Centriole maturation in the amoebae ofPhysarum polycephalum

Summary The precise geometry of pro-centriole formation has been studied inPhysarum polycephalum amoebae. The spatial references used were the posterior and the anterior kinetosomes which are unequivocally defined by the presence of the posterior para-kinetosomal structure, the microtubular array 4 and the microtubular arrays 1, 2, and 3. The observations made suggest that pro-centrioles follow a maturation process. A pro-centriole formed during the n^th cell cycle becomes the posterior kinetosome during the (n + 1)^th cell cycle and the anterior one during all the following cell cycles. Pro-centriole formation occurs late in the cell cycle. This observation disagrees with a role of pro-centriole formation in the regulation of S phase in contrast to what has been suggested in other eucaryotic cells.     

Migration of the cell nucleus during the amoebo-flagellate transformation ofPhysarum polycephalum is mediated by an actin-generated force that acts on the centrosome

Summary The mechanism of cell-nuclear migration during the amoebo-flagellate transformation inPhysarum polycephalum was examined by fluorescence microscopy after staining with a tubulinspecific antibody, rhodamine-conjugated phalloidin and 4,6-diamidino-2-phenylindole (DAPI). While the round amoeba cells changed to comma-shaped swarm cells within 20min after suspension in buffer, the cell nuclei moved from the central region of each cell to the periphery, each forming a sharp projection in the direction of movement. A centrosome also migrated from the center of the cell to the cell periphery. Since the centrosome was in close contact with the tip that protruded from the cell nucleus throughout the cellnuclear migration, the migration of the cell nucleus and the centrosome could be recognized as comigration. Then the flagella began to elongate from the centrosome and the cells became slender and polarized, adopting the so-called comma-shape. On the basis of these observations, the transformation process was classified into three steps: cell-nuclear migration, flagella formation and swarm maturation. The comigration of the cell nucleus and the centrosome was not inhibited by the anti-microtubule drug nocodazole (4 M) but it was inhibited by the anti-microfilament drug cytochalasin A (4 M), suggesting that the force of migration is generated by microfilaments. To investigate the role of the centrosome in this comigration in detail, we identified two aberrant strains, defective in swimming ability, from among various laboratory strains. The two strains, TM 4 and J, were found to have defects in cell-nuclear migration. Strain TM 4 had two types of irregular swarm cells: in one, only a part of the cell nucleus projected a thin filamentous structure; and in the other, no cell-nuclear migration occurred. Strain J had two centrosomes per cell and such swarm cells exhibited an attempt of cell-nuclear migration at two sites which corresponded to the position of the centrosome. The characteristics of these two strains indicate that the centrosome is essential for cell-nuclear migration. Our observations suggest that the cell-nuclear migration is mediated by actin-generated forces that act on the centrosome rather than on the cell nucleus itself.Abbreviations FITC fluorescein isothiocyanate - DAP 4,6-diamidino-2-phenylindole - PBS phosphate-buffered saline - KPB potassium phosphate buffer - MTOC microtubule organizing center     

Stabilization of monoasters by taxol in the amoebae ofPhysarum polycephalum (Myxomycetes)

Summary Although the plasmodial stage of the MyxomycetePhysarum polycephalum was unaffected with 200 M taxol, the amoebal stage was sensitive to 10 M taxol. The first effect of taxol resulted in an accumulation of cells blocked as a monopolar centrosphere surrounded by condensed chromosomes. In 79% of cases these monoasters contained two pairs of centrioles. The mitotic block in a monopolar stage in the presence of taxol delayed the occurrence of late mitotic events such as chromosome decondensation and formation of the nuclear envelope. Escape from the monopolar centrosphere stage and formation of multinucleated amoebae involved a transient monopolar reconstruction stage in which a long microtubular bundle interacted with a small chromosomal mass outside the monoaster.     

