Faithful chromosome segregation is an essential component of cell division in all organisms. The eukaryotic mitotic machinery uses the cytoskeleton to move specific chromosomal regions. To investigate the potential role of the actin-like MreB protein in bacterial chromosome segregation, we first demonstrate that MreB is the direct target of the small molecule A22. We then demonstrate that A22 completely blocks the movement of newly replicated loci near the origin of replication but has no qualitative or quantitative effect on the segregation of other loci if added after origin segregation. MreB selectively interacts, directly or indirectly, with origin-proximal regions of the chromosome, arguing that the origin-proximal region segregates via an MreB-dependent mechanism not used by the rest of the chromosome.

Achieving proper polarity is essential for cellular function. In bacteria, cell polarity has been observed by using both morphological and molecular markers; however, no general regulators of bacterial cell polarity have been identified. Here we investigate the effect on cell polarity of two cytoskeletal elements previously implicated in cell shape determination. We find that the actin-like MreB protein mediates global cell polarity in Caulobacter crescentus, although the intermediate filament-like CreS protein influences cell shape without affecting cell polarity. MreB is organized in an axial spiral that is dynamically rearranged during the cell cycle, and MreB dynamics may be critical for the determination of cell polarity. By examining depletion and overexpression strains, we demonstrate that MreB is required both for the polar localization of the chromosomal origin sequence and the dynamic localization of regulatory proteins to the correct cell pole. We propose that the molecular polarity inherent in an actin-like filament is translated into a mechanism for directing global cell polarity.T he bacterium Caulobacter crescentus is particularly well suited to studies of cell polarity because of its inherently asymmetric life cycle that yields, at each cell division, progeny that have different polar morphologies and cell fates (Fig. 1C). The larger ''stalked'' cell progeny has a cytoplasmic extension known as a stalk at one pole, and the smaller ''swarmer'' cell progeny has a flagellum and pili at one pole. Immediately after cell division, the stalked cell initiates DNA replication, grows, and synthesizes a flagellum and a pilus secretion apparatus at the pole opposite the stalk before dividing asymmetrically (1). After emerging from a brief G1 arrest, the swarmer cell sheds its flagellum and pili, develops a stalk at that same pole, and initiates DNA replication. This new stalked cell then proceeds through the same asymmetric life cycle as other stalked cells. The stalk is clearly identifiable by light microscopy, making it easy to monitor Caulobacter polarity throughout the cell cycle. In addition, several structural and regulatory proteins as well as the origin of replication have been shown to dynamically localize to the stalked pole, the swarmer pole, or both poles during the cell cycle (2). These proteins and chromosomal regions serve as molecular markers of cell polarity. Subcellularly localized molecules are not unique to Caulobacter. For example, chemoreceptors, histidine kinases, response regulators, and several chromosomal loci have specific addresses in a wide variety of bacteria, including those without obvious morphological asymmetry (3-7). In addition, virtually every eukaryotic cell has subcellularly localized proteins, such as secretory molecules localized to the bud tip in yeast and neurotransmitter receptors localized to neuronal synapses (8, 9).The mechanism by which Caulobacter cells achieve such exquisite polarity is unknown, and no global regulators of cell polarity have been identified i...

We have analyzed the spontaneous symmetry breaking and initiation of actin-based motility in keratocytes (fish epithelial cells). In stationary keratocytes, the actin network flow was inwards and radially symmetric. Immediately before motility initiation, the actin network flow increased at the prospective cell rear and reoriented in the perinuclear region, aligning with the prospective axis of movement. Changes in actin network flow at the cell front were detectable only after cell polarization. Inhibition of myosin II or Rho kinase disrupted actin network organization and flow in the perinuclear region and decreased the motility initiation frequency, whereas increasing myosin II activity with calyculin A increased the motility initiation frequency. Local stimulation of myosin activity in stationary cells by the local application of calyculin A induced directed motility initiation away from the site of stimulation. Together, these results indicate that large-scale actin–myosin network reorganization and contractility at the cell rear initiate spontaneous symmetry breaking and polarized motility of keratocytes.

Epithelial folding transforms simple sheets of cells into complex three-dimensional tissues and organs during animal development. Epithelial folding has mainly been attributed to mechanical forces generated by an apically localized actomyosin network, however, contributions of forces generated at basal and lateral cell surfaces remain largely unknown. Here we show that a local decrease of basal tension and an increased lateral tension, but not apical constriction, drive the formation of two neighboring folds in developing Drosophila wing imaginal discs. Spatially defined reduction of extracellular matrix density results in local decrease of basal tension in the first fold; fluctuations in F-actin lead to increased lateral tension in the second fold. Simulations using a 3D vertex model show that the two distinct mechanisms can drive epithelial folding. Our combination of lateral and basal tension measurements with a mechanical tissue model reveals how simple modulations of surface and edge tension drive complex three-dimensional morphological changes.

The actin homolog MreB contributes to bacterial cell shape. Here, we explore the role of the coexpressed MreC protein in Caulobacter and show that it forms a periplasmic spiral that is out of phase with the cytoplasmic MreB spiral. Both mreB and mreC are essential, and depletion of either protein results in a similar cell shape defect. MreB forms dynamic spirals in MreC-depleted cells, and MreC localizes helically in the presence of the MreB-inhibitor A22, indicating that each protein can form a spiral independently of the other. We show that the peptidoglycan transpeptidase Pbp2 also forms a helical pattern that partially colocalizes with MreC but not MreB. Perturbing either MreB (with A22) or MreC (with depletion) causes GFP-Pbp2 to mislocalize to the division plane, indicating that each is necessary but not sufficient to generate a helical Pbp2 pattern. We show that it is the division process that draws Pbp2 to midcell in the absence of MreB's regulation, because cells depleted of the tubulin homolog FtsZ maintain a helical Pbp2 localization in the presence of A22. By developing and employing a previously uncharacterized computational method for quantitating shape variance, we find that a FtsZ depletion can also partially rescue the A22-induced shape deformation. We conclude that MreB and MreC form spatially distinct and independently localized spirals and propose that MreB inhibits division plane localization of Pbp2, whereas MreC promotes lengthwise localization of Pbp2; together these two mechanism ensure a helical localization of Pbp2 and, thereby, the maintenance of proper cell morphology in Caulobacter.actin ͉ MreB ͉ MreC ͉ Pbp2 P rokaryotes exhibit a wide variety of cell shapes (including rods, spheres, spirals, squares, and stars), but the mechanisms by which these shapes are achieved are poorly understood. The extracellular peptidoglycan layer provides structural rigidity for bacterial cells and is of central importance in the establishment and maintenance of cell shape (1, 2). This layer is a meshwork of disaccharide chains (alternating N-acetylglucosamine and N-acetylmuramic acid sugars) cross-linked by short peptide bridges. Rod-shaped bacteria are believed to possess two peptidoglycan synthesis complexes with distinct activities: one for elongation along the cell length and the other for cell division (1, 3, 4). It is thought that maintenance of a regular rod shape requires the activities of these two complexes to be carefully balanced (3).The bacterial cytoskeleton also plays a role in the establishment and maintenance of cell shape (2). Homologs for the three major types of eukaryotic cytoskeletal elements have been identified in bacteria: the actin homolog is MreB, the tubulin homolog, FtsZ, and the intermediate filament homolog, Crescentin (5). Of these known cytoskeletal elements, MreB is the only one required to establish an underlying rod-like character. Mutations in mreB confer a spherical-like morphology to the normally rod-like cells of Escherichia coli, Bacillus subtilis, and Caulobact...