Cell migration directionality and speed are independently regulated by RasG and Gβ in Dictyostelium cells in electrotaxis.

Motile cells manifest increased migration speed and directionality in gradients of stimuli, including chemoattractants, electrical potential and substratum stiffness. Here, we demonstrate that Dictyostelium cells move directionally in response to an electric field (EF) with specific acceleration/deceleration kinetics of directionality and migration speed. Detailed analyses of the migration kinetics suggest that migration speed and directionality are separately regulated by Gβ and RasG, respectively, in EF-directed cell migration. Cells lacking Gβ, which is essential for all chemotactic responses in Dictyostelium, showed EF-directed cell migration with the same increase in directionality in an EF as wild-type cells. However, these cells failed to show induction of the migration speed upon EF stimulation as much as wild-type cells. Loss of RasG, a key regulator of chemoattractant-directed cell migration, resulted in almost complete loss of directionality, but similar acceleration/deceleration kinetics of migration speed as wild-type cells. These results indicate that Gβ and RasG are required for the induction of migration speed and directionality, respectively, in response to an EF, suggesting separation of migration speed and directionality even with intact feedback loops between mechanical and signaling networks

Cellular Ability to Sense Spatial Gradients in the Presence of Multiple Competitive Ligands

Many eukaryotic and prokaryotic cells can exhibit remarkable sensing ability under small gradient of chemical compound. In this study, we approach this phenomenon by considering the contribution of multiple ligands to the chemical kinetics within Michaelis-Menten model. This work was inspired by the recent theoretical findings from Bo Hu et al. [Phys. Rev. Lett. 105, 048104 (2010)], our treatment with practical binding energies and chemical potential provides the results which are consistent with experimental observations.Comment: 5 pages, 4 figure

cAMP activates adenylate and guanylate cyclase of Dictyostelium discoideum cells by binding to different classes of cell-surface receptors. A study with extracellular Ca2+

cAMP induces a transient increase of cAMP and cGMP levels in Dictyostelium discoideum cells. Fast binding experiments reveal three types of cAMP-binding site (S, H and L), which have different off-rates (t0.5, 0.7-15 s) and different affinities (Kd, 15-450 nM). A time- and cAMP-concentration-dependent transition of H- to L-sites occurs during the binding reaction. Extracellular Ca2+ had multiple effects on cAMP-binding sites. (i) The number of H + L-sites increased 2.5-fold, while the number of S-sites was not strongly affected. (ii) The Kd of the S-sites was reduced from 16 nM to 5 nM (iii) The conversion of H-sites to L-sites was inhibited up to 80%. The kinetics of the cAMP-induced cAMP accumulation was not strongly altered by Ca2+, but the amount of cAMP produced was inhibited up to 80%. The kinetics of the cAMP-induced cGMP accumulation was strongly altered; maximal levels were obtained sooner, and the Ka was reduced from 15 to 3.5 nM cAMP. Ca2+, Mg2+ and Mn2+ increased the number of binding sites, all with EC50 = 0.5 mM. The S-sites and the cGMP response were modified by equal Ca2+ concentrations and by higher concentrations of Mg2+ and Mn2+ (EC50 are respectively 0.4 mM, 2.5 mM and about 25 mM). The conversion of H- to L-sites and the cAMP response were specifically inhibited by Ca2+ with EC50 = 20 µM. It is concluded that cAMP activates guanylate cyclase through the S-sites; adenylate cyclase is activated by the H + L-sites, in which the appearance of the L-sites during the binding reaction represents the coupling of occupied surface cAMP receptors to adenylate cyclase.

Overexpression of the cAMP Receptor 1 in Growing Dictyostelium Cells

cAR1, the cAMP receptor expressed normally during the early aggregation stage of the Dictyostelium developmental program, has been expressed during the growth stage, when only low amounts of endogenous receptors are present. Transformants expressing cAR1 have 7-40 times over growth stage and 3-5-fold over aggregation stage levels of endogenous receptors. The high amounts of cAR1 protein expressed constitutively throughout early development did not drastically disrupt the developmental program; the onset of aggregation was delayed by 1-3 h, and then subsequent stages proceeded normally. The affinity of the expressed cAR1 was similar to that of the endogenous receptors in aggregation stage cells when measured either in phosphate buffer (two affinity states with K(d)'s of approximately 30 and 300 nM) or in 3 M ammonium sulfate (one affinity state with a K(d) of 2-3 nM). When expressed during growth, cAR1 did not appear to couple to its normal effectors since these cells failed to carry out chemotaxis or to elevate cGMP or cAMP levels when stimulated with cAMP. However, cAMP stimulated phosphorylation, and loss of ligand binding of cAR1 did occur. Like aggregation stage control cells, the cAR1 protein shifted in apparent molecular mass from 40 to 43 kDa and became highly phosphorylated when exposed to cAMP. In addition, the number of surface cAMP binding sites in cAR1 cells was reduced by over 80% during prolonged cAMP stimulation. These results define a useful system to express altered cARl proteins and examine their regulatory functions

Contact-controlled amoeboid motility induces dynamic cell trapping in 3D-microstructured surfaces.

