Flattening,movement and control of division of epithelial-like cells

The kinetics of cell division and movement in four epithelial-like cell lines, grown in continuously perfused culture medium, were studied by time-lapse cinemicrography. One line exhibited “contact regulation of cell division,” so that the rate of mitosis per cell decreased steadily as population density increased. In the other three lines mitosis was not controlled as a function of population density until the cells became very crowded. An explanation for this difference was sought in terms of the hypothesis that the rate of division depends on the area of the cell membrane. Cells of the contact-regulated line flattened uniformly on the substrate. Their motility was restrained by adhesion between their borders. As they crowded together, contact inhibition of cell overlap caused a steady decrease in average surface area per cell. All three of the non-controlled lines also had contact inhibition of overlap. Cells of two of them flattened on the substrate; but these cells had little mutual adhesion and were highly motile, so that they continually changed their shapes. The areas of their cell membranes were therefore not subject to a restraint that could control the rate of division. Cells of the fourth line remained rounded or only slightly flattened during culture growth, so that no change in cell membrane area occurred that could change the rate of division.     

A role for a novel centrosome cycle in asymmetric cell division

Tissue stem cells play a key role in tissue maintenance. Drosophila melanogaster central brain neuroblasts are excellent models for stem cell asymmetric division. Earlier work showed that their mitotic spindle orientation is established before spindle formation. We investigated the mechanism by which this occurs, revealing a novel centrosome cycle. In interphase, the two centrioles separate, but only one is active, retaining pericentriolar material and forming a "dominant centrosome." This centrosome acts as a microtubule organizing center (MTOC) and remains stationary, forming one pole of the future spindle. The second centriole is inactive and moves to the opposite side of the cell before being activated as a centrosome/MTOC. This is accompanied by asymmetric localization of Polo kinase, a key centrosome regulator. Disruption of centrosomes disrupts the high fidelity of asymmetric division. We propose a two-step mechanism to ensure faithful spindle positioning: the novel centrosome cycle produces a single interphase MTOC, coarsely aligning the spindle, and spindle-cortex interactions refine this alignment.     

Size and complexity among multicellular organisms

The diversity of specialized cell types ('complexity') is estimated for a wide range of multicellular organisms. Complexity increases with size, independently of phylogeny. This is interpreted in economic terms as the consequence of a greater degree of cooperative division of labour within larger entities. The rate of increase of complexity with size is less in the case of a cooperative division of labour (cell types within bodies) than in the analogous case of a competitive division of labour (species within communities). This is atttributed to the inutility of single specialized cells whose goods must be shared among all the many cells of a large organism. Major groups of organisms differ in complexity at given size: animals are more complex than plants, and phaeophytes are simpler than either.     

Mechanism of centrosome positioning during the wound response in BSC-1 cells

Locomoting cells are characterized by a pronounced external and internal anterior-posterior polarity. One of the events associated with cell polarization at the onset of locomotion is a shift of the centrosome, or MTOC, ahead of the nucleus. This position is believed to be of strategic importance for directional cell movement and cell polarity. We have used BSC-1 cells at the edge of an in vitro wound to clarify the causal relationship between MTOC position and the initiation of cell polarization. We find that pronounced cell polarization (the extension of a lamellipod) can take place in the absence of MTOC repositioning or microtubules. Conversely, MTOCs will reposition even after lamellar extension and cell polarization have occurred. Repositioning requires microtubules that extend to the cell periphery and is independent of selective detyrosination of microtubules extending towards the cell front. Significantly, MTOCs maintain, or at least attempt to maintain, a position at the cell's centroid. This is most clearly demonstrated in wounded monolayers of enucleated cells where the MTOC closely follows the centroid position. We suggest that the primary response to the would is the biased extension of a lamellipod, which can occur in the absence of microtubules and MTOC repositioning. Lamellipod extension leads to a shift of the cell's centroid towards the wound. The MTOC, in an attempt to maintain a position near the cell center, will follow. This will automatically put the MTOC ahead of the nucleus in the vast majority of cells. The nucleus as a reference for MTOC position may not be as meaningful as previously thought.     

