Role of murein lipoprotein in morphogenesis of the bacterial division septum: phenotypic similarity of lkyD and lpo mutants.

Phenotypes were compared in two different classes of mutants with defects in murein-lipoprotein (lkyD mutants of Salmonella typhimurium and an lpo mutant of Escherichia coli). Both mutations are associated with the same triad of phenotypic abnormalities, consisting of defective formation of the division septum, leakage of periplasmic proteins during growth, and increased sensitivity to several unrelated external toxic agents. The abnormality in septum formation consists of a defect in invagination of the outer membrane during formation of the nascent septum. The results suggest that formation of the murein-lipoprotein link plays an important role in differentiation of the division septum and perhaps also in maintaining the normal barrier function of the outer membrane.     

Preferential attachment of membrane glycoproteins to the cytoskeleton at the leading edge of lamella

The active forward movement of cells is often associated with the rearward transport of particles over the surfaces of their lamellae. Unlike the rest of the lamella, we found that the leading edge (within 0.5 microns of the cell boundary) is specialized for rearward transport of membrane-bound particles, such as Con A-coated latex microspheres. Using a single-beam optical gradient trap (optical tweezers) to apply restraining forces to particles, we can capture, move and release particles at will. When first bound on the central lamellar surface, Con A-coated particles would diffuse randomly; when such bound particles were brought to the leading edge of the lamella with the optical tweezers, they were often transported rearward. As in our previous studies, particle transport occurred with a concurrent decrease in apparent diffusion coefficient, consistent with attachment to the cytoskeleton. For particles at the leading edge of the lamella, weak attachment to the cytoskeleton and transport occurred with a half-time of 3 s; equivalent particles elsewhere on the lamella showed no detectable attachment when monitored for several minutes. Particles held on the cell surface by the laser trap attached more strongly to the cytoskeleton with time. These particles could escape a trapping force of 0.7 X 10(-6) dyne after 18 +/- 14 (sd) s at the leading edge, and after 64 +/- 34 (SD) s elsewhere on the lamella. Fluorescent succinylated Con A staining showed no corresponding concentration of general glycoproteins at the leading edge, but cytochalasin D-resistant filamentous actin was found at the leading edge. Our results have implications for cell motility: if the forces used for rearward particle transport were applied to a rigid substratum, cells would move forward. Such a mechanism would be most efficient if the leading edge of the cell contained preferential sites for attachment and transport.     

Morphogenesis of the bacterial division septum: identification of potential sites of division in lkyD mutants of Salmonella typhimurium.

It previously has been shown that lkyD mutants of Salmonella typhimurium form large blebs of outer membrane over the septal and polar regions of dividing cells. To determine whether the outer membrane blebs are formed over potential sites of division even in the absence of septal ingrowth, lkyD strains were studied under conditions in which ingrowth of inner membrane and murein was prevented by inactivation of the envA gene product. In aseptate filaments of the LkyD EnvA strain, outer membrane blebs occurred with the usual frequency and were preferentially located over regions where new septa were formed when cell division was subsequently permitted to resume. The results indicate that the outer membrane blebs of the LkyD strain are markers for potential sites of cell division, implying that an alteration in association of outer membrane and murein exists in these sites before the initiation of septal ingrowth. This localized change in cell envelope organization is independent of the septation-inducing effects of the envA gene product.     

Compartmentalization of the periplasmic space at division sites in gram-negative bacteria.

Phase-contrast and serial-section electron microscopy were used to study the patterns of localized plasmolysis that occur when cells of Salmonella typhimurium and Escherichia coli are exposed to hypertonic solutions of sucrose. In dividing cells the nascent septum was flanked by localized regions of periseptal plasmolysis. In randomly growing populations, plasmolysis bays that were not associated with septal ingrowth were clustered at the midpoint of the cell and at 1/4 and 3/4 cell lengths. The localized regions of plasmolysis were limited by continuous zones of adhesion that resembled the periseptal annular adhesion zones described previously in lkyD mutants of S. typhimurium (T. J. MacAlister, B. MacDonald, and L. I. Rothfield, Proc. Natl. Acad. Sci. USA 80:1372-1376, 1983). When cell division was blocked by growing divC(Ts) cells at elevated temperatures, the localized regions of plasmolysis were clustered along the aseptate filaments at positions that corresponded to sites where septum formation occurred when cell division was permitted to resume by a shift back to the permissive temperature. Taken together the results are consistent with a model in which extended zones of adhesion define localized compartments within the periplasmic space, predominantly located at future sites of cell division.     

