Two localization motifs mediate polar residence of FrzS during cell movement and reversals of Myxococcus xanthus

Myxococcus xanthus utilizes two motility systems for surface locomotion: A-motility and S-motility. S-motility is mediated by extension and retraction of type IV pili. Cells exhibiting S-motility periodically reverse by switching the assembly of type IV pili from the old leading pole to the new leading pole. These cellular reversals involve regulated pole-to-pole oscillations of the FrzS protein. We constructed and characterized in-frame deletion mutations in several FrzS domains to determine their roles in protein localization. We found that FrzS has distinct domains required for residence at the leading cell pole, pole-to-pole transport and lagging cell pole. Our results are consistent with a model whereby S-motility reversals are mediated by a protein translocation system that delivers motility proteins such as FrzS from the leading cell pole to the lagging cell pole.     

A myxobacterial S-motility protein dances with poles

Coordinated movement of packs of S-motile Myxococcus xanthus cells relies on extrusion and retraction of pili that are located at one cell pole. At regular intervals the pili switch their polar location and cells reverse direction. Recently, the FrzS S-motility protein was observed to localize predominantly to the piliated pole. In time, FrzS was redeployed to the opposite pole and its sequestration at the new site coincided with cell reversal. The C-terminal region of FrzS, a response regulator homolog, is rich in coiled-coil motifs and is required for dynamic localization and proper motility. These results raise the possibility that proper spatial control of FrzS has an important role in the regulation of cell reversal and S-motility.     

FrzS regulates social motility in Myxococcus xanthus by controlling exopolysaccharide production

Myxococcus xanthus Social (S) motility occurs at high cell densities and is powered by the extension and retraction of Type IV pili which bind ligands normally found in matrix exopolysaccharides (EPS). Previous studies showed that FrzS, a protein required for S-motility, is organized in polar clusters that show pole-to-pole translocation as cells reverse their direction of movement. Since the leading cell pole is the site of both the major FrzS cluster and type IV pilus extension/retraction, it was suggested that FrzS might regulate S-motility by activating pili at the leading cell pole. Here, we show that FrzS regulates EPS production, rather than type IV pilus function. We found that the frzS phenotype is distinct from that of Type IV pilus mutants such as pilA and pilT, but indistinguishable from EPS mutants, such as epsZ. Indeed, frzS mutants can be rescued by the addition of purified EPS, 1% methylcellulose, or co-culturing with wildtype cells. Our data also indicate that the cell density requirement in S-motility is likely a function of the ability of cells to construct functional multicellular clusters surrounding an EPS core.     

Coupling of protein localization and cell movements by a dynamically localized response regulator in Myxococcus xanthus

Myxococcus xanthus cells harbor two motility machineries, type IV pili (Tfp) and the A-engine. During reversals, the two machineries switch polarity synchronously. We present a mechanism that synchronizes this polarity switching. We identify the required for motility response regulator (RomR) as essential for A-motility. RomR localizes in a bipolar, asymmetric pattern with a large cluster at the lagging cell pole. The large RomR cluster relocates to the new lagging pole in parallel with cell reversals. Dynamic RomR localization is essential for cell reversals, suggesting that RomR relocalization induces the polarity switching of the A-engine. The analysis of RomR mutants shows that the output domain targets RomR to the poles and the receiver domain is essential for dynamic localization. The small GTPase MglA establishes correct RomR polarity, and the Frz two-component system regulates dynamic RomR localization. FrzS localizes with Tfp at the leading pole and relocates in an Frz-dependent manner to the opposite pole during reversals; FrzS and RomR localize and oscillate independently. The Frz system synchronizes these oscillations and thus the synchronous polarity switching of the motility machineries.     

