Orientation of spindle axis and distribution of plasma membrane proteins during cell division in polarized MDCKII cells

MDCKII cells differentiate into a simple columnar epithelium when grown on a permeable support; the monolayer is polarized for transport and secretion. Individual cells within the monolayer continue to divide at a low rate without disturbing the function of the epithelium as a barrier to solutes. This presents an interesting model for the study of mitosis in a differentiated epithelium which we have investigated by confocal immunofluorescence microscopy. We monitored the distribution of microtubules, centrioles, nucleus, tight junctions, and plasma membrane proteins that are specifically targeted to the apical and basolateral domains. The stable interphase microtubule cytoskeleton was rapidly disassembled at prophase onset and reassembled at cytokinesis. As the interphase microtubules disassembled at prophase, the centrioles moved from their interphase position at the apical membrane to the nucleus and acquired the ability to organize microtubule asters. Orientation of the spindle parallel to the plane of the monolayer occurred between late prophase and metaphase and persisted through cytokinesis. The cleavage furrow formed asymmetrically perpendicular to the plane of the monolayer initiating at the basolateral side and proceeding to the apical domain. The interphase microtubule network reformed after the centrioles migrated from the spindle poles to resume their interphase apical position. Tight junctions (ZO-1), which separate the apical from the basolateral domains, remained assembled throughout all phases of mitosis. E-cadherin and a 58-kD antigen maintained their basolateral plasma membrane distributions, and a 114- kD antigen remained polarized to the apical domain. These proteins were useful for monitoring the changes in shape of the mitotic cells relative to neighboring cells, especially during telophase when the cell shape changes dramatically. We discuss the changes in centriole position during the cell cycle, mechanisms of spindle orientation, and how the maintenance of polarized plasma membrane domains through mitosis may facilitate the rapid reformation of the polarized interphase cytoplasm.     

Development and functions of the cytoskeleton during ciliogenesis in metazoa

The different steps of ciliogenesis occurring in quail oviduct were compared to the ciliogenesis pattern described in other metazoan species. Centrioles are generated according to pathways that are found within the same cell: the centriolar and the acentriolar pathways. In the acentriolar pathway, centrioles are generated in the Golgi area, without contact with the preexisting centrioles of the centrosomes, and they migrate toward the apical membrane. The control of this polarized migration was studied by means of several drugs (colchicine, nocodazol, taxol, cytochalasin D, benzodiazepines) and immunocytochemistry. It was suggested that an actin-myosin system was involved in the migration of centrioles, whereas labile microtubules were not necessary. Basal bodies must dock with plasma membrane or cytoplasmic vesicles for the initiation of axonemal microtubule polymerization. This signal is necessary even in the presence of taxol.     

Cytoskeletal control of centrioles movement during the establishment of polarity in Madin-Darby canine kidney cells

The two centrioles that are localized close to each other and to the nucleus in single Madin-Darby Canine kidney cells (MDCK) move apart by distances as large as 13 microns after the establishment of extensive cellular junctions. Microfilaments, and possibly microtubules appear to be responsible for this separation. In fully polarized cells, the centrioles are localized just beneath the apical membrane. After disruption of intercellular junctions in low calcium medium, the centrioles move back towards the cell center. This process requires intact microtubules but happens even in the absence of microfilaments. These results indicate that the position of centrioles is determined by opposing forces produced by microtubules and microfilaments and suggest that the balance between these forces is modulated by the assembly of cellular junctions. Centriole separation appears to be an early event in the process that precedes their final positioning in the apical-most region of the polarized cell.     

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.     

Changes in organization and microtubule assembly activity of the centrosome during lymphocyte stimulation

Changes in the organization of centrosomes in mouse splenic T lymphocytes stimulated by concanavalin A (con A) were examined by electron microscopy of serial sections. In both resting and stimulated lymphocytes the single centrosome consists of a pair of centrioles, satellite bodies, and pericentriolar material. In resting cell centrosomes the satellite bodies are preferentially associated with, and appear to be attached by short stalks to, one of the centrioles. The satellite bodies are the primary sites of microtubule termination in the resting cell centrosome. During stimulation by con A there is a several-fold increase in microtubule content. This is correlated with an overall increase in centrosome size, an apparent increase in the size and in the number of satellite bodies, and a redistribution of satellite bodies to occupy a position between the two centrioles. Increased numbers of microtubules are detected terminating on the satellite bodies and in the pericentriolar material of the stimulated cell centrosome. Microtubule assembly from centrosomes in vitro was assessed by electron microscopy using detergent-permeabilized lymphocytes that had been pretreated to remove endogenous microtubules and supplied with purified bovine brain tubulin. These studies indicate that satellite bodies are major sites of microtubule assembly in both resting and stimulated cell centrosomes and show that the centrosomes of stimulated cells assemble more microtubules in vitro than resting cell centrosomes. This parallels the increase in microtubule content in intact lymphocytes stimulated by con A and suggests that the changes in centrosome organization and microtubule assembly capacity that occur during stimulation are causally related.     