Rate and topography of peptidoglycan synthesis during cell division in Escherichia coli: concept of a leading edge.

The rate at which the peptidoglycan of Escherichia coli is synthesized during the division cycle was studied with two methods. One method involved synchronization of E. coli MC4100 lysA cultures by centrifugal elutriation and subsequent pulse-labeling of the synchronously growing cultures with [meso-3H]diaminopimelic acid ([3H]Dap). The second method was autoradiography of cells pulse-labeled with [3H]Dap. It was found that the peptidoglycan is synthesized at a more or less exponentially increasing rate during the division cycle with a slight acceleration in this rate as the cells start to constrict. Apparently, polar cap formation requires synthesis of extra surface components, presumably to accommodate for a change in the surface-to-volume ratio. Furthermore, it was found that the pool size of Dap was constant during the division cycle. Close analysis of the topography of [3H]Dap incorporation at the constriction site revealed that constriction proceeded by synthesis of peptidoglycan at the leading edge of the invaginating cell envelope. During constriction, no reallocation of incorporation occurred, i.e., the incorporation at the leading edge remained high throughout the process of constriction. Impairment of penicillin-binding protein 3 by mutation or by the specific beta-lactam antibiotic furazlocillin did not affect [3H]Dap incorporation during initiation of constriction. However, the incorporation at the constriction site was inhibited in later stages of the constriction process. It is concluded that during division at least two peptidoglycan-synthesizing systems are operating sequentially.     

The actin-based nanomachine at the leading edge of migrating cells.

Two fundamental parameters of the highly dynamic, ultrathin lamellipodia of migrating fibroblasts have been determined-its thickness in living cells (176 +/- 14 nm), by standing-wave fluorescence microscopy, and its F-actin density (1580 +/- 613 microm of F-actin/microm(3)), via image-based photometry. In combination with data from previous studies, we have computed the density of growing actin filament ends at the lamellipodium margin (241 +/- 100/microm) and the maximum force (1.86 +/- 0.83 nN/microm) and pressure (10.5 +/- 4.8 kPa) obtainable via actin assembly. We have used cell deformability measurements (. J. Cell Sci. 44:187-200;. Proc. Natl. Acad. Sci. USA. 79:5327-5331) and an estimate of the force required to stall the polymerization of a single filament (. Proc. Natl. Acad. Sci. USA. 78:5613-5617;. Biophys. J. 65:316-324) to argue that actin assembly alone could drive lamellipodial extension directly.     

The structure and prenatal morphogenesis of the nasal septum in the rat

One hundred fetuses of the Sprague-Dawley rat were used: ten for each prenatal day, beginning with the twelfth day of gestation. Pregnant animals were sacrificed, fetuses removed and subsequently fixed in buffered formalin solution. Fetal heads were dehydrated, embedded in paraffin, and sectioned serially in the rostrocaudal direction at 10 to 15 μ. Serial sections from fetuses representing each day of gestation were stained with either H and E, Mallory's trichrome procedure, Gomori's reaction for alkaline phosphatase, or Steedman's alcian blue reaction. At the twelfth day, the primary nasal cavities were first observed. One day later, the nasobuccal membrane was established, and the vomeronasal organ invaginated into the nasal septum. Following the rupture of the membrane, at the fourteenth day, the nasal and buccal cavities remained in communication until the palatal shelves fused with the septum, at the seventeenth day. Prior to the thirteenth day, the septal skeleton is mesenchymal. The ossification in the vomer started at the sixteenth day and expanded progressively throughout prenatal life. First glandular primordia, one on each side of the septum, were observed during the sixteenth day, the number increased to five at term. The ducts ended in single blind sacs, before the eighteenth day, afterwards, the ducts presented an increasing number of collateral and terminal branches. There was no evidence of mucigen secretion from the septal glands during prenatal life. The initial stratified olfactory epithelium differentiated morphologically into a vestibular, respiratory, and an olfactory epithelium prior to the sixteenth prenatal day.     