Cell orientation of swimming bacteria: From theoretical simulation to experimental evaluation

The motion of small bacteria consists of two phases: relatively long runs alternate with intermittent stops, back-ups, or tumbles, depending on the species. In polar monotrichous bacteria, the flagellum is anchored at the cell pole inherited from the parent generation (old pole) and is surrounded by a chemoreceptor cluster. During forward swimming, the leading pole is always the pole recently formed in cell division (new pole). The flagella of the peritrichous bacterium Escherichia coli often form a bundle behind the old pole. Its cell orientation and receptor positioning during runs generally mimic that of monotrichous bacteria. When encountering a solid surface, peritrichous bacteria exhibit a circular motion with the leading pole dipping downward. Some polar monotrichous bacteria also perform circular motion near solid boundaries, but during back-ups. In this case, the leading pole points upward. Very little is known about behavior near milieu-air interfaces. Biophysical simulations have revealed some of the mechanisms underlying these phenomena, but leave many questions unanswered. Combining biophysics with molecular techniques will certainly advance our understanding of bacterial locomotion.     

Bacterial motility complexes require the actin‐like protein,MreB and the Ras homologue,MglA

Gliding motility in the bacterium Myxococcus xanthus uses two motility engines: S‐motility powered by type‐IV pili and A‐motility powered by uncharacterized motor proteins and focal adhesion complexes. In this paper, we identified MreB, an actin‐like protein, and MglA, a small GTPase of the Ras superfamily, as essential for both motility systems. A22, an inhibitor of MreB cytoskeleton assembly, reversibly inhibited S‐ and A‐motility, causing rapid dispersal of S‐ and A‐motility protein clusters, FrzS and AglZ. This suggests that the MreB cytoskeleton is involved in directing the positioning of these proteins. We also found that a ΔmglA motility mutant showed defective localization of AglZ and FrzS clusters. Interestingly, MglA–YFP localization mimicked both FrzS and AglZ patterns and was perturbed by A22 treatment, consistent with results indicating that both MglA and MreB bind to motility complexes. We propose that MglA and the MreB cytoskeleton act together in a pathway to localize motility proteins such as AglZ and FrzS to assemble the A‐motility machineries. Interestingly, M. xanthus motility systems, like eukaryotic systems, use an actin‐like protein and a small GTPase spatial regulator.     

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.     

A Dynamic Response Regulator Protein Modulates G-Protein-Dependent Polarity in the Bacterium Myxococcus xanthus

Migrating cells employ sophisticated signal transduction systems to respond to their environment and polarize towards attractant sources. Bacterial cells also regulate their polarity dynamically to reverse their direction of movement. In Myxococcus xanthus, a GTP-bound Ras-like G-protein, MglA, activates the motility machineries at the leading cell pole. Reversals are provoked by pole-to-pole switching of MglA, which is under the control of a chemosensory-like signal transduction cascade (Frz). It was previously known that the asymmetric localization of MglA at one cell pole is regulated by MglB, a GTPase Activating Protein (GAP). In this process, MglB specifically localizes at the opposite lagging cell pole and blocks MglA localization at that pole. However, how MglA is targeted to the leading pole and how Frz activity switches the localizations of MglA and MglB synchronously remained unknown. Here, we show that MglA requires RomR, a previously known response regulator protein, to localize to the leading cell pole efficiently. Specifically, RomR-MglA and RomR-MglB complexes are formed and act complementarily to establish the polarity axis, segregating MglA and MglB to opposite cell poles. Finally, we present evidence that Frz signaling may regulate MglA localization through RomR, suggesting that RomR constitutes a link between the Frz-signaling and MglAB polarity modules. Thus, in Myxococcus xanthus, a response regulator protein governs the localization of a small G-protein, adding further insight to the polarization mechanism and suggesting that motility regulation evolved by recruiting and combining existing signaling modules of diverse origins.     

Maternal Messenger RNA as a Determinant of Pole Cell Formation in Drosophila Embryos

Some polar plasm components are UV-sensitive. Messenger RNA extracted from oocytes or cleavage embryos can to induce pole cells in embryos that have been deprived of ability to form pole cells by UV-irradiation. This article reviews studies on the role of this mRNA in the developmental pathway leading to germ cell formation.     

