Role of Actin Microfilaments in Black Creek Canal Virus Morphogenesis

We have investigated the involvement of cytoskeletal proteins in the morphogenesis of Black Creek Canal virus (BCCV), a New World hantavirus. Immunofluorescent staining of BCCV-infected cells revealed a filamentous pattern of virus antigen, the appearance of which was sensitive to treatment with cytochalasin D, an actin microfilament-depolymerizing drug. Double immunofluorescence staining of BCCV-infected Vero cells with anti-BCCV nucleocapsid (N) monoclonal antibody and phalloidin revealed a colocalization of the BCCV N protein with actin microfilaments. A similar, though less prominent, filamentous pattern was observed in BHK21 cells transiently expressing the BCCV N protein alone but not in cells expressing the BCCV G1 and G2 glycoproteins. Moreover, the association of the N protein with actin microfilaments was confirmed by coimmunoprecipitation with β-actin-specific antibody. Treatment of the BCCV-infected Vero cells at 3 days postinfection with cytochalasin D decreased the yield of released BCCV by 94% relative to the yield from untreated cells. Pretreatment of Vero cells with cytochalasin D prior to and during BCCV adsorption and entry had no effect on the outcome of virus production. These results indicate that actin filaments may play an important role in hantavirus assembly and/or release.     

The Drosophila Actin Regulator ENABLED Regulates Cell Shape and Orientation during Gonad Morphogenesis

Organs develop distinctive morphologies to fulfill their unique functions. We used Drosophila embryonic gonads as a model to study how two different cell lineages, primordial germ cells (PGCs) and somatic gonadal precursors (SGPs), combine to form one organ. We developed a membrane GFP marker to image SGP behaviors live. These studies show that a combination of SGP cell shape changes and inward movement of anterior and posterior SGPs leads to the compaction of the spherical gonad. This process is disrupted in mutants of the actin regulator, enabled (ena). We show that Ena coordinates these cell shape changes and the inward movement of the SGPs, and Ena affects the intracellular localization of DE-cadherin (DE-cad). Mathematical simulation based on these observations suggests that changes in DE-cad localization can generate the forces needed to compact an elongated structure into a sphere. We propose that Ena regulates force balance in the SGPs by sequestering DE-cad, leading to the morphogenetic movement required for gonad compaction.     

Melatonin Inhibits Embryonic Salivary Gland Branching Morphogenesis by Regulating Both Epithelial Cell Adhesion and Morphology

Many organs, including salivary glands, lung, and kidney, are formed by epithelial branching during embryonic development. Branching morphogenesis occurs via either local outgrowths or the formation of clefts that subdivide epithelia into buds. This process is promoted by various factors, but the mechanism of branching morphogenesis is not fully understood. Here we have defined melatonin as a potential negative regulator or “brake” of branching morphogenesis, shown that the levels of it and its receptors decline when branching morphogenesis begins, and identified the process that it regulates. Melatonin has various physiological functions, including circadian rhythm regulation, free-radical scavenging, and gonadal development. Furthermore, melatonin is present in saliva and may have an important physiological role in the oral cavity. In this study, we found that the melatonin receptor is highly expressed on the acinar epithelium of the embryonic submandibular gland. We also found that exogenous melatonin reduces salivary gland size and inhibits branching morphogenesis. We suggest that this inhibition does not depend on changes in either proliferation or apoptosis, but rather relates to changes in epithelial cell adhesion and morphology. In summary, we have demonstrated a novel function of melatonin in organ formation during embryonic development.     

