A Mechanism for the Polarity Formation of Chemoreceptors at the Growth Cone Membrane for Gradient Amplification during Directional Sensing

Accurate response to external directional signals is essential for many physiological functions such as chemotaxis or axonal guidance. It relies on the detection and amplification of gradients of chemical cues, which, in eukaryotic cells, involves the asymmetric relocalization of signaling molecules. How molecular events coordinate to induce a polarity at the cell level remains however poorly understood, particularly for nerve chemotaxis. Here, we propose a model, inspired by single-molecule experiments, for the membrane dynamics of GABA chemoreceptors in nerve growth cones (GCs) during directional sensing. In our model, transient interactions between the receptors and the microtubules, coupled to GABA-induced signaling, provide a positive-feedback loop that leads to redistribution of the receptors towards the gradient source. Using numerical simulations with parameters derived from experiments, we find that the kinetics of polarization and the steady-state polarized distribution of GABA receptors are in remarkable agreement with experimental observations. Furthermore, we make predictions on the properties of the GC seen as a sensing, amplification and filtering module. In particular, the growth cone acts as a low-pass filter with a time constant ∼10 minutes determined by the Brownian diffusion of chemoreceptors in the membrane. This filtering makes the gradient amplification resistent to rapid fluctuations of the external signals, a beneficial feature to enhance the accuracy of neuronal wiring. Since the model is based on minimal assumptions on the receptor/cytoskeleton interactions, its validity extends to polarity formation beyond the case of GABA gradient sensing. Altogether, it constitutes an original positive-feedback mechanism by which cells can dynamically adapt their internal organization to external signals.     

Cell Division Resets Polarity and Motility for the Bacterium Myxococcus xanthus

Links between cell division and other cellular processes are poorly understood. It is difficult to simultaneously examine division and function in most cell types. Most of the research probing aspects of cell division has experimented with stationary or immobilized cells or distinctly asymmetrical cells. Here we took an alternative approach by examining cell division events within motile groups of cells growing on solid medium by time-lapse microscopy. A total of 558 cell divisions were identified among approximately 12,000 cells. We found an interconnection of division, motility, and polarity in the bacterium Myxococcus xanthus. For every division event, motile cells stop moving to divide. Progeny cells of binary fission subsequently move in opposing directions. This behavior involves M. xanthus Frz proteins that regulate M. xanthus motility reversals but is independent of type IV pilus “S motility.” The inheritance of opposing polarity is correlated with the distribution of the G protein RomR within these dividing cells. The constriction at the point of division limits the intracellular distribution of RomR. Thus, the asymmetric distribution of RomR at the parent cell poles becomes mirrored at new poles initiated at the site of division.     

Modelling Directional Guidance and Motility Regulation in Cell Migration

Although cell migration is an essential process in development, how cells reach their final destination is not well understood. Secreted molecules are known to have a migratory effect, but it remains unclear whether such molecules act as directional guidance cues or as motility regulators. There is potential to use signalling molecules in new medical therapies, so it is important to identify the exact role these molecules play. This paper focuses on distinguishing between inhibitory and repulsive effects produced by signalling molecules, based on recent experiments examining the effect of Slit, a secreted protein, on the migration of neurons from the brain. The primary role of Slit, whether it is an inhibitor or repellent of neurons, is in dispute. We present population-level continuum models and recast these in terms of transition probabilities governing individual cells. Various cell-sensing strategies are considered within this framework. The models are applied to the neuronal migration experiments. To resolve the particular role of Slit, simulations of the models characterising different cell-sensing strategies are compared at the population and individual cell level, providing two complementary perspectives on the system. Difficulties and limitations in deducing cell migration rules from time-lapse imaging are discussed.     

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation,Barrier Function,and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.     

