarity in the fission yeast Schizosaccharomyces pombe. These are simple rod-shaped cells that form cortical domains at cell tips for cell growth and at the cell middle for cytokinesis. In both cases, microtubule-based systems help to shape the cell by breaking symmetry, providing endogenous spatial cues to position these sites. The plus ends of dynamic microtubules deliver polarity factors to the cell tips, leading to local activation of the GTPase cdc42p and the actin assembly machinery. Microtubule bundles contribute to positioning the division plane through the nucleus and the cytokinesis factor mid1p. Recent advances illustrate how the spatial and temporal regulation of cell polarization integrates many elements, including historical landmarks, positive and negative controls, and competition between pathways.One of the ultimate goals in cell biology is to understand how cells are assembled. As in the development of multicellular organisms, single cells need to form distinct spatial domains with specific form, structure, and functions. How do cells organize themselves in space to form a specific shape and size?The fission yeast Schizosaccharomyces pombe is an attractive, simple unicellular model organism for studying cell morphogenesis. These are nonmotile cells with highly invariant shape 8–14 µm long and 3 µm in diameter. The relative simplicity of the cells and the powers of genetic approaches and live cell imaging facilitate rigorous and quantitative studies.Here, we review the current understanding of spatial regulation in fission yeast. The cell defines distinct cortical domains at each of the cell tips, along the sides of cells, and at the cell division plane. Each cortical domain is characterized by different sets of molecules, which impart distinct functions. In particular, as it proceeds through its cell cycle, the cell delineates distinct actin-rich cortical regions at cell tips for polarized cell growth and at the middle for cell division. In both cases, a self-organizing network of microtubules directly or indirectly contributes to the proper localization of these markers. In cell polarity, microtubule ends transport polarity factors to the plasma membrane, where they function to recruit protein complexes involved in actin assembly. In cytokinesis, a medial cortical site is marked by an interacting system of microtubules, the nucleus, and cell tip factors, and functions to organize actin filaments into a cytokinetic ring. This reliance on microtubules contrasts with polarity mechanisms in budding yeast in which spatial cues are dependent on septins and actin, but not microtubules. As many of these processes involve conserved proteins, this work in fission yeast contributes toward understanding the more complex microtubule-based regulation of cell migration, cytokinesis, and cell shape regulation in animal cells. This work in fission yeast thus provides a paradigm for how a self-organizing system can shape a cell.     

19.

Identification of CD63 as a tissue inhibitor of metalloproteinase-1 interacting cell surface protein   总被引：1，自引：0，他引：1  

Jung KK  Liu XW  Chirco R  Fridman R  Kim HR 《The EMBO journal》2006,25(17):3934-3942

This study identified CD63, a member of the tetraspanin family, as a TIMP-1 interacting protein by yeast two-hybrid screening. Immunoprecipitation and confocal microscopic analysis confirmed CD63 interactions with TIMP-1, integrin beta1, and their co-localizations on the cell surface of human breast epithelial MCF10A cells. TIMP-1 expression correlated with the level of active integrin beta1 on the cell surface independent of cell adhesion. While MCF10A cells within a three-dimensional (3D) matrigel matrix form polarized acinar-like structures, TIMP-1 overexpression disrupted breast epithelial cell polarization and inhibited caspase-mediated apoptosis in centrally located cells, necessary for the formation and maintenance of the hollow acinar-like structures. Small hairpin RNA (shRNA)-mediated CD63 downregulation effectively reduced TIMP-1 binding to the cell surface, TIMP-1 co-localization with integrin beta1, and consequently reversed TIMP-1-mediated integrin beta1 activation, cell survival signaling and apoptosis inhibition. CD63 downregulation also restored polarization and apoptosis of TIMP-1 overexpressing MCF10A cells within a 3D-matrigel matrix. Taken together, the present study identified CD63 as a cell surface binding partner for TIMP-1, regulating cell survival and polarization via TIMP-1 modulation of tetraspanin/integrin signaling complex.     

20.