Changes in organization and microtubule assembly activity of the centrosome during lymphocyte stimulation

Changes in the organization of centrosomes in mouse splenic T lymphocytes stimulated by concanavalin A (con A) were examined by electron microscopy of serial sections. In both resting and stimulated lymphocytes the single centrosome consists of a pair of centrioles, satellite bodies, and pericentriolar material. In resting cell centrosomes the satellite bodies are preferentially associated with, and appear to be attached by short stalks to, one of the centrioles. The satellite bodies are the primary sites of microtubule termination in the resting cell centrosome. During stimulation by con A there is a several-fold increase in microtubule content. This is correlated with an overall increase in centrosome size, an apparent increase in the size and in the number of satellite bodies, and a redistribution of satellite bodies to occupy a position between the two centrioles. Increased numbers of microtubules are detected terminating on the satellite bodies and in the pericentriolar material of the stimulated cell centrosome. Microtubule assembly from centrosomes in vitro was assessed by electron microscopy using detergent-permeabilized lymphocytes that had been pretreated to remove endogenous microtubules and supplied with purified bovine brain tubulin. These studies indicate that satellite bodies are major sites of microtubule assembly in both resting and stimulated cell centrosomes and show that the centrosomes of stimulated cells assemble more microtubules in vitro than resting cell centrosomes. This parallels the increase in microtubule content in intact lymphocytes stimulated by con A and suggests that the changes in centrosome organization and microtubule assembly capacity that occur during stimulation are causally related.     

A multicomponent assembly pathway contributes to the formation of acentrosomal microtubule arrays in interphase Drosophila cells

In animal cells, centrosomes nucleate microtubules that form polarized arrays to organize the cytoplasm. Drosophila presents an interesting paradox however, as centrosome-deficient mutant animals develop into viable adults. To understand this discrepancy, we analyzed behaviors of centrosomes and microtubules in Drosophila cells, in culture and in vivo, using a combination of live-cell imaging, electron microscopy, and RNAi. The canonical model of the cycle of centrosome function in animal cells states that centrosomes act as microtubule-organizing centers throughout the cell cycle. Unexpectedly, we found that many Drosophila cell-types display an altered cycle, in which functional centrosomes are only present during cell division. On mitotic exit, centrosomes disassemble producing interphase cells containing centrioles that lack microtubule-nucleating activity. Furthermore, steady-state interphase microtubule levels are not changed by codepleting both gamma-tubulins. However, gamma-tubulin RNAi delays microtubule regrowth after depolymerization, suggesting that it may function partially redundantly with another pathway. Therefore, we examined additional microtubule nucleating factors and found that Mini-spindles, CLIP-190, EB1, or dynein RNAi also delayed microtubule regrowth; surprisingly, this was not further prolonged when we codepleted gamma-tubulins. Taken together, these results modify our view of the cycle of centrosome function and reveal a multi-component acentrosomal microtubule assembly pathway to establish interphase microtubule arrays in Drosophila.     

Behavior of the cell center during epithelial cell spreading

In the epithelial cells of mouse embryo renal channels, centrioles are located near the plasma membrane of the apical part of the cell. In most of the cells an active centriole carries a cilium, which comes out into the channel lumen. In the epithelial cells, suspended after trypsinisation and in single cells adhering to the substrate, the centrioles are located near the nucleus, and the outcoming cilia are not observed. In the spread cells of epithelial islets, the centrioles are also found near the nucleus, and in most cases an active centriole carries a cilium, which comes out of the cytoplasm at the upper side of the cell. In the peripheral cells of the islet, centrioles are positioned between the nucleus and the active edge of the cell. In the epithelial cells in situ, a relatively small number of microtubules radiate from the active centrioles. In the suspended cells, the activation of microtubule formation is observed in the cell center. In the spread cells of the epithelial islets there occurs a further increase in the number of microtubules radiating from the active centrioles. In the peripheral cells which cause translocation of the epithelial islet in the culture, the number of microtubules, radiating from the centrioles does not differ significantly from that of the inner cells of the islet. The cell center of the epithelial cells does not seem to be actively involved in the locomotion of the epithelial cells in the culture.     