Overexpression of oskar directs ectopic activation of nanos and presumptive pole cell formation in Drosophila embryos.

In Drosophila, a small group of maternal effect genes, including oskar, defines a shared pathway leading to the provision of two determinants at the posterior pole of the embryo. One determinant is the posterior body patterning morphogen nanos, and the other directs germ cell formation. Overexpression of oskar causes the shared pathway to be hyperactivated, with excess nanos activity present throughout the embryo and a superabundance of posterior pole cells. In addition, presumptive pole cells appear at a novel anterior position. Strikingly, formation of these ectopic pole cells is enhanced in nanos mutants. This observation may reflect competition between nanos and the germ cell determinant for a shared and limiting precursor.     

The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions

In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate–bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA–MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein–cytoskeleton interactions are a universally conserved feature.     

Phosphorylation‐dependent localization of the response regulator FrzZ signals cell reversals in Myxococcus xanthus

The life cycle of Myxococcus xanthus includes co‐ordinated group movement and fruiting body formation, and requires directed motility and controlled cell reversals. Reversals are achieved by inverting cell polarity and re‐organizing many motility proteins. The Frz chemosensory pathway regulates the frequency of cell reversals. While it has been established that phosphotransfer from the kinase FrzE to the response regulator FrzZ is required, it is unknown how phosphorylated FrzZ, the putative output of the pathway, targets the cell polarity axis. In this study, we used Phos‐tag SDS‐PAGE to determine the cellular level of phospho‐FrzZ under different growth conditions and in Frz signalling mutants. We detected consistent FrzZ phosphorylation, albeit with a short half‐life, in cells grown on plates, but not from liquid culture. The available pool of phospho‐FrzZ correlated with reversal frequencies, with higher levels found in hyper‐reversing mutants. Phosphorylation was not detected in hypo‐reversing mutants. Fluorescence microscopy revealed that FrzZ is recruited to the leading cell pole upon phosphorylation and switches to the opposite pole during reversals. These results are consistent with the hypothesis that the Frz pathway modulates reversal frequency through a localized response regulator that targets cell polarity regulators at the leading cell pole.     

A Response Regulator Interfaces between the Frz Chemosensory System and the MglA/MglB GTPase/GAP Module to Regulate Polarity in Myxococcus xanthus

How cells establish and dynamically change polarity are general questions in cell biology. Cells of the rod-shaped bacterium Myxococcus xanthus move on surfaces with defined leading and lagging cell poles. Occasionally, cells undergo reversals, which correspond to an inversion of the leading-lagging pole polarity axis. Reversals are induced by the Frz chemosensory system and depend on relocalization of motility proteins between the poles. The Ras-like GTPase MglA localizes to and defines the leading cell pole in the GTP-bound form. MglB, the cognate MglA GTPase activating protein, localizes to and defines the lagging pole. During reversals, MglA-GTP and MglB switch poles and, therefore, dynamically localized motility proteins switch poles. We identified the RomR response regulator, which localizes in a bipolar asymmetric pattern with a large cluster at the lagging pole, as important for motility and reversals. We show that RomR interacts directly with MglA and MglB in vitro. Furthermore, RomR, MglA, and MglB affect the localization of each other in all pair-wise directions, suggesting that RomR stimulates motility by promoting correct localization of MglA and MglB in MglA/RomR and MglB/RomR complexes at opposite poles. Moreover, localization analyses suggest that the two RomR complexes mutually exclude each other from their respective poles. We further show that RomR interfaces with FrzZ, the output response regulator of the Frz chemosensory system, to regulate reversals. Thus, RomR serves at the functional interface to connect a classic bacterial signalling module (Frz) to a classic eukaryotic polarity module (MglA/MglB). This modular design is paralleled by the phylogenetic distribution of the proteins, suggesting an evolutionary scheme in which RomR was incorporated into the MglA/MglB module to regulate cell polarity followed by the addition of the Frz system to dynamically regulate cell polarity.     