The fusome organizes the microtubule network during oocyte differentiation in Drosophila     

Grieder NC  de Cuevas M  Spradling AC 《Development (Cambridge, England)》2000,127(19):4253-4264

Differentiation of the Drosophila oocyte takes place in a cyst of 16 interconnected germ cells and is dependent on a network of microtubules that becomes polarized as differentiation progresses (polarization). We have investigated how the microtubule network polarizes using a GFP-tubulin construct that allows germ-cell microtubules to be visualized with greater sensitivity than in previous studies. Unexpectedly, microtubules are seen to associate with the fusome, an asymmetric germline-specific organelle, which elaborates as cysts form and undergoes complex changes during cyst polarization. This fusome-microtubule association occurs periodically during late interphases of cyst divisions and then continuously in 16-cell cysts that have entered meiotic prophase. As meiotic cysts move through the germarium, microtubule minus ends progressively focus towards the center of the fusome, as visualized using a NOD-lacZ marker. During this same period, discrete foci rich in gamma tubulin that very probably correspond to migrating cystocyte centrosomes also associate with the fusome, first on the fusome arms and then in its center, subsequently moving into the differentiating oocyte. The fusome is required for this complex process, because microtubule network organization and polarization are disrupted in hts(1) mutant cysts, which lack fusomes. Our results suggest that the fusome, a specialized membrane-skeletal structure, which arises in early germ cells, plays a crucial role in polarizing 16-cell cysts, at least in part by interacting with microtubules and centrosomes.     

Structural change in the cell center during fibroblast polarization and movement

In polarizing and migrating 3T3/Balb mouse fibroblasts, the centrioles are located between the nucleus and the leading edge of the cell. In cells within the monolayer and in migrating cells, the centrioles have a random orientation towards the substrate. In polarized cells, that still remain in the monolayer, one centriole may be perpendicular to the substrate plane in 70% of cases. Upon polarization and migration of fibroblasts, the number of microtubules, which radiate from the centriolar region, increases. These data support a hypothesis that the number of microtubules in the cell centre characterizes the rate of their renovation in the cytoplasm. It is concluded that the cell centre is strongly involved in polarization and migration of fibroblasts.     

Cell biology of planar polarity transmission in the Drosophila wing

The coordination of epithelial planar polarization is a critical step in the formation of well-ordered tissues. The process has been extensively studied in Drosophila, where genetic analysis has identified a set of "tissue polarity" genes that serve to coordinate planar polarity of cells in the developing wings, bristles and eyes. In the last several years, it has emerged that six of these genes encode junctional proteins. In the wing epithelium, these proteins undergo a polarized redistribution, forming separate proximal and distal cortical domains within each cell. The mechanisms that mediate cortical polarization and cue its direction have been the subject of intense investigation. Cuing the orientation of cortical polarization appears to depend on the atypical Cadherins Fat and Dachsous, although these proteins do not become polarized themselves, nor do they colocalize with components of polarized cortical domains. Interestingly, these Cadherins also act at earlier developmental stages to polarize tissue growth along the proximal-distal axis and it will be interesting to see whether these processes are mechanistically related. Once the axis of polarization is determined, cortical polarity seems to be propagated, at least locally, by a cascade of direct cell-cell interactions mediated by the proximal and distal domains. The cell biological mechanisms leading to polarization are still unclear, but the process depends on the control of Protein Phosphatase 2A activity by its regulatory subunit, Widerborst. Interestingly, Widerborst is found on a planar web of microtubules with connections to apical junctions, suggesting that these microtubules may have an important function in polarizing the cortex.     

Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170

Linkage of microtubules to special cortical regions is essential for cell polarization. CLIP-170 binds to the growing ends of microtubules and plays pivotal roles in orientation. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts with CLIP-170. In Vero fibroblasts, IQGAP1 localizes at the polarized leading edge. Expression of carboxy-terminal fragment of IQGAP1, which includes the CLIP-170 binding region, delocalizes GFP-CLIP-170 from the tips of microtubules and alters the microtubule array. Activated Rac1/Cdc42, IQGAP1, and CLIP-170 form a tripartite complex. Furthermore, expression of an IQGAP1 mutant defective in Rac1/Cdc42 binding induces multiple leading edges. These results indicate that Rac1/Cdc42 marks special cortical spots where the IQGAP1 and CLIP-170 complex is targeted, leading to a polarized microtubule array and cell polarization.     