Microtubules regulate disassembly of epithelial apical junctions

Background  

Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion.     

Identification and function of the centrosome centromatrix

Centrosomes direct the organization of microtubules in animal cells. However, in the absence of centrosomes, cytoplasm has the potential to organize microtubules and assemble complex structures such as anastral spindles. During cell replication or following fertilization, centrioles that are incapable of organizing microtubules into astral arrays are introduced into this complex cytoplasmic environment. These centrioles become associated with pericentriolar material responsible for centrosome-dependent microtubule nucleation, and thus the centrosome matures to ultimately become a dominant microtubule organizing center that serves as a central organizer of cell cytoplasm. We describe the identification of a novel structure within the pericentriolar material of centrosomes called the centromatrix. The centromatrix is a salt-insoluble filamentous scaffold to which subunit structures that are necessary for microtubule nucleation and abundant in the cytoplasm bind. We propose that the centromatrix serves to concentrate and focus these subunits to form the microtubule organizing center. Since binding of these subunits to the centromatrix does not require nucleotides, we propose a model for centrosome assembly which predicts that the assembly of the centromatrix is a rate-limiting step in centrosome assembly and maturation.     

The Centrosomal Linker and Microtubules Provide Dual Levels of Spatial Coordination of Centrosomes

The centrosome is the principal microtubule organizing center in most animal cells. It consists of a pair of centrioles surrounded by pericentriolar material. The centrosome, like DNA, duplicates exactly once per cell cycle. During interphase duplicated centrosomes remain closely linked by a proteinaceous linker. This centrosomal linker is composed of rootletin filaments that are anchored to the centrioles via the protein C-Nap1. At the onset of mitosis the linker is dissolved by Nek2A kinase to support the formation of the bipolar mitotic spindle. The importance of the centrosomal linker for cell function during interphase awaits characterization. Here we assessed the phenotype of human RPE1 C-Nap1 knockout (KO) cells. The absence of the linker led to a modest increase in the average centrosome separation from 1 to 2.5 μm. This small impact on the degree of separation is indicative of a second level of spatial organization of centrosomes. Microtubule depolymerisation or stabilization in C-Nap1 KO cells dramatically increased the inter-centrosomal separation (> 8 μm). Thus, microtubules position centrosomes relatively close to one another in the absence of linker function. C-Nap1 KO cells had a Golgi organization defect with a two-fold expansion of the area occupied by the Golgi. When the centrosomes of C-Nap1 KO cells showed considerable separation, two spatially distinct Golgi stacks could be observed. Furthermore, migration of C-Nap1 KO cells was slower than their wild type RPE1 counterparts. These data show that the spatial organization of centrosomes is modulated by a combination of centrosomal cohesion and microtubule forces. Furthermore a modest increase in centrosome separation has major impact on Golgi organization and cell migration.     

Microtubule organization and function in epithelial cells

Microtubules are essential for many aspects of polarity in multicellular organisms, ranging from the asymmetric distribution of cell-fate determinants in the one-cell embryo to the transient polarity generated in migrating fibroblasts. Epithelial cells exhibit permanent cell polarity characterized by apical and basolateral surface domains of distinct protein and lipid composition that are segregated by tight junctions. They are also endowed with a microtubule network that reflects the asymmetry of their cell surface: microtubule minus-ends face the apical- and microtubule plus-ends the basal domain. Strikingly, the formation of distinct surface domains during epithelial differentiation is accompanied by the re-organization of microtubules from a uniform array focused at the centrosome to the noncentrosomal network that aligns along the apico-basolateral polarity axis. The significance of this coincidence for epithelial morphogenesis and the signaling mechanisms that drive microtubule repolymerization in developing epithelia remain major unresolved questions that we are only beginning to address. Studies in cultured polarized epithelial cells have established that microtubules serve as tracks that facilitate targeted vesicular transport. Novel findings suggest, moreover, that microtubule-based transport promotes protein sorting, and even the generation of transport carriers in the endo- and exocytic pathways.     