Rapid Lytic Granule Convergence to the MTOC in Natural Killer Cells Is Dependent on Dynein But Not Cytolytic Commitment

Natural killer cells are lymphocytes specialized to participate in host defense through their innate ability to mediate cytotoxicity by secreting the contents of preformed secretory lysosomes (lytic granules) directly onto a target cell. This form of directed secretion requires the formation of an immunological synapse and occurs stepwise with actin reorganization preceding microtubule-organizing center (MTOC) polarization to the synapse. Because MTOC polarization to the synapse is required for polarization of lytic granules, we attempted to define their interrelationship. We found that compared with the time required for MTOC polarization, lytic granules converged to the MTOC rapidly. The MTOC-directed movement of lytic granules was independent of actin and microtubule reorganization, dependent on dynein motor function, occurred before MTOC polarization, and did not require a commitment to cytotoxicity. This defines a novel paradigm for rapid MTOC-directed transport as a prerequisite for directed secretion, one that may prepare, but not commit cells for precision secretory function.     

Electron microscopic study of the process of intermediate cell division in the cyst of the coccidian Sarcocystis muris

The intermediate cell is a third definitely outlined morpho-functional type of cells within sarcocysts, in addition to the two other well known ones--metrocytes and merozoites (Fedoseenko, Levit, 1979; Beyer et al., 1981). The intermediate cell divides by endodyogeny, the nuclear division being accomplished by semi-closed pleuromitosis. In the dividing nuclei, centrioles and extranuclear bundle of microtubules connecting two pairs of centrioles are seen in addition to centrocones and associated semi-spindles. Pro-, ana- and telophases of mitosis have been followed. The microtubule organizing center (MTOC) seen in the cytoplasm of the intermediate cell is represented by the polar ring with microtubules originating from it. The MTOC is involved in the division of both the nucleus and the cytoplasm. The formation of the polar ring (MTOC) from the Golgi-adjunct has been first discovered and followed in the course of the intermediate cell division.     

Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells

The microtubule-organizing center (MTOC) is reoriented between the nucleus and the leading edge in many migrating cells and contributes to directional migration. Models suggest that the MTOC is moved to its position during reorientation. By direct imaging of wound-edge fibroblasts after triggering MTOC reorientation with soluble factors, we found instead that the nucleus moved away from the leading edge to reorient the MTOC, while the MTOC remained stationary. Rearward nuclear movement was coupled with actin retrograde flow and was regulated by a pathway involving Cdc42, MRCK, myosin, and actin. Nuclear movement was unaffected by the inhibition of dynein, Par6, or PKCzeta, yet these components were essential for MTOC reorientation, as they maintained the MTOC at the cell centroid. These results show that nuclear repositioning is an initial polarizing event in migrating cells and that the positions of the nucleus and the MTOC are established by separate regulatory pathways.     

The use of immunostaining to quantify x-ray and heat-induced multiple microtubule organizing centers in normal and transformed cells

Microtubule organizing centers (MTOC) in control, irradiated and heated C3H 10T1/2 mouse embryo cells and two radiation-transformed sublines, R1 and R25, were made visible by indirect immunofluorescence using antibody against tubulin. The MTOC were reformed by 5-min incubation in fresh medium after the microtubules were depolymerized with nocodazole. The R1 line had a different distribution of MTOC/cell than the parent 10T1/2 line or R25, which had similar distributions. After irradiation, multiple MTOC appeared in the normal and radiation-transformed cells irradiated to 10 Gy and incubated for 24 or 48 h. The multiple foci of microtubule reformation in the irradiated cells indicate that radiation damage is expressed in structural elements in the cytoplasm. After heat treatment of the three cell lines (43 degrees C for 93 min and 45 degrees C for 25 min), the MTOC were disrupted and many cells did not have visible organizing centers at 24 or 48 h, while others had a large number of small centers of microtubule reformation. The distribution of MTOC/cell seen in R25 cells after the treatment had similar patterns to those of the 10T1/2 line rather than to those of the other radiation-transformed line, R1. Thus, the radiation or heat response seen in the MTOC is not dependent upon cell transformation.     

