Regulation of dynamic polarity switching in bacteria by a Ras‐like G‐protein and its cognate GAP

The rod‐shaped cells of the bacterium Myxococcus xanthus move uni‐directionally and occasionally undergo reversals during which the leading/lagging polarity axis is inverted. Cellular reversals depend on pole‐to‐pole relocation of motility proteins that localize to the cell poles between reversals. We show that MglA is a Ras‐like G‐protein and acts as a nucleotide‐dependent molecular switch to regulate motility and that MglB represents a novel GTPase‐activating protein (GAP) family and is the cognate GAP of MglA. Between reversals, MglA/GTP is restricted to the leading and MglB to the lagging pole defining the leading/lagging polarity axis. For reversals, the Frz chemosensory system induces the relocation of MglA/GTP to the lagging pole causing an inversion of the leading/lagging polarity axis. MglA/GTP stimulates motility by establishing correct polarity of motility proteins between reversals and reversals by inducing their pole‐to‐pole relocation. Thus, the function of Ras‐like G‐proteins and their GAPs in regulating cell polarity is found not only in eukaryotes, but also conserved in bacteria.     

Wolbachia bacteria reside in host Golgi-related vesicles whose position is regulated by polarity proteins

Wolbachia pipientis are intracellular symbiotic bacteria extremely common in various organisms including Drosophila melanogaster, and are known for their ability to induce changes in host reproduction. These bacteria are present in astral microtubule-associated vesicular structures in host cytoplasm, but little is known about the identity of these vesicles. We report here that Wolbachia are restricted only to a group of Golgi-related vesicles concentrated near the site of membrane biogenesis and minus-ends of microtubules. The Wolbachia vesicles were significantly mislocalized in mutant embryos defective in cell/planar polarity genes suggesting that cell/tissue polarity genes are required for apical localization of these Golgi-related vesicles. Furthermore, two of the polarity proteins, Van Gogh/Strabismus and Scribble, appeared to be present in these Golgi-related vesicles. Thus, establishment of polarity may be closely linked to the precise insertion of Golgi vesicles into the new membrane addition site.     

Global cell sorting is mediated by local cell-cell interactions in the C. elegans embryo

The Caenorhabditis elegans embryo achieves pattern formation by sorting cells into coherent regions before morphogenesis is initiated. The sorting of cells is coupled to their fate. Cells move extensively relative to each other to reach their correct position in the body plan. Analyzing the mechanism of cell sorting in in vitro culture experiments using 4D microscopy, we show that all AB-derived cells sort only according to their local neighbors, and that all cells are able to communicate with each other. The directions of cell movement do not depend on a cellular polarity but only on local cell-cell interactions; in experimental situations, this allows even the reversal of the polarity of whole regions of the embryo. The work defines a new mechanism of pattern formation we call "cell focusing".     

Planar polarity and short-range polarization in Drosophila embryos

Planar cell polarity is a common and probably universal feature of epithelial cells throughout their life. It is not only visible in the external parts of adult animals and plants, but also present in newborn cells such as in the primary Drosophila epithelium. It controls not only cell shape and differentiation, but also cell motility, cell shape changes and it directs how animals are shaped. In this review, we report how planar cell polarity arises in Drosophila embryos and thereby illustrate how general and extensive planar polarity is during development, from the very beginning to the end. We present the main features of planar cell polarization in Drosophila embryos, in particular the fact that it occurs over a short range of just a few cell diameters, and within a very short time window. We contrast these with other systems, such as the adult Drosophila wing where planar cell polarity occurs at longer range.     