Self-association of PAR-3-mediated by the conserved N-terminal domain contributes to the development of epithelial tight junctions

PAR-3 is a scaffold-like PDZ-containing protein that forms a complex with PAR-6 and atypical protein kinase C (PAR-3-atypical protein kinase C-PAR-6 complex) and contributes to the establishment of cell polarity in a wide variety of biological contexts. In mammalian epithelial cells, it localizes to tight junctions, the most apical end of epithelial cell-cell junctions, and contributes to the formation of functional tight junctions. However, the mechanism by which PAR-3 localizes to tight junctions and contributes to their formation remains to be clarified. Here we show that the N-terminal conserved region, CR1-(1-86), and the sequence 937-1,024 are required for its recruitment to the most apical side of the cell-cell contact region in epithelial Madin-Darby canine kidney cells. We also show that CR1 self-associates to form an oligomeric complex in vivo and in vitro. Further, overexpression of CR1 in Madin-Darby canine kidney cells disturbs the distribution of atypical protein kinase C and PAR-6 as well as PAR-3 and delays the formation of functional tight junctions. These results support the notion that the CR1-mediated self-association of the PAR-3-containing protein complex plays a role during the formation of functional tight junctions.     

Association of centrioles with clusters of apical vesicles in mitotic thyroid epithelial cells. Are centrioles involved in directing secretion?

Summary The ultrastructure of thyroid epithelial cells in mitosis has been investigated. A spatial association is described between clusters of apical vesicles (believed to contain thyroglobulin destined for secretion into the follicular lumen) and centrioles, in late prophase and late telophase cells. Quantitative techniques demonstrate the statistical significance of this association and suggest that it is not related to proximity of the Golgi apparatus or to the location of the centriole in the cell, which changes considerably during these phases of mitosis. The physical basis for this association remains uncertain, but microtubules emanating from the pericentriolar area may be involved.In interphase cells, centrioles are located very close to the follicular lumen, where the majority of apical vesicles are also found. The association of centrioles with clusters of apical vesicles also in mitotic cells suggests that in interphase cells the apically located centrioles may serve as a focus for apical vesicles, helping to direct these secretory vesicles toward the follicular lumen and to maintain cellular polarization. Previous studies demonstrating that centrioles can act as microtubule organizing centers in interphase cells and studies linking microtubules and secretion also tend to support this hypothesis.The author is grateful to Drs. Jan Wolff, Lars E. Ericson, and Seymour H. Wollman for useful discussions and to Mr. Franklin E. Reed for expert technical assistance.     

Centrosome‐mediated microtubule remodeling during axon formation in human iPSC‐derived neurons

Axon formation critically relies on local microtubule remodeling and marks the first step in establishing neuronal polarity. However, the function of the microtubule‐organizing centrosomes during the onset of axon formation is still under debate. Here, we demonstrate that centrosomes play an essential role in controlling axon formation in human‐induced pluripotent stem cell (iPSC)‐derived neurons. Depleting centrioles, the core components of centrosomes, in unpolarized human neuronal stem cells results in various axon developmental defects at later stages, including immature action potential firing, mislocalization of axonal microtubule‐associated Trim46 proteins, suppressed expression of growth cone proteins, and affected growth cone morphologies. Live‐cell imaging of microtubules reveals that centriole loss impairs axonal microtubule reorganization toward the unique parallel plus‐end out microtubule bundles during early development. We propose that centrosomes mediate microtubule remodeling during early axon development in human iPSC‐derived neurons, thereby laying the foundation for further axon development and function.     

CAMSAP3-dependent microtubule dynamics regulates Golgi assembly in epithelial cells

The Golgi assembly pattern varies among cell types. In fibroblast cells, the Golgi apparatus concentrates around the centrosome that radiates microtubules; whereas in epithelial cells, whose microtubules are mainly noncentrosomal, the Golgi apparatus accumulates around the nucleus independently of centrosome. Little is known about the mechanisms behind such cell type-specific Golgi and microtubule organization. Here, we show that the microtubule minus-end binding protein Nezha/CAMSAP3 (calmodulin-regulated spectrin-associated protein 3) plays a role in translocation of Golgi vesicles in epithelial cells. This function of CAMSAP3 is supported by CG-NAP (centrosome and Golgi localized PKN-associated protein) through their binding. Depletion of either one of these proteins similarly induces fragmentation of Golgi membranes. Furthermore, we find that stathmin-dependent microtubule dynamics is graded along the radial axis of cells with highest activity at the perinuclear region, and inhibition of this gradient disrupts perinuclear distribution of the Golgi apparatus. We propose that the assembly of the Golgi apparatus in epithelial cells is induced by a multi-step process, which includes CAMSAP3-dependent Golgi vesicle clustering and graded microtubule dynamics.     