Interplay between microtubule dynamics and intracellular organization

Microtubules are hollow tubes essential for many cellular functions such as cell polarization and migration, intracellular trafficking and cell division. They are polarized polymers composed of α and β tubulin that are, in most cells, nucleated at the centrosome at the center of the cell. Microtubule plus-ends are oriented towards the periphery of the cell and explore the cytoplasm in a very dynamic manner. Microtubule alternate between phases of growth and shrinkage in a manner described as dynamic instability. Their dynamics is highly regulated by multiple factors: tubulin post-translational modifications such as detyrosination or acetylation, and microtubule-associated proteins, among them the plus-tip tracking proteins. This regulation is necessary for microtubule functions in the cell. In this review, we will focus on the role of microtubules in intracellular organization. After an overview of the mechanisms responsible for the regulation of microtubule dynamics, the major roles of microtubules dynamics in organelle positioning and organization in interphase cells will be discussed. Conversely, the role of certain organelles, like the nucleus and the Golgi apparatus as microtubule organizing centers will be reviewed. We will then consider the role of microtubules in the establishment and maintenance of cell polarity using few examples of cell polarization: epithelial cells, neurons and migrating cells. In these cells, the microtubule network is reorganized and undergoes specific and local regulation events; microtubules also participate in the intracellular reorganization of different organelles to ensure proper cell differentiation.     

Transmembrane control of laminin, lectin receptors and surface villi in teratocarcinoma-derived endodermal cells

Distribution of laminin on the surface of teratocarcinoma-derived parietal endoderm cells was studied by immuno-histochemical staining of the fixed specimen using affinity-purified anti-laminin antibody. Laminin was distributed on the basal surface of the cells, while treatment either with colchicine or with cytochalasin D (CD) resulted in a severely polarized distribution; laminin was seen only at one end of the cell. Treatment with both the reagents did not cause the severe polarization. Receptors for lectins and cell surface villi were polarized by treatment with CD but not by treatment with colchicine. These results suggest that laminin--or its cell surface receptor--is linked to both microfilament and microtubules and that the mode of transmembrane control for laminin is different from certain other cell surface components of the cells.     

Microtubule targeting of substrate contacts promotes their relaxation and dissociation.

We recently showed that substrate contact sites in living fibroblasts are specifically targeted by microtubules (Kaverina, I., K. Rottner, and J.V. Small. 1998. J. Cell Biol. 142:181-190). Evidence is now provided that microtubule contact targeting plays a role in the modulation of substrate contact dynamics. The results are derived from spreading and polarized goldfish fibroblasts in which microtubules and contact sites were simultaneously visualized using proteins conjugated with Cy-3, rhodamine, or green fluorescent protein.For cells allowed to spread in the presence of nocodazole the turnover of contacts was retarded, as compared with controls and adhesions that were retained under the cell body were dissociated after microtubule reassembly. In polarized cells, small focal complexes were found at the protruding cell front and larger adhesions, corresponding to focal adhesions, at the retracting flanks and rear. At retracting edges, multiple microtubule contact targeting preceded contact release and cell edge retraction. The same effect could be observed in spread cells, in which microtubules were allowed to reassemble after local disassembly by the application of nocodazole to one cell edge. At the protruding front of polarized cells, focal complexes were also targeted and as a result remained either unchanged in size or, more rarely, were disassembled. Conversely, when contact targeting at the cell front was prevented by freezing microtubule growth with 20 nM taxol and protrusion stimulated by the injection of constitutively active Rac, peripheral focal complexes became abnormally enlarged. We further found that the local application of inhibitors of myosin contractility to cell edges bearing focal adhesions induced the same contact dissociation and edge retraction as observed after microtubule targeting.Our data are consistent with a mechanism whereby microtubules deliver localized doses of relaxing signals to contact sites to retard or reverse their development. We propose that it is via this route that microtubules exert their well-established control on cell polarity.     