Synthesis of peptidoglycan and membrane during the division cycle of rod-shaped, gram-negative bacteria.

A modified procedure for determining the pattern of peptidoglycan synthesis during the division cycle has allowed the measurement of the rate of side wall synthesis during the division cycle without the contribution due to pole formation. As predicted by a model proposing that the surface growth of the cell is regulated by mass increase, we find a decrease in side wall synthesis in the latter half of the division cycle. This supports the proposal that, upon invagination, pole growth accommodates a significant proportion of the increasing cell mass and that residual side wall growth occurs in response to the residual mass increase not accommodated by pole volume. The observed side wall synthesis patterns support the proposal that mass increase is a major, and possibly sole, regulator of bacterial surface increase. Membrane synthesis during the division cycle of the gram-negative, rod-shaped bacteria Escherichia coli and Salmonella typhimurium has also been measured with similar methods. The rate of membrane synthesis--measured by incorporation of radioactive glycerol or palmitate relative to simultaneous labeling with radioactive leucine--exhibits the same pattern as peptidoglycan synthesis. The results are compatible with a model of cell surface growth containing the following elements. (i) During the period of the division cycle prior to invagination, growth of the cell occurs predominantly in the side wall and the cell grows only in length. (ii) When invagination begins, pole growth accommodates some cytoplasmic increase, leading to a concomitant decrease in side wall synthesis. (iii) Surface synthesis increases relative to mass synthesis during the last part of the division cycle because of pole formation. It is proposed here that membrane synthesis passively follows the pattern of peptidoglycan synthesis during the division cycle.     

Membrane-murein attachment at the leading edge of the division septum: a second membrane-murein structure associated with morphogenesis of the gram-negative bacterial division septum.

Electron microscopy of plasmolyzed cells of Salmonella typhimurium revealed a continuous zone of membrane-murein attachment at the leading edge of the division septum at all stages of septal invagination. The membrane-murein attachment site had a characteristic ultrastructural appearance and remained as a bacterial birth scar at the new pole of each of the two daughter cells after cell separation. The continuous zone of membrane-murein attachment at the leading septal edge represents the second organelle based on a topologically ordered domain of membrane-murein adhesion to be described at the site of cell division.     

Cofilin determines the migration behavior and turning frequency of metastatic cancer cells

We have investigated the effects of inhibiting the expression of cofilin to understand its role in protrusion dynamics in metastatic tumor cells, in particular. We show that the suppression of cofilin expression in MTLn3 cells (an apolar randomly moving amoeboid metastatic tumor cell) caused them to extend protrusions from only one pole, elongate, and move rectilinearly. This remarkable transformation was correlated with slower extension of fewer, more stable lamellipodia leading to a reduced turning frequency. Hence, the loss of cofilin caused an amoeboid tumor cell to assume a mesenchymal-type mode of movement. These phenotypes were correlated with the loss of uniform chemotactic sensitivity of the cell surface to EGF stimulation, demonstrating that to chemotax efficiently, a cell must be able to respond to chemotactic stimulation at any region on its surface. The changes in cell shape, directional migration, and turning frequency were related to the re-localization of Arp2/3 complex to one pole of the cell upon suppression of cofilin expression.     

The Two Poles of the Lymphocyte: Specialized Cell Compartments for Migration and Recruitment