Oriented adhesion of Escherichia coli to polystyrene particles

The adhesion of nonflagellated Escherichia coli strain K-12 to polystyrene (PS) latex spheres or glass capillaries has been observed by using several techniques. Attention was focused on the orientation of the rod-shaped bacteria as they adhered to the surfaces in 100 mM phosphate-buffered saline. Data show that PS particles adhered to the ends of the bacteria more than 90% of the time. Moreover, the PS particles adhered to one end only, never to both. Similarly, for experiments with bacteria adhering to glass, the bacteria adhered on their ends. In order to determine whether the end of a bacterium had a different charge density from that of the middle, rotational electrophoresis experiments were used. These experiments indicated no measurable charge nonuniformity. In order to examine how strongly adhered the bacteria were to the PS particles, differential electrophoresis was used. Almost always, bacteria were found to be irreversibly adhered to the PS spheres. The cause of the oriented adhesion is not likely due to surface lipopolysaccharides (LPS), since the three strains of K-12 that were used, each having a different length of LPS, showed similar behavior. The results are discussed in terms of bacterial cell polarity. The data indicate that nanodomains on the bacterial ends are important for adhesion and that the time scale for irreversible adhesion is short.     

Cell polarity and epithelial oncogenesis

Pathologists have long recognized that tumour formation in epithelia leads to disruption of normal epithelial cell polarity. Despite this, few studies have taken advantage of new information on the biogenesis of cell polarity to analyse the process of epithelial oncogenesis. Recent studies of epithelial cell lines now indicate that the pattern of breakdown of polarity during oncogenesis may reflect the way in which normal epithelial cells achieve polarity. These results suggest not only a novel way to study the development of polarity in vitro, but also new ideas for the early detection of cancer.     

The polarising role of cell adhesion molecules in early development

Polarising a cell or an embryo is a crucial and recurrent event during development, as it is important for cell differentiation and migration. Cells can become polarised along their apical-basal axis and also within the plane of the tissue layer to which they belong. The embryo develops three axes: the anteroposterior, the dorsoventral and the left-right axis. Recent work indicates instructive roles for cell adhesion molecules in establishing not only apical-basal polarity but also planar cell polarity and, surprisingly, in the generation of left-right asymmetry in vertebrates. Signalling cascades that regulate polarity formation seem to be conserved among different organisms, thereby raising the intriguing question of whether this also holds true for the cell adhesion molecules.     

Oriented Adhesion of Escherichia coli to Polystyrene Particles

The adhesion of nonflagellated Escherichia coli strain K-12 to polystyrene (PS) latex spheres or glass capillaries has been observed by using several techniques. Attention was focused on the orientation of the rod-shaped bacteria as they adhered to the surfaces in 100 mM phosphate-buffered saline. Data show that PS particles adhered to the ends of the bacteria more than 90% of the time. Moreover, the PS particles adhered to one end only, never to both. Similarly, for experiments with bacteria adhering to glass, the bacteria adhered on their ends. In order to determine whether the end of a bacterium had a different charge density from that of the middle, rotational electrophoresis experiments were used. These experiments indicated no measurable charge nonuniformity. In order to examine how strongly adhered the bacteria were to the PS particles, differential electrophoresis was used. Almost always, bacteria were found to be irreversibly adhered to the PS spheres. The cause of the oriented adhesion is not likely due to surface lipopolysaccharides (LPS), since the three strains of K-12 that were used, each having a different length of LPS, showed similar behavior. The results are discussed in terms of bacterial cell polarity. The data indicate that nanodomains on the bacterial ends are important for adhesion and that the time scale for irreversible adhesion is short.     

GTPases in bacterial cell polarity and signalling

In bacteria, large G domain GTPases have well-established functions in translation, protein translocation, tRNA modification and ribosome assembly. In addition, bacteria also contain small Ras-like GTPases consisting of stand-alone G domains. Recent data have revealed that small Ras-like GTPases as well as large G domain GTPases in bacteria function in the regulation of cell polarity, signal transduction and possibly also in cell division. The small Ras-like GTPase MglA together with its cognate GAP MglB regulates cell polarity in Myxococcus xanthus, and the small Ras-like GTPase CvnD9 in Streptomyces coelicolor is involved in signal transduction. Similarly, the large GTPase FlhF together with the ATPase FlhG regulates the localization and number of flagella in polarly flagellated bacteria. Moreover, large dynamin-like GTPases in bacteria may function in cell division. Thus, the function of GTPases in bacteria may be as pervasive as in eukaryotes.     