Myosin-V Opposes Microtubule-Based Cargo Transport and Drives Directional Motility on Cortical Actin

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Highlights? Myosin-V alternates between active and passive actin-binding modes ? Myosin-V opposes kinesin-propelled cargo redistribution ? Myosin-V anchors kinesin-propelled cargo in actin-rich areas ? Myosin-V can drive medium-range directional transport at the cell periphery     

Bacterial Quorum Sensing Molecule N-3-Oxo-Dodecanoyl-L-Homoserine Lactone Causes Direct Cytotoxicity and Reduced Cell Motility in Human Pancreatic Carcinoma Cells

In spite of chemotherapeutic and surgical advances, pancreatic cancer continues to have a dismal prognosis. Metastasis due to tumor cell migration remains the most critical challenge in treating pancreatic cancer, and conventional chemotherapy is rarely curative. In the quest for more novel molecules to fight this disease, we tested the hypothesis that the Pseudomonas aeruginosa quorum sensing signal molecule N-3-oxo-dodecanoyl-L-homoserine lactone (O-DDHSL) would be cytotoxic to and reduce mobility of pancreatic carcinoma cells (Panc-1 and Aspc-1). Results showed a decrease in cell viability from apoptosis, diminished colony formation, and inhibition of migration of the evaluated pancreatic carcinoma cell lines. Also, cell viability decreased in the presence of O-DDHSL when cells were grown in matrigel basement membrane matrix. While messenger RNA for IQGAP-1 decreased in Panc-1 and HPDE cells upon exposure to O-DDHSL, no change was observed in Aspc-1 cells. Cofilin mRNA expression was found to be increased in both HPDE and Panc-1 cells with marginal decrease in Aspc-1 cells. RhoC, a Rho-family GTPase involved in cell motility, increased in the presence of O-DDHSL, suggesting a possible compensatory response to alteration in other migration associated genes. Our results indicate that O-DDHSL could be an effective biomolecule in eukaryotic systems with multimodal function for essential molecular targeting in pancreatic cancer.     

An Experimental and Computational Study of the Effect of ActA Polarity on the Speed of Listeria monocytogenes Actin-based Motility

Listeria monocytogenes is a pathogenic bacterium that moves within infected cells and spreads directly between cells by harnessing the cell's dendritic actin machinery. This motility is dependent on expression of a single bacterial surface protein, ActA, a constitutively active Arp2,3 activator, and has been widely studied as a biochemical and biophysical model system for actin-based motility. Dendritic actin network dynamics are important for cell processes including eukaryotic cell motility, cytokinesis, and endocytosis. Here we experimentally altered the degree of ActA polarity on a population of bacteria and made use of an ActA-RFP fusion to determine the relationship between ActA distribution and speed of bacterial motion. We found a positive linear relationship for both ActA intensity and polarity with speed. We explored the underlying mechanisms of this dependence with two distinctly different quantitative models: a detailed agent-based model in which each actin filament and branched network is explicitly simulated, and a three-state continuum model that describes a simplified relationship between bacterial speed and barbed-end actin populations. In silico bacterial motility required a cooperative restraining mechanism to reconstitute our observed speed-polarity relationship, suggesting that kinetic friction between actin filaments and the bacterial surface, a restraining force previously neglected in motility models, is important in determining the effect of ActA polarity on bacterial motility. The continuum model was less restrictive, requiring only a filament number-dependent restraining mechanism to reproduce our experimental observations. However, seemingly rational assumptions in the continuum model, e.g. an average propulsive force per filament, were invalidated by further analysis with the agent-based model. We found that the average contribution to motility from side-interacting filaments was actually a function of the ActA distribution. This ActA-dependence would be difficult to intuit but emerges naturally from the nanoscale interactions in the agent-based representation.     

Hugl1 and Hugl2 in Mammary Epithelial Cells: Polarity,Proliferation, and Differentiation