Alternative routes for the establishment of surface polarity during compaction of the mouse embryo

During the process of compaction, mouse 8-cell blastomeres flatten upon each other and polarize along an axis perpendicular to cell contacts. If the process of flattening is prevented, polarization can still occur, but does so in a lower proportion of cells than for control populations, and without the normal contact-directed orientation. We compared contact-directed and noncontact-directed processes to see if they involve common mechanisms. In nonflattened cells, surface polarization was favored in cells with nuclei located close to the cell surface, and the positions of surface poles and of nuclei tended to coincide. We present evidence that microtubules are involved in the development of microvillous poles associated with nuclei. In contrast it is known that polarization of microvilli occurs in the absence of microtubules if blastomeres are allowed to flatten. We conclude that surface polarization of mouse blastomeres can be accomplished by at least two alternative routes. One requires flattening but is independent of microtubules, and another can occur without flattening but involves a microtubule-mediated interaction between the nucleus and the cell cortex. It seems that both these pathways operate in the undisturbed embryo.     

Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid

In this study, we examined the contribution of microtubules to epithelial morphogenesis in primary thyroid cell cultures. Thyroid follicles consist of a single layer of polarized epithelial cells surrounding a closed compartment, the follicular lumen. Freshly isolated porcine thyroid cells aggregate and reorganize to form follicles when grown in primary cultures. Follicular reorganization is principally a morphogenetic process that entails the assembly of biochemically distinct apical and basolateral membrane domains, delimited by tight junctions. The establishment of cell surface polarity during folliculogenesis coincided with the polarized redistribution of microtubules, predominantly in the developing apical poles of cells. Disruption of microtubule integrity using either colchicine or nocodazole caused loss of defined apical membrane domains, tight junctions and follicular lumina. Apical membrane and tight junction markers became randomly distributed at the outer surfaces of aggregates. In contrast, the basolateral surface markers, E-cadherin and Na(+),K(+)-ATPase, remained correctly localized at sites of cell-cell contact and at the free surfaces of cell aggregates. These findings demonstrate that microtubules play a necessary role in thyroid epithelial morphogenesis. Specifically, microtubules are essential to preserve the correct localization of apical membrane components within enclosed cellular aggregates, a situation that is also likely to pertain where lumina must be formed from solid aggregates of epithelial precursors.     

The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium

Studies of the developing trophectoderm in the mouse embryo have shown that extensive cellular remodeling occurs during epithelial formation. In this investigation, confocal immunofluorescence microscopy is used to examine the three-dimensional changes in cellular architecture that take place during the polarization of a terminally differentiated epithelial cell line. Madin-Darby canine kidney cells were plated at a low density on permeable filter supports. Antibodies that specifically recognize components of the tight junction, adherens junction, microtubules, centrosomes, and the Golgi complex were used to study the spatial remodeling of the cytoarchitecture during the formation of the polarized cell layer. The immunofluorescence data were correlated with establishment of functional tight junctions as measured by transepithelial resistance and back-exchange of the cell surface, labeled with metabolites of the fluorescent lipid analogue N-(7-[4- nitrobenzo-2-oxa-1,3-diazole]) aminocaproyl sphingosine. 1 d after plating, single cells had microtubules, radiating from a broad region, that contained the centrosomes and the Golgi complex. 2 d after plating, the cells had grown to confluence and had formed functional tight junctions close to the substratum. The centrioles had split and no longer organized the microtubules which were running above and below the nucleus. The Golgi complex had spread around the nucleus. By the fifth day after plating, the final polarized state had been achieved. The junctional complex had moved greater than 10 microns upward from its basal location. The centrioles were together below the apical membrane, and the Golgi complex formed a ribbon-like convoluted structure located in the apical region above the nucleus. The microtubules were organized in an apical web and in longitudinal microtubule bundles in the apical-basal axis of the columnar cell. The longitudinal microtubules were arranged with their minus ends spread over the apical region of the cell and their plus ends toward the basal region. These findings show that there is an extensive remodeling of epithelial cytoarchitecture after formation of cell-cell contacts. Reorganization of the microtubule network results in functional polarization of the cytoplasm.     