Information processing for the organization of chemotactic behavior ofPhysarum polycephalum studied by micro-thermography

Summary The spatial and temporal pattern of oscillating temperatures on the cell surface of a plasmodial strand ofPhysarum polycephalum was measured with a sensitive thermal image camera. The longitudinal tension of the strand was studied simultaneously. In the absence of chemical stimulation, the phases of the temperature oscillation observed at various portions of the strand were entrained with almost coincidental phase. The temperature and tension oscillation were synchronized, although the phase difference between them was occasionally changed. With local chemical stimulation, the phase of the temperature oscillation advanced in the portion to which the plasmodium would be induced to migrate. The phases between temperature and tension oscillations then became constant. The mechanism by which the plasmodium processes local information of chemical stimulus to global information for the migration is discussed.     

Periodic events and cell cycle regulation in the plasmodia ofPhysarum polycephalum

The multinucleated plasmodia ofPhysarum polycephalum, a myxomycete, have been extensively used in cell cycle studies. The natural synchrony of mitosis and DNA synthesis, easy culture methods, the ready fusions obtainable between plasmodia, and the amenability to phase specific studies, employing physical and chemical perturbers, are some of the attractive features of this organism. Because of the absence of a Gl phase in the plasmodia, there is a crowding of cell cycle specific marker events at the G2/M boundary, which reflect features of both the G2/M and the Gl/S boundaries of a typical eukaryotic cell. Prominent among these are the synthesis and overall activity of thymidine kinase, the co-triggering of tubulin and histone genes, translation of their mRNA, the organization and duplication of the microtubular organizing centres of the mitotic spindle and the triggering of cdc 2 kinase activity. These above events have not only served as good markers to monitor the progress of the plasmodial cell cycle, but have also been fairly thoroughly analysed by means of specific perturbers such as DNA synthesis inhibitors, antimicrotubular drugs, UV-irradiation, heat-shock etc. Along with fusion studies, these perturbation studies have been helpful in the formulation of various models on regulation of mitosis. These above aspects as well as prospects for future studies employing this organism are discussed This paper is dedicated to the memory of the late Prof. S C K Nair, formerly University Professor of Physics.     

Effect of microtubule-disrupting drugs on protein and RNA synthesis in Physarum polycephalum amoebae

The effects of the microtubule-disrupting drugs, colchicine, vinblastine, podophyllotoxin, griseofulvin, and lumicolchicine (10^-5 M), on protein and RNA synthesis were studied in Physarum polycephalum amoebae in culture. All, except lumicolchicine, were found to simultaneously reduce the rate of protein synthesis and stimulate RNA synthesis. These results parallel the effects seen in cells exposed to heat shock. Treatment of the cells with a microfilament-disrupting drug, cytochalasin B (10 g/ml in ethanol), resulted in a reduced rate of protein synthesis after 2 h compared to a similar effect by vinblastine in 5–15 min. A morphological abnormality, microtubule paracystals, were seen associated with centrioles in vinblastine-treated cells in which protein synthesis had been reduced by 50%. Vinblastine and podophyllotoxin were shown to interfere with the recovery of protein synthesis after inhibition by low or elevated temperatures. The possible role of microtubules in regulating the translational response of a cell to an external environmental stimulus is discussed.     

Caldesmon affects actin organization at the leading edge and inhibits cell migration

The effect of the suppression of expression of the actin-binding protein caldesmon on the motility of nonmuscle cells has been studied. A more than a fivefold decrease in the content of this protein in cells by RNA interference led to the disturbance of the formation of actin stress fibers and acceleration of cell migration to the zone of injury of the monolayer. A stimulation of stationary cells by serum induced more than 1,5-fold accumulation of stress fibers only in control cells, but not in caldesmon-deficient cells. Similarly, the accumulation of actin filaments was observed in actively migrating cells of only wild type, but not in the cells with low caldesmon content. These changes occurred mainly at the leading edge of the migrating cell where the distinct structure of actin filaments was not seen in the absence of caldesmon. It was assumed that caldesmon inhibits cell migration due to the stabilization of actin in filaments and a decrease in the dynamics of monomeric actin at the leading edge of the migrating cell.     

NMR assignment of PN2-3, a tubulin interaction subdomain of the CPAP protein

We report the NMR assignment of the PN2-3 subdomain of the CPAP protein. It has been previously shown that this motif interacts with tubulin, inhibits microtubule nucleation from the centrosome and depolymerizes taxol-stabilized microtubules. Marie-Jeanne Clément and Philippe Savarin contributed equally.     