The Pollard lab at Salk: moving the leading edge forward

This essay will review the years that the Pollard lab was at the Salk Institute in the last half of the 1990s. It was a highly productive time both in research and in training. For me personally, it shaped my career for the better in ways I am still discovering.     

Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose.

The crystal structure of a family-III cellulose-binding domain (CBD) from the cellulosomal scaffoldin subunit of Clostridium thermocellum has been determined at 1.75 A resolution. The protein forms a nine-stranded beta sandwich with a jelly roll topology and binds a calcium ion. conserved, surface-exposed residues map into two defined surfaces located on opposite sides of the molecule. One of these faces is dominated by a planar linear strip of aromatic and polar residues which are proposed to interact with crystalline cellulose. The other conserved residues are contained in a shallow groove, the function of which is currently unknown, and which has not been observed previously in other families of CBDs. On the basis of modeling studies combined with comparisons of recently determined NMR structures for other CBDs, a general model for the binding of CBDs to cellulose is presented. Although the proposed binding of the CBD to cellulose is essentially a surface interaction, specific types and combinations of amino acids appear to interact selectively with glucose moieties positioned on three adjacent chains of the cellulose surface. The major interaction is characterized by the planar strip of aromatic residues, which align along one of the chains. In addition, polar amino acid residues are proposed to anchor the CBD molecule to two other adjacent chains of crystalline cellulose.     

Crystal structure of the bacterial cell division inhibitor MinC

Bacterial cell division requires accurate selection of the middle of the cell, where the bacterial tubulin homologue FtsZ polymerizes into a ring structure. In Escherichia coli, site selection is dependent on MinC, MinD and MINE: MinC acts, with MinD, to inhibit division at sites other than the midcell by directly interacting with FTSZ: Here we report the crystal structure to 2.2 A of MinC from Thermotoga maritima. MinC consists of two domains separated by a short linker. The C-terminal domain is a right-handed beta-helix and is involved in dimer formation. The crystals contain two different MinC dimers, demonstrating flexibility in the linker region. The two-domain architecture and dimerization of MinC can be rationalized with a model of cell division inhibition. MinC does not act like SulA, which affects the GTPase activity of FtsZ, and the model can explain how MinC would select for the FtsZ polymer rather than the monomer.     

Weak force stalls protrusion at the leading edge of the lamellipodium

Protrusion, the first step of cell migration, is driven by actin polymerization coupled to adhesion at the cell's leading edge. Polymerization and adhesive forces have been estimated, but the net protrusion force has not been measured accurately. We arrest the leading edge of a moving fish keratocyte with a hydrodynamic load generated by a fluid flow from a micropipette. The flow arrests protrusion locally as the cell approaches the pipette, causing an arc-shaped indentation and upward folding of the leading edge. The effect of the flow is reversible upon pipette removal and dependent on the flow direction, suggesting that it is a direct effect of the external force rather than a regulated cellular response. Modeling of the fluid flow gives a surprisingly low value for the arresting force of just a few piconewtons per micrometer. Enhanced phase contrast, fluorescence, and interference reflection microscopy suggest that the flow does not abolish actin polymerization and does not disrupt the adhesions formed before the arrest but rather interferes with weak nascent adhesions at the very front of the cell. We conclude that a weak external force is sufficient to reorient the growing actin network at the leading edge and to stall the protrusion.     

Axon guidance receptors direct growth cone pathfinding: rivalry at the leading edge

One of the earliest steps in the development of the central and peripheral nervous systems is the initiation of axon outgrowth from newly born neurons. Nascent axons then navigate towards their specific targets to establish the intricate network of axon projections found within the mature central nervous system. In doing so, the projecting axons must continually reassess their spatial environment and accurately select the correct pathways among the maze of possible routes. A variety of molecular navigational systems governing axon pathfinding have now been identified. Understanding how these individual molecular guidance systems operate at the level of a single axon, and, how these different systems work in concert to initiate and steer axonal migration is a major goal in developmental neurobiology.     