The Rho-mDia1 pathway regulates cell polarity and focal adhesion turnover in migrating cells through mobilizing Apc and c-Src

Directed cell migration requires cell polarization and adhesion turnover, in which the actin cytoskeleton and microtubules work critically. The Rho GTPases induce specific types of actin cytoskeleton and regulate microtubule dynamics. In migrating cells, Cdc42 regulates cell polarity and Rac works in membrane protrusion. However, the role of Rho in migration is little known. Rho acts on two major effectors, ROCK and mDia1, among which mDia1 produces straight actin filaments and aligns microtubules. Here we depleted mDia1 by RNA interference and found that mDia1 depletion impaired directed migration of rat C6 glioma cells by inhibiting both cell polarization and adhesion turnover. Apc and active Cdc42, which work together for cell polarization, localized in the front of migrating cells, while active c-Src, which regulates adhesion turnover, localized in focal adhesions. mDia1 depletion impaired localization of these molecules at their respective sites. Conversely, expression of active mDia1 facilitated microtubule-dependent accumulation of Apc and active Cdc42 in the polar ends of the cells and actin-dependent recruitment of c-Src in adhesions. Thus, the Rho-mDia1 pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and delivering Apc/Cdc42 and c-Src to their respective sites of action.     

Regulation of cell migration by dynamic microtubules

Microtubules define the architecture and internal organization of cells by positioning organelles and activities, as well as by supporting cell shape and mechanics. One of the major functions of microtubules is the control of polarized cell motility. In order to support the asymmetry of polarized cells, microtubules have to be organized asymmetrically themselves. Asymmetry in microtubule distribution and stability is regulated by multiple molecular factors, most of which are microtubule-associated proteins that locally control microtubule nucleation and dynamics. At the same time, the dynamic state of microtubules is key to the regulatory mechanisms by which microtubules regulate cell polarity, modulate cell adhesion and control force-production by the actin cytoskeleton. Here, we propose that even small alterations in microtubule dynamics can influence cell migration via several different microtubule-dependent pathways. We discuss regulatory factors, potential feedback mechanisms due to functional microtubule-actin crosstalk and implications for cancer cell motility.     

Analogous ultrastructure and surface properties during capping and phagocytosis in leukocytes

Ultrastructural analyses have revealed striking similarities between Concanavalin A capping and phagocytosis in leukocytes. Both processes involve extensive membrane movement to form a protuberance or pseudopods; a dense network of microfilaments is recruited into both the protuberance and the pseudopods; microtubules are disassembled either generally (capping) or in the local region of the pseudopods (phagocytosis); and cells generally depleted of microtubules by colchicine show polarized phagocytosis via the microfilament-rich protuberance rather than uniform peripheral ingestion of particles via individual pseudopods. Cap formation can thus be viewed as occurring as an exaggeration of the same ultrastructural events that mediate phagocytosis. Similar changes in cell surface topography also accompany capping and phagocytosis. Thus, in nonfixed cells, Concanavalin A-receptor complexes aggregate into the region of the protuberance in colchicine-treated leukocytes (conventional capping) or into the region of pseudopod formation in phagocytizing leukocytes. In the latter case, the movement of lectin-receptor complexes occurs from membrane overlying peripheral microtubules into filament-rich pseudopods that exclude microtubules. These data provide evidence against a role for microtubules as "anchors" for lectin receptors. Rather, they indicate a preferential movement of cell surface Concanavalin A-receptor complexes towards areas of extensive (the protuberance) or localized (pseudopods) microfilament concentration. In conventional capping, Concanavalin A must be added to the colchicine-treated cells before fixation in order to demonstrate movement of receptors from a diffuse distribution into the protuberance. However, Convanavalin A receptors are enriched in the membrane associated with phagocytic particles as compared to the remaining membrane. This particle-induced redistribution of receptors is particularly prominent in colchicine-treated cells that phagocytize and are then fixed and Concanavalin A labeled; both lectin receptors and beads are concentrated over the protuberance. Thus, the final analogy between conventionally capped and phagocytic cells is that in both cases the properties of the plasma membrane in regions of microfilament concentration are modified by Concanavalin A itself (capping) or by the phagocytized particle, to limit locally the diffusion of Concanavalin A receptors.     