Aiming for the top: non-cell autonomous control of shoot stem cells in Arabidopsis

Journal of Plant Research - In multicellular organisms, not all cells are created equal. Instead, organismal complexity is achieved by specialisation and division of labour between distinct cell...     

Microtubule detachment from the microtubule-organizing center as a key event in the complete turnover of microtubules in cells

Microtubule (MT) response to different steady state temperatures and to rapid shifts in temperature was studied quantitatively in large, thin cells (LT-cells) from the goldfish scale. MT number and total tubulin concentration per cell were found to be fairly constant in cells from the same fish, regardless of cell size but between fish, could differ by a factor of two. The total tubulin concentration was similar to that found in mammalian tissue culture cells and the proportion in MT form increased with increasing steady state temperature. Total MT length quickly and exponentially decreased when cells were rapidly chilled to approximately -3 degrees C. In contrast, the average length of the MTs bound to the MT organizing center (MTOC) did not significantly change. Free MTs were generated during chilling and had an average length roughly half that of bound MTs. These observations suggest that 1) there is a functional block to rapid depolymerization at the unattached end of the MTOC bound MTs and 2) depolymerization of the MT occurs from the originally bound end only after its release from the MTOC. The presence of free MTs in a wide variety of cells suggests that these two features may be characteristic of steady state MTs in other cells. When the temperature of the LT-cells was abruptly raised, the number of MTs initiated on the MTOC rapidly increased and reached a brief steady state long before the MTs completely elongated. Many MTs then apparently detached from the MTOC and depolymerized before a final steady state was reached. When cells containing newly polymerized MTs were chilled to approximately -3 degrees C, the MTs detached from the MTOC more rapidly than those starting from steady state. Furthermore, the block to depolymerization at the unattached end was not complete. These observations suggest that newly formed, non-steady state MTs are different from the older, steady state MTs.     

Sporogenesis in Eusporangiate Ferns: I. Monoplastidic Meiosis in Angiopteris (Marattiales)

Angiopteris (Marattiales) undergoes the more primitive form of monoplastidic meiosis, while other ferns have evolved the polyplastidic type typical of seed plants. In monoplastidic cell division, the single plastid divides and serves as site of the microtubule organizing center (MTOC) for spindle formation resulting in coordinated division of plastid, nucleus, and cytoplasm. In plants with polyplastidic cell division, the MTOC is diffuse and generally perinuclear. Monoplastidic cell division is seen as a plesiomorphic feature that was inherited from algal ancestors containing a single plastid and modified through evolution. Monoplastidic meiosis occurs in all groups of bryophytes (although in only a few hepatics), Isoetes, Selaginella, certain generic segregates of Lycopodium, and in members of the Marattiales. It is not known to occur in psilophytes, Equisetum, leptosporangiate ferns, or seed plants. Received 30 January 2001/ Accepted in revised form 24 April 2001     

Cdc42 mediates nucleus movement and MTOC polarization in Swiss 3T3 fibroblasts under mechanical shear stress

Nucleus movement is essential during nucleus positioning for tissue growth and development in eukaryotic cells. However, molecular regulators of nucleus movement in interphase fibroblasts have yet to be identified. Here, we report that nuclei of Swiss 3T3 fibroblasts undergo enhanced movement when subjected to shear flows. Such movement includes both rotation and translocation and is dependent on microtubule, not F-actin, structure. Through inactivation of Rho GTPases, well-known mediators of cytoskeleton reorganization, we demonstrate that Cdc42, not RhoA or Rac1, controls the extent of nucleus translocation, and more importantly, of nucleus rotation in the cytoplasm. In addition to generating nuclei movement, we find that shear flows also causes repositioning of the MTOC in the direction of flow. This behavior is also controlled by Cdc42 via the Par6/protein kinase Czeta pathway. These results are the first to establish Cdc42 as a molecular regulator of not only shear-induced MTOC polarization in Swiss 3T3 fibroblasts, but also of shear-induced microtubule-dependent nucleus movement. We propose that the movements of MTOC and nucleus are coupled chemically, because they are both regulated by Cdc42 and dependent on microtubule structure, and physically, possibly via Hook/SUN family homologues similar to those found in Caenorhabditis elegans.     