The centriole cycle in the amoebae of the myxomycetePhysarum polycephalum

Summary In the amoebae of the myxomycetePhysarum polycephalum, procentrioles are formed on the anterior and posterior centrioles in early prophase. Although the relative position of the parental and procentrioles is fixed, all relative positions of the daughter and parental centrioles were observed. During the different stages of mitosis daughter centrioles elongate and acquire anterior satellites, one of the characteristic features of the anterior centrioles. All other anterior morphological characteristics appear only in telophase and early reconstruction stages. In contrast to the parental posterior centrioles, which do not change morphologically during the successive mitotic stages, the parental anterior centrioles lose their morphological characteristics in late prophase and early prometaphase and then acquire the morphological features characteristic of the posterior centrioles. Thus, the following maturation scheme is suggested: a procentriole becomes an anterior centriole during the first mitosis and a posterior centriole during the second mitosis. Since posterior features are maintained during mitosis, the posterior centriole corresponds to the final state of centriole maturation.     

The role of the cell nucleus in mechanotransduction

Mechanical forces are known to influence cellular processes with consequences at the cellular and physiological level. The cell nucleus is the largest and stiffest organelle, and it is connected to the cytoskeleton for proper cellular function. The connection between the nucleus and the cytoskeleton is in most cases mediated by the linker of nucleoskeleton and cytoskeleton (LINC) complex. Not surprisingly, the nucleus and the associated cytoskeleton are implicated in multiple mechanotransduction pathways important for cellular activities. Herein, we review recent advances describing how the LINC complex, the nuclear lamina, and nuclear pore complexes are involved in nuclear mechanotransduction. We will also discuss how the perinuclear actin cytoskeleton is important for the regulation of nuclear mechanotransduction. Additionally, we discuss the relevance of nuclear mechanotransduction for cell migration, development, and how nuclear mechanotransduction impairment leads to multiple disorders.     

Intermediate-affinity LFA-1 binds alpha-actinin-1 to control migration at the leading edge of the T cell

T lymphocytes use LFA-1 to migrate into lymph nodes and inflammatory sites. To investigate the mechanisms regulating this migration, we utilize mAbs selective for conformational epitopes as probes for active LFA-1. Expression of the KIM127 epitope, but not the 24 epitope, defines the extended conformation of LFA-1, which has intermediate affinity for ligand ICAM-1. A key finding is that KIM127-positive LFA-1 forms new adhesions at the T lymphocyte leading edge. This LFA-1 links to the cytoskeleton through alpha-actinin-1 and disruption at the level of integrin or actin results in loss of cell spreading and migratory speed due to a failure of attachment at the leading edge. The KIM127 pattern contrasts with high-affinity LFA-1 that expresses both 24 and KIM127 epitopes, is restricted to the mid-cell focal zone and controls ICAM-1 attachment. Identification of distinctive roles for intermediate- and high-affinity LFA-1 in T lymphocyte migration provides a biological function for two active conformations of this integrin for the first time.     

PKD is recruited to sites of actin remodelling at the leading edge and negatively regulates cell migration

Protein kinase D (PKD) has been implicated in the regulation of cell shape, adhesion, and migration. At the leading edge of migrating cells active PKD co-localizes with F-actin, Arp3 and cortactin. Platelet derived growth factor (PDGF) activates PKD and recruits the kinase to the leading edge, suggesting a role for PKD in actin remodelling. In support of this, PKD directly interacts with F-actin and phosphorylates cortactin in vitro. Interference with PKD function by overexpression of a dominant negative PKD or by PKD-specific siRNA enhanced cell migration, whereas cells overexpressing PKD wild type displayed reduced migratory potential. Taken together, these data reveal a negative regulatory function of PKD in cell migration.     