The Cell Adhesion Molecule L1 Is Developmentally Regulated in the Renal Epithelium and Is Involved in Kidney Branching Morphogenesis

We immunopurified a surface antigen specific for the collecting duct (CD) epithelium. Microsequencing of three polypeptides identified the antigen as the neuronal cell adhesion molecule L1, a member of the immunoglobulin superfamily. The kidney isoform showed a deletion of exon 3. L1 was expressed in the mesonephric duct and the metanephros throughout CD development. In the adult CD examined by electron microscopy, L1 was not expressed on intercalated cells but was restricted to CD principal cells and to the papilla tall cells. By contrast, L1 appeared late in the distal portion of the elongating nephron in the mesenchymally derived epithelium and decreased during postnatal development. Immunoblot analysis showed that expression, proteolytic cleavage, and the glycosylation pattern of L1 protein were regulated during renal development. L1 was not detected in epithelia of other organs developing by branching morphogenesis. Addition of anti-L1 antibody to kidney or lung organotypic cultures induced dysmorphogenesis of the ureteric bud epithelium but not of the lung. These results suggest a functional role for L1 in CD development in vitro. We further postulate that L1 may be involved in the guidance of developing distal tubule and in generation and maintenance of specialized cell phenotypes in CD.     

Cell Constriction: Contractile Role of Microfilaments in Division and Development

The role of microfilaments in causing cell constrictions isdiscussed from a comparative point of view. Morphologicallysimilar microfilaments occur in the contractile ring of dividingcells and in the apices of neural plate cells during neurulation.New evidence is presented regarding the distribution and orientationof apical microfilaments in neural plates of chicks and salamanderembryos. These findings complement what is known about neurulationin frogs and cell cleavage in a variety of cells. In all cases,cell constriction occurs precisely and exclusively at thoseplanes in which circular arrays of microfilaments are found.A sliding mechanism of microfilament contractility is discussed,as are possible mechanisms involved in filament alignment. Attentionis given to the cell surface as a substratum for microfilamentassembly. New evidence is presented regarding the early morphologicaldetermination of the neural plate in Xenopus, even before microfilamentsare clearly evident or invagination begins.     

Signaling in Cell Differentiation and Morphogenesis

All the information to make a complete, fully functional living organism is encoded within the genome of the fertilized oocyte. How is this genetic code translated into the vast array of cellular behaviors that unfold during the course of embryonic development, as the zygote slowly morphs into a new organism? Studies over the last 30 years or so have shown that many of these cellular processes are driven by secreted or membrane-bound signaling molecules. Elucidating how the genetic code is translated into instructions or signals during embryogenesis, how signals are generated at the correct time and place and at the appropriate level, and finally, how these instructions are interpreted and put into action, are some of the central questions of developmental biology. Our understanding of the causes of congenital malformations and disease has improved substantially with the rapid advances in our knowledge of signaling pathways and their regulation during development. In this article, I review some of the signaling pathways that play essential roles during embryonic development. These examples show some of the mechanisms used by cells to receive and interpret developmental signals. I also discuss how signaling pathways downstream from these signals are regulated and how they induce specific cellular responses that ultimately affect cell fate and morphogenesis.     

Hydraulic Properties of MDCK Cell Epithelium

The water permeability of the apical and basolateral cell membranes and the compliance of the lateral intercellular spaces (LIS) of MDCK monolayers were measured on confluent cultures grown on permeable supports. Cell membrane water permeabilities were determined, using quantitative differential interference light microscopy, from the rate of cell volume decrease after exposure to a hyperosmotic bathing solution. Both membranes exhibited osmotic water permeabilities (P_OSM) of ∼10 μm/sec, comparable to that of unmodified lipid bilayers. The compliance of the cell membranes forming the lateral intercellular space (LIS) between cells was determined from the pressure-volume relation. Confocal microscopy of fluorescent labeling of the basolateral cell membranes was used to delineate the LIS geometry as transepithelial hydrostatic pressure was varied. The LIS were poorly deformable as a function of transepithelial hydrostatic pressure until a pressure of ≥8 cm H₂O (basolateral > apical) was reached where catastrophic failure of intercellular connections occurred. The compliance of the LIS was calculated from the geometry changes at pressures <8 cm H₂O and ranged from 0.05–0.11 cm H₂O⁻¹, comparable to that previously predicted in mathematical models of the rat proximal tubule. Received: 10 January 1996/Revised: 9 May 1996     