Loss of epithelial polarity is described as a hallmark of epithelial cancer. To determine the role of Hugl1 and Hugl2 expression in the breast, we investigated their localization in human mammary duct tissue and the effects of expression modulation in normal and cancer cell lines on polarity, proliferation and differentiation. Expression of Hugl1 and Hugl2 was silenced in both MCF10A cells and Human Mammary Epithelial Cells and cell lines were grown in 2-D on plastic and in 3-D in Matrigel to form acini. Cells in monolayer were compared for proliferative and phenotypic changes while acini were examined for differences in size, ability to form a hollow lumen, nuclear size and shape, and localization of key domain-specific proteins as a measure of polarity. We detected overlapping but distinct localization of Hugl1 and Hugl2 in the human mammary gland, with Hugl1 expressed in both luminal and myoepithelium and Hugl2 largely restricted to myoepithelium. On a plastic surface, loss of Hugl1 or Hugl2 in normal epithelium induced a mesenchymal phenotype, and these cells formed large cellular masses when grown in Matrigel. In addition, loss of Hugl1 or Hugl2 expression in MCF10A cells resulted in increased proliferation on Matrigel, while gain of Hugl1 expression in tumor cells suppressed proliferation. Loss of polarity was also observed with knockdown of either Hugl1 or Hugl2, with cells growing in Matrigel appearing as a multilayered epithelium, with randomly oriented Golgi and multiple enlarged nuclei. Furthermore, Hugl1 knock down resulted in a loss of membrane identity and the development of cellular asymmetries in Human Mammary Epithelial Cells. Overall, these data demonstrate an essential role for both Hugl1 and Hugl2 in the maintenance of breast epithelial polarity and differentiated cell morphology, as well as growth control.     

Wdpcp,a PCP Protein Required for Ciliogenesis,Regulates Directional Cell Migration and Cell Polarity by Direct Modulation of the Actin Cytoskeleton

Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet–Biedl/Meckel–Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration.     

Effects of n-Alkylamines on the Motility and Viability of Heterosigma akashiwo Cells

The cytotoxic effects of positively charged liposomes and theircomponents on Heterosigma akashiwo cells were investigated.Positively charged liposomes reduced cell motility and eventuallycaused cytolysis. Negatively charged and non-charged liposomeshad little effect on cell motility and/or cell viability. Damagewas also induced by a single application of some n-alkyl-amines.Among n-alkylamines tested, laurylamine (C₁₂) was most effectivein reducing motility and causing cytolysis of cells. The extentof the deleterious effects increased with increasing concentrationsof laurylamine and with the duration of treatment. The extentof damage to cells by laurylamine changed periodically duringthe cell cycle. The effects of laurylamine began to increaseat the forth hour of the light period and began to decreaseat the first hour of the dark period, under condition of 12hours of light and 12 hours of darkness. Laurie and myristicacids, which each have a carboxyl group in place of the aminogroup of the corresponding alkylamines, laurylamine and myristylamine,had little effect on the cells. (Received December 21, 1988; Accepted March 29, 1989)     

Quantification of Cell Edge Velocities and Traction Forces Reveals Distinct Motility Modules during Cell Spreading

Actin-based cell motility and force generation are central to immune response, tissue development, and cancer metastasis, and understanding actin cytoskeleton regulation is a major goal of cell biologists. Cell spreading is a commonly used model system for motility experiments – spreading fibroblasts exhibit stereotypic, spatially-isotropic edge dynamics during a reproducible sequence of functional phases: 1) During early spreading, cells form initial contacts with the surface. 2) The middle spreading phase exhibits rapidly increasing attachment area. 3) Late spreading is characterized by periodic contractions and stable adhesions formation. While differences in cytoskeletal regulation between phases are known, a global analysis of the spatial and temporal coordination of motility and force generation is missing. Implementing improved algorithms for analyzing edge dynamics over the entire cell periphery, we observed that a single domain of homogeneous cytoskeletal dynamics dominated each of the three phases of spreading. These domains exhibited a unique combination of biophysical and biochemical parameters – a motility module. Biophysical characterization of the motility modules revealed that the early phase was dominated by periodic, rapid membrane blebbing; the middle phase exhibited continuous protrusion with very low traction force generation; and the late phase was characterized by global periodic contractions and high force generation. Biochemically, each motility module exhibited a different distribution of the actin-related protein VASP, while inhibition of actin polymerization revealed different dependencies on barbed-end polymerization. In addition, our whole-cell analysis revealed that many cells exhibited heterogeneous combinations of motility modules in neighboring regions of the cell edge. Together, these observations support a model of motility in which regions of the cell edge exhibit one of a limited number of motility modules that, together, determine the overall motility function. Our data and algorithms are publicly available to encourage further exploration.     