Leukocytes navigate by compass: roles of PI3Kgamma and its lipid products

Morphologic polarity is necessary for the motility of mammalian cells. In leukocytes responding to a chemoattractant, this polarity is regulated by activities of small Rho guanosine triphosphatases (Rho GTPases) and the phosphoinositide 3-kinases (PI3Ks). Moreover, in neutrophils, lipid products of PI3Ks appear to regulate activation of Rho GTPases, are required for cell motility and accumulate asymmetrically to the plasma membrane at the leading edge of polarized cells. By spatially regulating Rho GTPases and organizing the leading edge of the cell, PI3Ks and their lipid products could play pivotal roles not only in establishing leukocyte polarity but also as compass molecules that tell the cell where to crawl.     

Fission yeast cell morphogenesis: identification of new genes and analysis of their role during the cell cycle

To identify new genes involved in the control of cell morphogenesis in the fission yeast Schizosaccharomyces pombe we have visually screened for temperature-sensitive mutants that show defects in cell morphology. We have isolated and characterized 64 mutants defining 19 independent genes, 10 of which have not been previously described. One class of mutants, defining 12 orb genes, become round and show a complete loss of cell polarity. A second class of mutants exhibits branched or bent morphologies. These mutants show defects in either selection of the growth site, defining two tea genes, or in the maintenance of growth direction, defining five ban genes. Immunofluorescence analysis of these morphological mutants shows defects in the organization of the microtubule and actin cytoskeleton. These defects include shortened, bundled, and asymmetrically localized microtubules and enlarged and mislocalized actin patches. Analysis of the mutant phenotypes has allowed us to order the genes into four groups according to their function during the cell cycle: genes required for the maintenance of cell polarity throughout the cell cycle; genes necessary only for the reestablishment of cell polarity after mitosis and not for maintaining cell polarity once it is established; genes essential for the transition from monopolar to bipolar growth and genes that severe as ''polarity markers''.     

Cystic fibrosis airway epithelial cell polarity and bacterial flagellin determine host response to Pseudomonas aeruginosa

The role of epithelial polarity and bacterial factors in the control of the innate immune response of airway epithelial cells to Pseudomonas aeruginosa PAK was investigated using a human, nasal cystic fibrosis (DeltaF508/DeltaF508) epithelial cell line CF15 grown as confluent layers on permeable supports. Addition of PAK to the basal surface of CF15 layers caused significant expression changes in 1525 different genes (out of 12 625 examined), including the cytokines IL-6, IL-8, IL-1beta and TNF-alpha, as well as genes associated with leucocyte adhesion, antibacterial factors, and NF-kappaB signalling. Confocal microscopy showed that nuclear migration of NF-kappaB in all CF15 cells was preceded by PAK binding to the basal and lateral surfaces of some cells. Addition of PAK to the apical surface of CF15 monolayers elicited changes in expression of only 602 genes, including 256 not affected during basolateral PAK exposure. Over time, cytokine expression during apical PAK was similar to that exhibited by basal PAK, but the magnitudes during apical treatment were much smaller with little/no nuclear migration of NF-kappaB in CF15 cells. Furthermore, these responses depended on the presence of flagellin, but not pili on the bacteria. Thus, P. aeruginosa triggered a strong innate immune response that depended on the apical versus basolateral polarity of CF15 cells and the presence of flagellin on the bacteria.     