The posttranslational modification of tubulin undergoes a switch from detyrosination to acetylation as epithelial cells become polarized

Tubulin posttranslational modifications (PTMs) have been suggested to provide navigational cues for molecular motors to deliver cargo to spatially segregated subcellular domains, but the molecular details of this process remain unclear. Here we show that in Madin-Darby Canine Kidney (MDCK) epithelial cells, microtubules express several tubulin PTMs. These modifications, however, are not coordinated, and cells have multiple subpopulations of microtubules that are marked by different combinations of PTMs. Furthermore these subpopulations show differential sensitivity to both drug- and cold-induced depolymerization, suggesting that they are functionally different as well. The composition and distribution of modified microtubules change as cells undergo the morphogenesis associated with polarization. Two-dimensionally polarized spreading cells have more detyrosinated microtubules that are oriented toward the leading edge, but three-dimensionally polarized cells have more acetylated microtubules that are oriented toward the apical domain. These data suggest that the transition from 2D polarity to 3D polarity involves both a reorganization of the microtubule cytoskeleton and a change in tubulin PTMs. However, in both 2D polarized and 3D polarized cells, the modified microtubules are oriented to support vectorial cargo transport to areas of high need.     

Regulation of microtubules in cell migration

Directional cell migration is a fundamental process in all organisms that is stringently regulated during tissue development, chemotaxis and wound healing. Migrating cells have a polarized morphology with an asymmetrical distribution of signaling molecules and the cytoskeleton. Microtubules are indispensable for the directional migration of certain cells. Recent studies have shown that Rho family GTPases, which are key regulators of cell migration, affect microtubules, in addition to the actin cytoskeleton and adhesion. Rho family GTPases capture and stabilize microtubules through their effectors at the cell cortex, leading to a polarized microtubule array; in turn, microtubules modulate the activities of Rho family GTPases. In this article, we discuss how a polarized microtubule array is established and how microtubules facilitate cell migration.     

Shaping Fission Yeast with Microtubules

For cell morphogenesis, the cell must establish distinct spatial domains at specified locations at the cell surface. Here, we review the molecular mechanisms of cell polarity in the fission yeast Schizosaccharomyces pombe. These are simple rod-shaped cells that form cortical domains at cell tips for cell growth and at the cell middle for cytokinesis. In both cases, microtubule-based systems help to shape the cell by breaking symmetry, providing endogenous spatial cues to position these sites. The plus ends of dynamic microtubules deliver polarity factors to the cell tips, leading to local activation of the GTPase cdc42p and the actin assembly machinery. Microtubule bundles contribute to positioning the division plane through the nucleus and the cytokinesis factor mid1p. Recent advances illustrate how the spatial and temporal regulation of cell polarization integrates many elements, including historical landmarks, positive and negative controls, and competition between pathways.One of the ultimate goals in cell biology is to understand how cells are assembled. As in the development of multicellular organisms, single cells need to form distinct spatial domains with specific form, structure, and functions. How do cells organize themselves in space to form a specific shape and size?The fission yeast Schizosaccharomyces pombe is an attractive, simple unicellular model organism for studying cell morphogenesis. These are nonmotile cells with highly invariant shape 8–14 µm long and 3 µm in diameter. The relative simplicity of the cells and the powers of genetic approaches and live cell imaging facilitate rigorous and quantitative studies.Here, we review the current understanding of spatial regulation in fission yeast. The cell defines distinct cortical domains at each of the cell tips, along the sides of cells, and at the cell division plane. Each cortical domain is characterized by different sets of molecules, which impart distinct functions. In particular, as it proceeds through its cell cycle, the cell delineates distinct actin-rich cortical regions at cell tips for polarized cell growth and at the middle for cell division. In both cases, a self-organizing network of microtubules directly or indirectly contributes to the proper localization of these markers. In cell polarity, microtubule ends transport polarity factors to the plasma membrane, where they function to recruit protein complexes involved in actin assembly. In cytokinesis, a medial cortical site is marked by an interacting system of microtubules, the nucleus, and cell tip factors, and functions to organize actin filaments into a cytokinetic ring. This reliance on microtubules contrasts with polarity mechanisms in budding yeast in which spatial cues are dependent on septins and actin, but not microtubules. As many of these processes involve conserved proteins, this work in fission yeast contributes toward understanding the more complex microtubule-based regulation of cell migration, cytokinesis, and cell shape regulation in animal cells. This work in fission yeast thus provides a paradigm for how a self-organizing system can shape a cell.     