The endosomal adaptor protein APPL1 impairs the turnover of leading edge adhesions to regulate cell migration

Cell migration is a complex process that requires the integration of signaling events that occur in distinct locations within the cell. Adaptor proteins, which can localize to different subcellular compartments, where they bring together key signaling proteins, are emerging as attractive candidates for controlling spatially coordinated processes. However, their function in regulating cell migration is not well understood. In this study, we demonstrate a novel role for the adaptor protein containing a pleckstrin-homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (APPL1) in regulating cell migration. APPL1 impairs migration by hindering the turnover of adhesions at the leading edge of cells. The mechanism by which APPL1 regulates migration and adhesion dynamics is by inhibiting the activity of the serine/threonine kinase Akt at the cell edge and within adhesions. In addition, APPL1 significantly decreases the tyrosine phosphorylation of Akt by the nonreceptor tyrosine kinase Src, which is critical for Akt-mediated cell migration. Thus, our results demonstrate an important new function for APPL1 in regulating cell migration and adhesion turnover through a mechanism that depends on Src and Akt. Moreover, our data further underscore the importance of adaptor proteins in modulating the flow of information through signaling pathways.     

Microtubule cytoskeleton and morphogenesis in the amoebae of the myxomycete Physarum polycephalum

Summary— The amoebae of the myxomycete Physarum polycephalum are of interest in order to analyze the morphogenesis of the microtubule and microfilament cytoskeleton during cell cycle and flagellation. The amoebal interphase microtubule cytoskeleton consists of 2 distinct levels of organization, which correspond to different physiological roles. The first level is composed of the 2 kinetosomes or centrioles and their associated structures. The anterior and posterior kinetosomes forming the anterior and posterior flagella are morphologically distinguishable. Each centriole plays a role in the morphogenesis of its associated satellites and specific microtubule arrays. The 2 distinct centrioles correspond to the 2 successive maturation stages of the pro-centrioles which are built during prophase. The second level of organization consists of a prominent microtubule organizing center (mtoc 1) to which the anterior centriole is attached at least during interphase. This mtoc plays a role in the formation of the mitotic pole. These observations based on ultrastructural and physiological analyses of the amoebal cystoskeleton are now being extended to the biochemical level. The complex formed by the 2 centrioles and the mtoc 1 has been purified without modifying the microtubule-nucleating activity of the mtoc 1. Several microtubule-associated proteins have been characterized by their ability to bind taxol-stabilized microtubules. Their functions (e.g., microtubule assembly, protection of microtubules against dilution or cold treatment, phosphorylating and ATPase activities) are under investigation. These biochemical approaches could allow in vitro analysis of the morphogenesis of the amoebal microtubule cytoskeleton.     

The Golgi apparatus and cell polarity: Roles of the cytoskeleton,the Golgi matrix,and Golgi membranes

Membrane trafficking plays a crucial role in cell polarity by directing lipids and proteins to specific subcellular locations in the cell and sustaining a polarized state. The Golgi apparatus, the master organizer of membrane trafficking, can be subdivided into three layers that play different mechanical roles: a cytoskeletal layer, the so-called Golgi matrix, and the Golgi membranes. First, the outer regions of the Golgi apparatus interact with cytoskeletal elements, mainly actin and microtubules, which shape, position, and orient the organelle. Closer to the Golgi membranes, a matrix of long coiled–coiled proteins not only selectively captures transport intermediates but also participates in signaling events during polarization of membrane trafficking. Finally, the Golgi membranes themselves serve as active signaling platforms during cell polarity events. We review here the recent findings that link the Golgi apparatus to cell polarity, focusing on the roles of the cytoskeleton, the Golgi matrix, and the Golgi membranes.     

Experimental modification of structures at the periphery of the liver cell nucleus

Summary This paper deals with the morphological effects of various experimental treatments on structures at the periphery of isolated liver cell nuclei. The results demonstrate that the outer and inner nuclear membranes, the pore-annulus complex, the fibrous lamina, and the perinuclear chromatin layer are interconnected, and together constitute an integrated cortex surrounding the nucleus in these interphase cells. Treatment with Triton X100 removes both the outer and inner nuclear membranes.Work supported by the U. S. Atomic Energy Commission.This work was started before the death of Dr. Wassermann on June 16, 1969; this paper is respectfully dedicated to his memory.     