Quantitative analysis of germline mitosis in adult C. elegans

Certain aspects of the distal gonad of C. elegans are comparable to niche/stem cell systems in other organisms. The distal tip cell (DTC) caps a blind-ended tube; only the distal germ cells maintain proliferation in response to signaling from the DTC via the GLP-1/Notch signaling pathway in the germ line. Fruitful comparison between this system and other stem cell systems is limited by a lack of basic information regarding germ cell division behavior in C. elegans. Here, we explore the spatial pattern of cell division frequency in the adult C. elegans germ line relative to distance from the distal tip. We mapped the positions of actively dividing germline nuclei in over 600 fixed gonad preparations including the wild type and a gain-of-function ligand-responsive GLP-1 receptor mutant with an extended mitotic zone. One particularly surprising observation from these data is that the frequency of cell divisions is lower in distal-most cells-cells that directly contact the distal tip cell body-relative to cells further proximal, a difference that persists in the gain-of-function GLP-1 mutant. These results suggest that cell division frequency in the distal-most cells may be suppressed or otherwise controlled in a complex manner. Further, our data suggest that the presence of an active cell division influences the probability of observing simultaneous cell divisions in the same gonad arm, and that simultaneous divisions tend to cluster spatially. We speculate that this system behaves similarly to niche/stem cell/transit amplifying cell systems in other organisms.     

MTOC reorientation occurs during FcgammaR-mediated phagocytosis in macrophages

Cell polarization is essential for targeting signaling elements and organelles to active plasma membrane regions. In a few specialized cell types, cell polarity is enhanced by reorientation of the MTOC and associated organelles toward dynamic membrane sites. Phagocytosis is a highly polarized process whereby particles >0.5 microm are internalized at stimulated regions on the cell surface of macrophages. Here we provide detailed evidence that the MTOC reorients toward the site of particle internalization during phagocytosis. We visualized MTOC proximity to IgG-sRBCs in fixed RAW264.7 cells, during live cell imaging using fluorescent chimeras to label the MTOC and using frustrated phagocytosis assays. MTOC reorientation in macrophages is initiated by FcgammaR ligation and is complete within 1 h. Polarization of the MTOC toward the phagosome requires the MT cytoskeleton and dynein motor activity. cdc42, PI3K, and mPAR-6 are all important signaling molecules for MTOC reorientation during phagocytosis. MTOC reorientation was not essential for particle internalization or phagolysosome formation. However Golgi reorientation in concert with MTOC reorientation during phagocytosis implicates MTOC reorientation in antigen processing events in macrophages.     

The machinery of mitochondrial fusion, division, and distribution, and emerging connections to apoptosis

Mitochondrial undergo regulated fusion and division in many organisms and cell types, and each event is mediated by a different complex of proteins each containing at least one large GTPase. The mitochondrial fusion and division molecular machinery is in large part conserved; recent studies show a functional connection between some of these proteins and the apoptotic cascade. Mitochondria also undergo directed movement in cells, and the gene products that attach and propel mitochondria along cytoskeletal elements (actin filaments in some organisms, microtubules in others) are becoming gradually elucidated.     