Changes in Cell and Organ Shape during Early Development of the Ocular Lens

It is currently fashionable to attribute changes in organ shapeduring development to the actions of microtubules and microfilamentson individual cells of the organ in question. In the case ofthe eye lens it has been proposed that cellular elongation underthe influence of microtubules and/or apical contraction by microfilamentsare responsible for the remodeling of the originally low cuboidalectoderm into the tall and wedge-shaped presumptive lens cells.Invagination of the lens is thought to follow automatically.These ideas cannot account for certain observations on lensmorphogenesis, such as the relatively fixed diameter of theorgan rudiment during early development, which is incompatiblewith the supposed contraction of the rudiment. We found that the area of contact between presumptive lens andoptic cup does become fixed after a few hours of "induction."There is a remarkable correlation in time between this fixation,and the process of lens cell elongation and increase in lenscell density. We calculated that the latter two can, in fact,be accounted for by population pressure caused by continuedcell division within the defined area of the lens rudiment.A mathematical model along these lines was developed, whichexplains lens invagination on the basis of cell number and size,extent of the area of contact between ectoderm and optic cup,and cell population doubling times. We hypothesize that the prevention of lateral cell spreadingwithin the lens territory, after the contact area becomes fixed,is a function of the build-up in extracellular materials inthis area during the "induction period." Both lens rudimentand presumptive retina contribute to this extracellular matrix.     

Curling and Local Shape Changes of Red Blood Cell Membranes Driven by Cytoskeletal Reorganization

Human red blood cells (RBCs) lack the actin-myosin-microtubule cytoskeleton that is responsible for shape changes in other cells. Nevertheless, they can display highly dynamic local deformations in response to external perturbations, such as those that occur during the process of apical alignment preceding merozoite invasion in malaria. Moreover, after lysis in divalent cation-free media, the isolated membranes of ruptured ghosts show spontaneous inside-out curling motions at the free edges of the lytic hole, leading to inside-out vesiculation. The molecular mechanisms that drive these rapid shape changes are unknown. Here, we propose a molecular model in which the spectrin filaments of the RBC cortical cytoskeleton control the sign and dynamics of membrane curvature depending on whether the ends of the filaments are free or anchored to the bilayer. Computer simulations of the model reveal that curling, as experimentally observed, can be obtained either by an overall excess of weakly-bound filaments throughout the cell, or by the flux of such filaments toward the curling edges. Divalent cations have been shown to arrest the curling process, and Ca²⁺ ions have also been implicated in local membrane deformations during merozoite invasion. These effects can be replicated in our model by attributing the divalent cation effects to increased filament-membrane binding. This process converts the curl-inducing loose filaments into fully bound filaments that arrest curling. The same basic mechanism can be shown to account for Ca²⁺-induced local and dynamic membrane deformations in intact RBCs. The implications of these results in terms of RBC membrane dynamics under physiological, pathological, and experimental conditions is discussed.     

Cell-Based Multi-Parametric Model of Cleft Progression during Submandibular Salivary Gland Branching Morphogenesis

Cleft formation during submandibular salivary gland branching morphogenesis is the critical step initiating the growth and development of the complex adult organ. Previous experimental studies indicated requirements for several epithelial cellular processes, such as proliferation, migration, cell-cell adhesion, cell-extracellular matrix (matrix) adhesion, and cellular contraction in cleft formation; however, the relative contribution of each of these processes is not fully understood since it is not possible to experimentally manipulate each factor independently. We present here a comprehensive analysis of several cellular parameters regulating cleft progression during branching morphogenesis in the epithelial tissue of an early embryonic salivary gland at a local scale using an on lattice Monte-Carlo simulation model, the Glazier-Graner-Hogeweg model. We utilized measurements from time-lapse images of mouse submandibular gland organ explants to construct a temporally and spatially relevant cell-based 2D model. Our model simulates the effect of cellular proliferation, actomyosin contractility, cell-cell and cell-matrix adhesions on cleft progression, and it was used to test specific hypotheses regarding the function of these parameters in branching morphogenesis. We use innovative features capturing several aspects of cleft morphology and quantitatively analyze clefts formed during functional modification of the cellular parameters. Our simulations predict that a low epithelial mitosis rate and moderate level of actomyosin contractility in the cleft cells promote cleft progression. Raising or lowering levels of contractility and mitosis rate resulted in non-progressive clefts. We also show that lowered cell-cell adhesion in the cleft region and increased cleft cell-matrix adhesions are required for cleft progression. Using a classifier-based analysis, the relative importance of these four contributing cellular factors for effective cleft progression was determined as follows: cleft cell contractility, cleft region cell-cell adhesion strength, epithelial cell mitosis rate, and cell-matrix adhesion strength.     