Diffusion of beta-lactam antibiotics through oligomeric or monomeric porin channels of some gram-negative bacteria

The penetration of anionic beta-lactam antibiotics through porins was evaluated as a mechanism of drug resistance. The major proteins with porin activity were purified from the outer membranes of six bacteria. Three of the six porins were oligomeric porins. The molecular weights of their monomers were 37 kDa from Photobacterium damsela, 42 kDa from Serratia liquefaciens, and 36 kDa from E. coli B. The other three porins were heat-modifiable monomeric porins with molecular weights of 43 kDa from Porphyromonas asaccharolytica and Acinetobacter baumannii, and 37 kDa from Escherichia coli K12.Comparison of the six porin proteins revealed that, independent of their aggregation state, their amino acid content is similar but not identical. All have double the amount of negatively charged amino acids compared with positively charged amino acids. They have a similar polarity and polarity index. Two of the six tested bacteria do not produce beta-lactamase. These two bacteria were sensitive to the different beta-lactams tested. The other four bacteria were resistant to all or to several beta-lactams.A modified liposome swelling method was used for determining the rate of penetration of charged beta-lactam antibiotics. Zwitterionic beta-lactams were found to penetrate into liposomes at a rate that more or less fits their molecular weight, whether the porins are monomeric or oligomeric. The penetration rates of negatively charged beta-lactams are different for oligomeric and monomeric porins. Negatively charged beta-lactams penetrate through oligomeric porins better than estimated by their molecular weight, whereas monomeric porins are less penetrable to negatively charged beta-lactams than estimated by their molecular weight. The contribution of all types of porins to the susceptibility of bacteria to beta-lactam antibiotics (zwitterionic or negatively charged) is apparently doubtful. The porins may decrease or increase bacterial penetration rates to beta-lactams, and only the existence of a potential beta-lactamase that can destroy the penetrating drug will cause resistance.     

Growth of Gram-negative bacteria in the presence of organic solvents

The growth behavior of Gram-negative bacteria when exposed to high concentrations (50% v/v) of water-insoluble organic solvents was investigated. The solvents were chosen according to their polarity values as denoted by a logarithmically expressed parameter log P, where P is the partition coefficient of a given solvent in an equimolar mixture of octanol and water. The cell growth was measured by the number of colonies developed on a solid agar medium in direct contact with the solvents. All 31 strains tested showed characteristic growth patterns. The survival and subsequent growth of bacteria increased with the increase in the log P value and was found to be strain specific. For all the strains, 100% cell growth was reached from 0% within 0.1–0.4 log P units. Log P₅₀ values, defined as the log P values at which 50% of the cells form colonies, were determined for each bacterial strain. On the whole, Pseudomonas strains were found to be more resistant to apolar solvents than all other bacteria tested. This resistance was dependent not only on the polarities but also on the toxic nature of different organic solvents, the cell membrane components, and to a limited extent, the growth medium. A tenfold increase in the Mg²⁺ concentration in the growth medium enhanced the solvent resistance of E. coli but had no such effect on Pseudomonads. In general, different growth temperatures had no impact on the solvent resistance of the Gram-negative bacteria tested.     

Multiple Wnts redundantly control polarity orientation in Caenorhabditis elegans epithelial stem cells

During development, cell polarization is often coordinated to harmonize tissue patterning and morphogenesis. However, how extrinsic signals synchronize cell polarization is not understood. In Caenorhabditis elegans, most mitotic cells are polarized along the anterior-posterior axis and divide asymmetrically. Although this process is regulated by a Wnt-signaling pathway, Wnts functioning in cell polarity have been demonstrated in only a few cells. We analyzed how Wnts control cell polarity, using compound Wnt mutants, including animals with mutations in all five Wnt genes. We found that somatic gonadal precursor cells (SGPs) are properly polarized and oriented in quintuple Wnt mutants, suggesting Wnts are dispensable for the SGPs' polarity, which instead requires signals from the germ cells. Thus, signals from the germ cells organize the C. elegans somatic gonad. In contrast, in compound but not single Wnt mutants, most of the six seam cells, V1-V6 (which are epithelial stem cells), retain their polarization, but their polar orientation becomes random, indicating that it is redundantly regulated by multiple Wnt genes. In contrast, in animals in which the functions of three Wnt receptors (LIN-17, MOM-5, and CAM-1) are disrupted--the stem cells are not polarized and divide symmetrically--suggesting that the Wnt receptors are essential for generating polarity and that they function even in the absence of Wnts. All the seam cells except V5 were polarized properly by a single Wnt gene expressed at the cell's anterior or posterior. The ectopic expression of posteriorly expressed Wnts in an anterior region and vice versa rescued polarity defects in compound Wnt mutants, raising two possibilities: one, Wnts permissively control the orientation of polarity; or two, Wnt functions are instructive, but which orientation they specify is determined by the cells that express them. Our results provide a paradigm for understanding how cell polarity is coordinated by extrinsic signals.     