Identification of CD63 as a tissue inhibitor of metalloproteinase-1 interacting cell surface protein

This study identified CD63, a member of the tetraspanin family, as a TIMP-1 interacting protein by yeast two-hybrid screening. Immunoprecipitation and confocal microscopic analysis confirmed CD63 interactions with TIMP-1, integrin beta1, and their co-localizations on the cell surface of human breast epithelial MCF10A cells. TIMP-1 expression correlated with the level of active integrin beta1 on the cell surface independent of cell adhesion. While MCF10A cells within a three-dimensional (3D) matrigel matrix form polarized acinar-like structures, TIMP-1 overexpression disrupted breast epithelial cell polarization and inhibited caspase-mediated apoptosis in centrally located cells, necessary for the formation and maintenance of the hollow acinar-like structures. Small hairpin RNA (shRNA)-mediated CD63 downregulation effectively reduced TIMP-1 binding to the cell surface, TIMP-1 co-localization with integrin beta1, and consequently reversed TIMP-1-mediated integrin beta1 activation, cell survival signaling and apoptosis inhibition. CD63 downregulation also restored polarization and apoptosis of TIMP-1 overexpressing MCF10A cells within a 3D-matrigel matrix. Taken together, the present study identified CD63 as a cell surface binding partner for TIMP-1, regulating cell survival and polarization via TIMP-1 modulation of tetraspanin/integrin signaling complex.     

The fusome organizes the microtubule network during oocyte differentiation in Drosophila

Differentiation of the Drosophila oocyte takes place in a cyst of 16 interconnected germ cells and is dependent on a network of microtubules that becomes polarized as differentiation progresses (polarization). We have investigated how the microtubule network polarizes using a GFP-tubulin construct that allows germ-cell microtubules to be visualized with greater sensitivity than in previous studies. Unexpectedly, microtubules are seen to associate with the fusome, an asymmetric germline-specific organelle, which elaborates as cysts form and undergoes complex changes during cyst polarization. This fusome-microtubule association occurs periodically during late interphases of cyst divisions and then continuously in 16-cell cysts that have entered meiotic prophase. As meiotic cysts move through the germarium, microtubule minus ends progressively focus towards the center of the fusome, as visualized using a NOD-lacZ marker. During this same period, discrete foci rich in gamma tubulin that very probably correspond to migrating cystocyte centrosomes also associate with the fusome, first on the fusome arms and then in its center, subsequently moving into the differentiating oocyte. The fusome is required for this complex process, because microtubule network organization and polarization are disrupted in hts(1) mutant cysts, which lack fusomes. Our results suggest that the fusome, a specialized membrane-skeletal structure, which arises in early germ cells, plays a crucial role in polarizing 16-cell cysts, at least in part by interacting with microtubules and centrosomes.