Pervasive migration of organellar DNA to the nucleus in plants

A surprisingly large number of plant nuclear DNA sequences inferred to be remnants of chloroplast and mitochondrial DNA migration events were detected through computer-assisted database searches. Nineteen independent organellar DNA insertions, with a median size of 117 by (range of 38 to >785 bp), occur in the proximity of 15 nuclear genes. One fragment appears to have been passed through a RNA intermediate, based on the presence of an edited version of the mitochondrial gene in the nucleus. Tandemly arranged fragments from disparate regions of organellar genomes and from different organellar genomes indicate that the fragments joined together from an intracellular pool of RNA and/or DNA before they integrated into the nuclear genome. Comparisons of integrated sequences to genes lacking the insertions, as well as the occurrence of coligated fragments, support a model of random integration by end joining. All transferred sequences were found in noncoding regions, but the positioning of organellar-derived DNA in introns, as well as regions 5 and 3 to nuclear genes, suggests that the random integration of organellar DNA has the potential to influence gene expression patterns. A semiquantitative estimate was performed on the amount of organellar DNA being transferred and assimilated into the nucleus. Based on this database survey, we estimate that 3–7% of the plant nuclear genomic sequence files contain organellar-derived DNA. The timing and the magnitude of genetic flux to the nuclear genome suggest that random integration is a substantial and ongoing process for creating sequence variation.Correspondence to: J.L. Blanchard     

The effect of cell sedimentation on measuring chondrocyte population migration using a Boyden chamber

The Boyden chamber assay provides a convenient method of assessing cell migration and measuring cell motility coefficients at the population level. Previous models of this assay completely ignore cell sedimentation in the suspension, assuming that all cells have already settled on the filter surface before commencing migration within the filter. However, ignoring cell sedimentation could lead to poor data interpretation because the time required for cells to settle through the suspension is close to the incubation period of only a few hours. This study models the Boyden chamber assay by incorporating the cell settling process to account for the cells remaining in the upper well when other cells migrate in the filter. The simulations in this study elucidate the experiments in the literature that test the haptotactic and chemotactic responses of rabbit chondrocytes to type II collagen. This study determines the cell population random motility, as well as the haptotaxis and chemotaxis coefficients, by fitting the experimental data. Results show that the chemotactic motility coefficient is 100 times greater than the haptotactic coefficient, and the equilibrium collagen-receptor dissociation constant is about 10-fold the haptotactic counterpart. Diffusion causes the soluble collagen gradients in the chemotactic case to decline over time, while the coated collagen gradients in the haptotactic assay are likely to remain fixed. As a result, the chemotactic case exhibits a lower number of migrated cells than the haptotactic assay. This study also demonstrates the influences of the dimensionless parameters that control cell behavior in the Boyden assay, providing a reference for future experiment designs.     

Interferon-gamma inhibits T84 epithelial cell migration by redirecting transcytosis of beta1 integrin from the migrating leading edge

Intestinal inflammation is associated with epithelial damage and formation of mucosal wounds. Epithelial cells migration is required for wound closure. In inflammatory status, migrating epithelial cells are exposed to proinflammatory cytokines such as IFN-gamma. However, influence of such cytokines on intestinal epithelial wound closure remains unknown. The present study was designed to investigate the effect of IFN-gamma on migration of model T84 intestinal epithelial cells and recovery of epithelial wounds. IFN-gamma significantly inhibited rate of T84 cell migration and closure of epithelial wounds. This effect was accompanied by the formation of large aberrant lamellipodia at the leading edge as well as significant decrease in the number of beta(1) integrin containing focal adhesions. IFN-gamma exposure increased endocytosis of beta(1) integrin and shifted its accumulation from early/recycling endosomes at the leading edge to a yet unidentified compartment at the cell base. This redirection in beta(1) integrin transcytosis was inhibited by depolymerization of microtubules with nocodazole and was unaffected by stabilization of microtubules with docetaxel. These results suggest that IFN-gamma attenuates epithelial wound closure by microtubule-dependent redirection of beta(1) integrin transcytosis from the leading edge of migrating cells thereby inhibiting adequate turnover of focal adhesion complexes and cell migration.