Size-dependent accumulation of mRNA at the leading edge of chicken embryo fibroblasts

β-actin mRNA localizes to the leading edge of a living chicken embryo fibroblast. Recently we proposed that the mRNA maintains its localization at the leading edge by utilizing the heterogeneity of cytoplasmic microstructure (Yamagishi et al., 2009 [10]). In this study, we observed the intracellular distribution of β-actin mRNA variants to elucidate the mechanism of mRNA localization at the leading edge. We found that the degree of localization correlated positively with the molecular mass of the mRNA variants. We further demonstrated that the molecular mass-dependent localization was found even with dextrans, which have no biological function. The dependency of localization on molecular mass suggested that the barrier effect caused by the physical obstruction of the cytoplasmic microstructure is one of the major factors controlling mRNA localization in motile fibroblasts.     

Integrin-mediated protein kinase A activation at the leading edge of migrating cells

cAMP-dependent protein kinase A (PKA) is important in processes requiring localized cell protrusion, such as cell migration and axonal path finding. Here, we used a membrane-targeted PKA biosensor to reveal activation of PKA at the leading edge of migrating cells. Previous studies show that PKA activity promotes protrusion and efficient cell migration. In live migrating cells, membrane-associated PKA activity was highest at the leading edge and required ligation of integrins such as α4β1 or α5β1 and an intact actin cytoskeleton. α4 integrins are type I PKA-specific A-kinase anchoring proteins, and we now find that type I PKA is important for localization of α4β1 integrin-mediated PKA activation at the leading edge. Accumulation of 3′ phosphorylated phosphoinositides [PtdIns(3,4,5)P₃] products of phosphatidylinositol 3-kinase (PI3-kinase) is an early event in establishing the directionality of migration; however, polarized PKA activation did not require PI3-kinase activity. Conversely, inhibition of PKA blocked accumulation of a PtdIns(3,4,5)P₃-binding protein, the AKT-pleckstrin homology (PH) domain, at the leading edge; hence, PKA is involved in maintaining cell polarity during migration. In sum, we have visualized compartment-specific PKA activation in migrating cells and used it to reveal that adhesion-mediated localized activation of PKA is an early step in directional cell migration.     

Activation of soluble guanylyl cyclase at the leading edge during Dictyostelium chemotaxis

Dictyostelium contains two guanylyl cyclases, GCA, a 12-transmembrane enzyme, and sGC, a homologue of mammalian soluble adenylyl cyclase. sGC provides nearly all chemoattractant-stimulated cGMP formation and is essential for efficient chemotaxis toward cAMP. We show that in resting cells the major fraction of the sGC-GFP fusion protein localizes to the cytosol, and a small fraction is associated to the cell cortex. With the artificial substrate Mn2+/GTP, sGC activity and protein exhibit a similar distribution between soluble and particulate fraction of cell lysates. However, with the physiological substrate Mg2+/GTP, sGC in the cytosol is nearly inactive, whereas the particulate enzyme shows high enzyme activity. Reconstitution experiments reveal that inactive cytosolic sGC acquires catalytic activity with Mg2+/GTP upon association to the membrane. Stimulation of cells with cAMP results in a twofold increase of membrane-localized sGC-GFP, which is accompanied by an increase of the membrane-associated guanylyl cyclase activity. In a cAMP gradient, sGC-GFP localizes to the anterior cell cortex, suggesting that in chemotacting cells, sGC is activated at the leading edge of the cell.     

Demonstration of cell division by septation in a variety of gram-negative rods.

Through use of an initial fixative employing a combination of crotonaldehyde and glutaraldehyde, septa were preserved in thin sections of dividing cells of strains of Pseudomonas aeruginosa, Salmonella typhimurium, Shigella sonnei, and Escherichia coli when grown at 30 C in a dilute basal medium. The same procedures, however, revealed only a constrictive division process in Proteus vulgaris and Erwinia sp. This adds to the evidence that septation, although difficult to demonstrate, is the process of cell division in the enteric gram-negative rods and the pseudomonads and that constriction is a fixation artifact in these organisms.