Effects of the herbicide isopropyl-N-phenyl carbamate on microtubules and MTOCs in lines of Nicotiana sylvestris resistant and sensitive to its action

To clarify the mechanism of isopropyl-N-phenyl carbamate (IPC) action on higher plant cells the sensitivity of microtubules (cortical network and mitotic arrays) and microtubule organizing centers to IPC treatment (30 microM) in IPC-resistant and sensitive Nicotiana sylvestris lines was studied. It was clearly demonstrated that IPC does not depolymerize plant MTs but causes the MTOC damage in cells, which results in MTOC fragmentation, splitting of the spindle poles and in abnormal division spindle formation. It was also found that IPC-resistance of mutant N. sylvestris line correlates not with tubulin resistance to IPC action but possibly with resistance of one of the proteins involved in MTOC composition.     

The position of the microtubule-organizing center relative to the nucleus is independent of the direction of cell migration in Dictyostelium discoideum

Dictyostelium amoebae can migrate in several different modes. We tested for correlations of the direction of cell locomotion with the relative positions of the nucleus and microtubule-organizing center (MTOC). Five cases were analyzed on electron micrographs with a microcomputer. Each mode of movement showed characteristic locations of the MTOC relative to the nucleus; however, they differed in the various cases. In randomly migrating interphase amoebae, the number of cells with the MTOC located behind the nucleus was twice as great as those with the MTOC located ahead of the nucleus. During chemotactic migration toward folic acid, cells with the MTOC behind the nucleus were more numerous, with a concomitant reduction of anterior MTOCs. When amoebae aggregated on agar plates, a posterior location of the MTOC was most strikingly favored, whereas in cells aggregating under submerged conditions, the MTOC was indifferently anterior or posterior to the nucleus. (It may be significant that EDTA-resistant cell-cell adhesion was fully expressed in the former cells, but weaker in the latter.) Finally, in the case of chemotactically migrating cells from dissociated pseudoplasmodia, which adhere by means of other molecules, the MTOC was consistently ahead of the nucleus. Thus the MTOC shows no necessary preferential position anterior or posterior to the nucleus; its position, rather, correlates with the type of migration and perhaps with the nature of cell-cell adhesion.     

The Obligate Human Pathogen, Neisseria gonorrhoeae, Is Polyploid

We show using several methodologies that the Gram-negative, diplococcal-bacterium Neisseria gonorrhoeae has more than one complete genome copy per cell. Gene dosage measurements demonstrated that only a single replication initiation event per chromosome occurs per round of cell division, and that there is a single origin of replication. The region containing the origin does not encode any genes previously associated with bacterial origins of replication. Quantitative PCR results showed that there are on average three genome copies per coccal cell unit. These findings allow a model for gonococcal DNA replication and cell division to be proposed, in which a minimum of two chromosomal copies exist per coccal unit within a monococcal or diplococcal cell, and these chromosomes replicate in unison to produce four chromosomal copies during cell division. Immune evasion via antigenic variation is an important mechanism that allows these organisms to continually infect a high risk population of people. We propose that polyploidy may be necessary for the high frequency gene conversion system that mediates pilin antigenic variation and the propagation of N. gonorrhoeae within its human hosts.     

The Obligate Human Pathogen, Neisseria gonorrhoeae, Is Polyploid

We show using several methodologies that the Gram-negative, diplococcal-bacterium Neisseria gonorrhoeae has more than one complete genome copy per cell. Gene dosage measurements demonstrated that only a single replication initiation event per chromosome occurs per round of cell division, and that there is a single origin of replication. The region containing the origin does not encode any genes previously associated with bacterial origins of replication. Quantitative PCR results showed that there are on average three genome copies per coccal cell unit. These findings allow a model for gonococcal DNA replication and cell division to be proposed, in which a minimum of two chromosomal copies exist per coccal unit within a monococcal or diplococcal cell, and these chromosomes replicate in unison to produce four chromosomal copies during cell division. Immune evasion via antigenic variation is an important mechanism that allows these organisms to continually infect a high risk population of people. We propose that polyploidy may be necessary for the high frequency gene conversion system that mediates pilin antigenic variation and the propagation of N. gonorrhoeae within its human hosts.