A Positive Feedback between Growth and Polarity Provides Directional Persistency and Flexibility to the Process of Tip Growth

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Fascin, an Actin-bundling Protein, Induces Membrane Protrusions and Increases Cell Motility of Epithelial Cells

Fascin is an actin-bundling protein that is found in membrane ruffles, microspikes, and stress fibers. The expression of fascin is greatly increased in many transformed cells, as well as in specialized normal cells including neuronal cells and antigen-presenting dendritic cells. A morphological characteristic common to these cells expressing high levels of fascin is the development of many membrane protrusions in which fascin is predominantly present. To examine whether fascin contributes to the alterations in microfilament organization at the cell periphery, we have expressed fascin in LLC-PK1 epithelial cells to levels as high as those found in transformed cells and in specialized normal cells. Expression of fascin results in large changes in morphology, the actin cytoskeleton, and cell motility: fascin-transfected cells form an increased number of longer and thicker microvilli on apical surfaces, extend lamellipodia-like structures at basolateral surfaces, and show disorganization of cell–cell contacts. Cell migration activity is increased by 8–17 times when assayed by modified Boyden chamber. Microinjection of a fascin protein into LLC-PK1 cells causes similar morphological alterations including the induction of lamellipodia at basolateral surfaces and formation of an increased number of microvilli on apical surfaces. Furthermore, microinjection of fascin into REF-52 cells, normal fibroblasts, induces the formation of many lamellipodia at all regions of cell periphery. These results together suggest that fascin is directly responsible for membrane protrusions through reorganization of the microfilament cytoskeleton at the cell periphery.     

Interaction between Natural Selection for Heterozygotes and Directional Selection

Significant correlations between allelic frequencies and environmental variables in a number of insect species have been demonstrated by multivariate techniques. Since many environmental variables show a strong relationship to geographic location and since gene flow between populations can also produce patterns of gene frequencies which are related to the geographic location, both selection and gene-flow hypotheses are consistent with the observed correlations. The genetic variables can be corrected for geographic location and so for linear gene-flow patterns. If, after correction, the genetic variables still show significant correlations with similarly corrected environmental variables, then these correlations are consistent with hypotheses of selection but not of gene flow. The data of Johnson and Schaffer (1973) have been reanalyzed using the method of canonical correlation after correction for geographical location by means of multiple regression. Five of the nine loci studied exhibit significant canonical correlations. These results, under the assumption of linear gene flow, support hypotheses of selective action of environmental variables in the genotype-environment relationships observed.     

Interaction Between Neutrophils and 4-Hydroxyalkenals and Consequences on Neutrophil Motility

The lipid peroxidation product 4-hydroxynonenal (HNE) and homologous aldehydes have been found to possess chemotactic activity for rat neutrophil leukocytes in the micromolar to picomolar range, depending on the compound. Such an activity is displayed only in the presence of albumin. The mechanisms by which aldehydes could interact with neutrophils are discussed. II is proposed that albumin acts as a carrier for the aldehyde and releases them to a neutrophil receptor. At concentrations around 10^?4M, 4-hydroxyal-kenals have been found to exert toxic effects on a number of cells, including a strong depression of neutrophil motility. Finally, HNE has been found at chemotactic concentrations in the inflammatory site. The possibility that HNE is involved in the neutrophil influx into the inflammatory site is considered.     

Quorum Sensing Controls Swarming Motility of Burkholderia glumae through Regulation of Rhamnolipids

Burkholderia glumae is a plant pathogenic bacterium that uses an acyl-homoserine lactone-mediated quorum sensing system to regulate protein secretion, oxalate production and major virulence determinants such as toxoflavin and flagella. B. glumae also releases surface-active rhamnolipids. In Pseudomonas aeruginosa and Burkholderia thailandensis, rhamnolipids, along with flagella, are required for the social behavior called swarming motility. In the present study, we demonstrate that quorum sensing positively regulates the production of rhamnolipids in B. glumae and that rhamnolipids are necessary for swarming motility also in this species. We show that a rhlA- mutant, which is unable to produce rhamnolipids, loses its ability to swarm, and that this can be complemented by providing exogenous rhamnolipids. Impaired rhamnolipid production in a quorum sensing-deficient B. glumae mutant is the main factor responsible for its defective swarming motility behaviour.