Chemotaxis, the directed migration of leukocytes towards a chemoattractant gradient, is a key phenomenon in the immune response. During lymphocyte-endothelial and – extracellular matrix interactions, chemokines induce the polarization of T lymphocytes. with generation of specialized cell compartments. The chemokine receptors involved in detection of the chemoattractant gradients concentrate at the leading edge (advancing front or anterior pole) of the cell. The adhesion molecules ICAM- 1, -3, CD44 and CD43 redistribute to the uropod, an appendage at the posterior pole of migrating T lymphocyte that protrudes from the contact area with endothelial or extracellular matrix substrates. Whereas chemokine receptors sense the direction of migration, the uropod is involved in the recruitment of bystander leukocytes through LFA-1/ICAM-dependent cell cell interactions. While β-actin concentrates preferentially at the cell's leading edge, the motor protein myosin II and a microtubule organizing center (MTOC) are packed in the uropod. The actin-binding protein moesin, which belongs to the ERM family of ezrin, radixin and moesin, redistributes to the distal portion of uropods and physically interacts with ICAM-3, CD44 and CD43, thus acting as a physical link between the membrane molecules and the actin cytoskeleton. Moreover, the moesin-ICAM-3 association correlates with the degree of cell polarity. The redistribution of the chemokine receptors and adhesion molecules to opposite poles of the cell in response to a chemoattractant gradient may guide cell migration and cell-cell interactions during lymphoid cell trafficking in immune and inflammatory responses.     

Self‐organized partitioning of dynamically localized proteins in bacterial cell division

How cells manage to get equal distribution of their structures and molecules at cell division is a crucial issue in biology. In principle, a feedback mechanism could always ensure equality by measuring and correcting the distribution in the progeny. However, an elegant alternative could be a mechanism relying on self‐organization, with the interplay between system properties and cell geometry leading to the emergence of equal partitioning. The problem is exemplified by the bacterial Min system that defines the division site by oscillating from pole to pole. Unequal partitioning of Min proteins at division could negatively impact system performance and cell growth because of loss of Min oscillations and imprecise mid‐cell determination. In this study, we combine live cell and computational analyses to show that known properties of the Min system together with the gradual reduction of protein exchange through the constricting septum are sufficient to explain the observed highly precise spontaneous protein partitioning. Our findings reveal a novel and effective mechanism of protein partitioning in dividing cells and emphasize the importance of self‐organization in basic cellular processes.     

Neuronal (bi)polarity as a self-organized process enhanced by growing membrane

Early in vitro and recent in vivo studies demonstrated that neuronal polarization occurs by the sequential formation of two oppositely located neurites. This early bipolar phenotype is of crucial relevance in brain organization, determining neuronal migration and brain layering. It is currently considered that the place of formation of the first neurite is dictated by extrinsic cues, through the induction of localized changes in membrane and cytoskeleton dynamics leading to deformation of the cells' curvature followed by the growth of a cylindrical extension (neurite). It is unknown if the appearance of the second neurite at the opposite pole, thus the formation of a bipolar cell axis and capacity to undergo migration, is defined by the growth at the first place, therefore intrinsic, or requires external determinants. We addressed this question by using a mathematical model based on the induction of dynamic changes in one pole of a round cell. The model anticipates that a second area of growth can spontaneously form at the opposite pole. Hence, through mathematical modeling we prove that neuronal bipolar axis of growth can be due to an intrinsic mechanism.     

Structure of the pilus assembly protein TadZ from Eubacterium rectale: implications for polar localization

The tad (tight adherence) locus encodes a protein translocation system that produces a novel variant of type IV pili. The pilus assembly protein TadZ (called CpaE in Caulobacter crescentus) is ubiquitous in tad loci, but is absent in other type IV pilus biogenesis systems. The crystal structure of TadZ from Eubacterium rectale (ErTadZ), in complex with ATP and Mg²⁺, was determined to 2.1 Å resolution. ErTadZ contains an atypical ATPase domain with a variant of a deviant Walker‐A motif that retains ATP binding capacity while displaying only low intrinsic ATPase activity. The bound ATP plays an important role in dimerization of ErTadZ. The N‐terminal atypical receiver domain resembles the canonical receiver domain of response regulators, but has a degenerate, stripped‐down ‘active site’. Homology modelling of the N‐terminal atypical receiver domain of CpaE indicates that it has a conserved protein–protein binding surface similar to that of the polar localization module of the social mobility protein FrzS, suggesting a similar function. Our structural results also suggest that TadZ localizes to the pole through the atypical receiver domain during an early stage of pili biogenesis, and functions as a hub for recruiting other pili components, thus providing insights into the Tad pilus assembly process.