Myxospore Formation in Myxococcus xanthus: Chemical Changes in the Cell Wall During Cellular Morphogenesis

Vegetative cells of Myxococcus xanthus (strain FB) were induced to form myxospores by the glycerol induction technique. Several structural changes took place in the peptidoglycan during myxospore formation. The percent of the peptidoglycan comprised of monomer (disaccharide peptide) decreased from about 20% to approximately 7%. The proportion of the total diaminopimelic acid possessing a free amino group decreased about 11%. A carbohydrate containing only glucose was found to be bound, possibly covalently, to the vegetative cell and myxospore peptidoglycan. The amount of carbohydrate relative to peptidoglycan decreased by two-thirds during myxospore formation. None of the above changes in the peptidoglycan were observed in a mutant (strain GNI) of M. xanthus which was unable to convert to myxospores when incubated in the glycerol induction medium, or in the parental wild type (FB) when it was incubated in induction medium lacking the myxospore inducer, glycerol.     

Origins of Escherichia coli Growth Rate and Cell Shape Changes at High External Osmolality

In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope, followed by an active recovery. After recovery, and if the external osmolality remains high, cells have been shown to grow more slowly, smaller, and at reduced turgor pressure. Despite the fact that the active recovery is a key stress response, the nature of these changes and how they relate to each other is not understood. Here, we use fluorescence imaging of single cells during hyperosmotic shocks, combined with custom made microfluidic devices, to show that cells fully recover their volume to the initial, preshock value and continue to grow at a slower rate immediately after the recovery. We show that the cell envelope material properties do not change after hyperosmotic shock, and that cell shape recovers along with cell volume. Taken together, these observations indicate that the turgor pressure recovers to its initial value so that reduced turgor is not responsible for the reduced growth rate observed immediately after recovery. To determine the point at which the reduction in cell size and turgor pressure occurs after shock, we measured the volume of E. coli cells at different stages of growth in bulk cultures. We show that cell volume reaches the same maximal level irrespective of the osmolality of the media. Based on these measurements, we propose that turgor pressure is used as a feedback variable for osmoregulatory pumps instead of being directly responsible for the reduction in growth rates. Reestablishment of turgor to its initial value might ensure correct attachment of the inner membrane and cell wall needed for cell wall biosynthesis.     

Directional Cell Migration,but Not Proliferation,Drives Hair Placode Morphogenesis

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Changes in Cell Size and Shape Associated with Changes in the Replication Time of the Chromosome of Escherichia coli

Average cell mass is shown to be inversely related to the concentration of thymine in the growth medium of a thy⁻ strain of Escherichia coli. The kinetics of the transition from one steady-state average cell mass to another was followed in an attempt to determine the relationship between the chromosome replication time and the time between completion of a round of chromosome replication and the subsequent cell division. Differences in average cell mass are shown to be associated with similar differences in average cell volume. Changes in volume associated with changes in thymine concentration are shown to be due primarily to differences in the width of cells. It is proposed that extension in length of the cell envelope occurs at a linear rate which is proportional to the growth rate and which doubles at the time of termination of rounds of replication. Changes in volume not associated with a change in growth rate are therefore accommodated by a change in cell width. Conditions are described under which average cell mass can continue to increase in successive generations and no steady-state average cell mass is achieved.