Four-dimensional imaging of filter-grown polarized epithelial cells

Understanding how epithelial cells generate and maintain polarity and function requires live cell imaging. In order for cells to become fully polarized, it is necessary to grow them on a permeable membrane filter; however, the translucent filter obstructs the microscope light path required for quantitative live cell imaging. Alternatively, the membrane filter may be excised but this eliminates selective access to apical and basolateral surfaces. Conversely, epithelial cells cultured directly on glass exhibit different phenotypes and functions from filter grown cells. Here, we describe a new method for culturing polarized epithelial cells on a Transwell filter insert that allows superior live cell imaging with spatial and temporal image resolution previously unachievable using conventional methods. Cells were cultured on the underside of a filter support. Epithelial cells grown in this inverted configuration exhibit a fully polarized architecture, including the presence of functional tight junctions. This new culturing system permits four-dimensional (three spatial dimension over time) imaging of endosome and Golgi apparatus dynamics, and permits selective manipulation of the apical and basolateral surfaces. This new technique has wide applicability for visualization and manipulation of polarized epithelial cells.     

Centriole inheritance

Early cell biologists perceived centrosomes to be permanent cellular structures. Centrosomes were observed to reproduce once each cycle and to orchestrate assembly a transient mitotic apparatus that segregated chromosomes and a centrosome to each daughter at the completion of cell division. Centrosomes are composed of a pair of centrioles buried in a complex pericentriolar matrix. The bulk of microtubules in cells lie with one end buried in the pericentriolar matrix and the other extending outward into the cytoplasm. Centrioles recruit and organize pericentriolar material. As a result, centrioles dominate microtubule organization and spindle assembly in cells born with centrosomes. Centrioles duplicate in concert with chromosomes during the cell cycle. At the onset of mitosis, sibling centrosomes separate and establish a bipolar spindle that partitions a set of chromosomes and a centrosome to each daughter cell at the completion of mitosis and cell division. Centriole inheritance has historically been ascribed to a template mechanism in which the parental centriole contributed to, if not directed, assembly of a single new centriole once each cell cycle. It is now clear that neither centrioles nor centrosomes are essential to cell proliferation. This review examines the recent literature on inheritance of centrioles in animal cells.Key words: centrosome, centriol, spindle, mitosis, microtubule, cell cycle, checkpoints     

Endocytosis of the apical junctional complex: mechanisms and possible roles in regulation of epithelial barriers

Tight junctions (TJ) and adherens junctions (AJ) regulate cell-cell adhesion and barrier function of simple polarized epithelia. These junctions are positioned in the apical end of the lateral plasma membrane and form the so-called apical junctional complex (AJC). Although initially seen as purely structural features, the AJC is now known to play important roles in cell differentiation and proliferation. The AJC is a highly dynamic entity, undergoing rapid remodeling during normal epithelial morphogenesis and under pathologic conditions. There is growing evidence that remodeling of the AJC is mediated by internalization of junctional proteins. This review summarizes what is known about endocytic pathways, intracellular destinations and signaling cascades involved in internalization of AJC proteins. Potential biological roles for AJC endocytosis in maintaining functional apical junctions, reversible opening of epithelial barrier and disruption of intercellular adhesion are also discussed.     

Microtubule-organizing centers abnormal in number, structure, and nucleating activity in x-irradiated mammalian cells

Microtubule-organizing centers (MTOCs) in x-irradiated cells were visualized by immunofluorescence using antibody against tubulin. From two to ten reassembly sites of microtubules appeared after microtubule depolymerization at low temperature in an irradiated mitotic cell, in contrast to nonirradiated mitotic cells, which predominantly show 2 MTOCs. A time-course examination of MTOCs in synchronously cultured cells revealed that the multiple MTOCs appeared not immediately after irradiation but at the time of mitosis. Those multiple MTOCs formed at mitosis were inherited by the daughter cells in the next generation. The structure and capacity of the centrosomes to nucleate microtubules in vitro were then examined by electron microscopy of whole-mount preparations as well as by dark-field microscopy. About 70-80% of the centrosomes derived from nonirradiated cells were composed of a pair of centrioles and pericentriolar material, which initiated greater than 100 microtubules. The fraction of fully active complete centrosomes decreased with time of incubation after irradiation. These were replaced by disintegrated centrosomal components such as dissociated centrioles and pericentriolar cloud, a nucleating site with a single centriole, or only an amorphous structure of pericentriolar cloud. Assembly of less than 20 microtubules onto the amorphous cloud without centrioles was seen in 54% of the initiating sites in mitotic cells 2 d after irradiation. These results suggest that x-irradiation causes disintegration of centrosomes at mitosis when the structural and functional reorganization of centrosomes is believed to occur.