Epithelial cell polarity: what flies can teach us about cancer

Epithelial cells are polarized along their apical-basal axis. Much of the cellular machinery that goes into establishing and maintaining epithelial cell polarity is evolutionarily conserved. Model organisms, including the fruit fly, Drosophila melanogaster, are thus particularly useful for the study of cell polarity. Work in Drosophila has identified several important components of the polarity machinery and has also established the surprising existence of a secondary cell polarity pathway required only under conditions of energetic stress. This work has important implications for the understanding of human cancer. Most cancers are epithelial in origin, and the loss of cell polarity is a critical step towards malignancy. Thus a better understanding of how polarity is established and maintained in epithelial cells will help us to understand the process of malignant transformation and may lead to improved therapies. In the present chapter we discuss the current understanding of how epithelial cell polarity is regulated and the known associations between polarity factors and cancer.     

Mediterranean views on epithelial polarity

Developmental and cell biologists viewed polarity through each other's eyes at the EMBO workshop on Epithelial Polarity in Development and Disease, March 27-31 2004, in Carry-le-Rouet, France, a small village west of Marseille on the rocky Mediterranean coast. The presentations highlighted our growing understanding, not only of the molecular mechanisms underlying polarity and the conservation of polarity complexes from worms to mammals, but also the diverse roles that epithelial polarity has during development.     

Correlation of axenic linkage groups with the position of the microtubule-organizing center in aggregating Dictyostelium.

Positioning of the microtubule-organizing center (MTOC) in Dictyostelium discoideum was found to be genetically regulated. We examined the wild-type strain NC-4 cells independently maintained in different laboratories, freshly recovered cells from spores stocked for over 20 years, the temperature-sensitive growth mutant HU49 isolated from NC-4, as well as strain V-12 which is the opposite mating-type to NC-4. During aggregation on nonnutrient agar plates, all these strains showed similar cell polarity, as defined by the alignment of the nucleus ahead of the MTOC. By contrast, in Ax2 and Ax3, axenic strains carrying axenic mutations on linkage groups II and III, the MTOC was usually positioned ahead of the nucleus. Cells containing axenic linkage group II but not III positioned the MTOC ahead of the nucleus. Conversely cell polarity of strains including axenic linkage group III but not II was similar to that of wild-type cells. Thus axenic linkage group II, probably axeC or other linked gene(s) not yet identified, is responsible for the location of the MTOC anterior to the nucleus during aggregation. The anterior positioning of the MTOCs was prevented by growth on bacteria in cells carrying both axenic linkage groups, but not in those carrying only axenic linkage group II.     

Planar cell polarity and a potential role for a Wnt morphogen gradient in stereociliary bundle orientation in the mammalian inner ear

The planar cell polarity (PCP) pathway, a noncanonical Wnt signaling pathway, is crucial for embryonic development in all animals as it is responsible for the regulation of coordinated orientation of structures within the plane of the various epithelia. In the mammalian cochlea, one of the best examples of planar polarity in vertebrates, stereociliary bundles located on mechanosensory hair cells within the sensory epithelium are all uniformly polarized. Generation of this polarity is important for hair cell mechanotransduction and auditory perception as stereociliary bundles are only sensitive to vibrations in their single plane of polarization. We describe the two step developmental process that results in the generation of planar polarity in the mammalian inner ear. Furthermore, we review evidence for the role of Wnt signaling, and the possible generation of a Wnt gradient, in planar polarity.