On flat substrates, several cell types exhibit amoeboid migration, which is characterized by restless stochastic successions of pseudopod protrusions. The orientation and frequency of new membrane protrusions characterize efficient search modes, which can respond to external chemical stimuli as observed during chemotaxis in amoebae. To quantify the influence of mechanical stimuli induced by surface topography on the migration modes of the amoeboid model organism Dictyostelium discoideum, we apply high resolution motion analysis in microfabricated pillar arrays of defined density and geometry. Cell motion is analyzed by a two-state motility-model, distinguishing directed cellular runs from phases of isotropic migration that are characterized by randomly oriented cellular protrusions. Cells lacking myosin II or cells deprived of microtubules show significantly different behavior concerning migration velocities and migrational angle distribution, without pronounced attraction to pillars. We conclude that microtubules enhance cellular ability to react with external 3D structures. Our experiments on wild-type cells show that the switching from randomly formed pseudopods to a stabilized leading pseudopod is triggered by contact with surface structures. These alternating processes guide cells according to the available surface in their 3D environment, which we observed dynamically and in steady-state situations. As a consequence, cells perform "home-runs" in low-density pillar arrays, crawling from pillar to pillar, with a characteristic dwell time of 75 s. At the boundary between a flat surface and a 3D structured substrate, cells preferentially localize in contact with micropillars, due to the additionally available surface in the microstructured arrays. Such responses of cell motility to microstructures might open new possibilities for cell sorting in surface structured arrays

Chemotaxis: a feedback-based computational model robustly predicts multiple aspects of real cell behaviour

The mechanism of eukaryotic chemotaxis remains unclear despite intensive study. The most frequently described mechanism acts through attractants causing actin polymerization, in turn leading to pseudopod formation and cell movement. We recently proposed an alternative mechanism, supported by several lines of data, in which pseudopods are made by a self-generated cycle. If chemoattractants are present, they modulate the cycle rather than directly causing actin polymerization. The aim of this work is to test the explanatory and predictive powers of such pseudopod-based models to predict the complex behaviour of cells in chemotaxis. We have now tested the effectiveness of this mechanism using a computational model of cell movement and chemotaxis based on pseudopod autocatalysis. The model reproduces a surprisingly wide range of existing data about cell movement and chemotaxis. It simulates cell polarization and persistence without stimuli and selection of accurate pseudopods when chemoattractant gradients are present. It predicts both bias of pseudopod position in low chemoattractant gradients and-unexpectedly-lateral pseudopod initiation in high gradients. To test the predictive ability of the model, we looked for untested and novel predictions. One prediction from the model is that the angle between successive pseudopods at the front of the cell will increase in proportion to the difference between the cell's direction and the direction of the gradient. We measured the angles between pseudopods in chemotaxing Dictyostelium cells under different conditions and found the results agreed with the model extremely well. Our model and data together suggest that in rapidly moving cells like Dictyostelium and neutrophils an intrinsic pseudopod cycle lies at the heart of cell motility. This implies that the mechanism behind chemotaxis relies on modification of intrinsic pseudopod behaviour, more than generation of new pseudopods or actin polymerization by chemoattractant

Phosphoinositide signaling plays a key role in cytokinesis

To perform the vital functions of motility and division, cells must undergo dramatic shifts in cell polarity. Recent evidence suggests that polarized distributions of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, which are clearly important for regulating cell morphology during migration, also play an important role during the final event in cell division, which is cytokinesis. Thus, there is a critical interplay between the membrane phosphoinositides and the cytoskeletal cortex that regulates the complex series of cell shape changes that accompany these two processes

PAWS1controls cytoskeletal dynamics and cell migration through association with the SH3 adaptor CD2AP

Our previous studies of PAWS1 (protein associated with SMAD1; also known as FAM83G) have suggested that this molecule has roles beyond BMP signalling. To investigate these roles, we have used CRISPR/Cas9 to generate PAWS1-knockout U2OS osteosarcoma cells. Here, we show that PAWS1 plays a role in the regulation of the cytoskeletal machinery, including actin and focal adhesion dynamics, and cell migration. Confocal microscopy and live cell imaging of actin in U2OS cells indicate that PAWS1 is also involved in cytoskeletal dynamics and organization. Loss of PAWS1 causes severe defects in F-actin organization and distribution as well as in lamellipodial organization, resulting in impaired cell migration. PAWS1 interacts in a dynamic fashion with the actin/cytoskeletal regulator CD2AP at lamellae, suggesting that its association with CD2AP controls actin organization and cellular migration. Genetic ablation of CD2AP from U2OS cells instigates actin and cell migration defects reminiscent of those seen in PAWS1-knockout cells.This article has an associated First Person interview with the first authors of the paper.</p

Lubricating Bacteria Model for Branching growth of Bacterial Colonies

Various bacterial strains (e.g. strains belonging to the genera Bacillus, Paenibacillus, Serratia and Salmonella) exhibit colonial branching patterns during growth on poor semi-solid substrates. These patterns reflect the bacterial cooperative self-organization. Central part of the cooperation is the collective formation of lubricant on top of the agar which enables the bacteria to swim. Hence it provides the colony means to advance towards the food. One method of modeling the colonial development is via coupled reaction-diffusion equations which describe the time evolution of the bacterial density and the concentrations of the relevant chemical fields. This idea has been pursued by a number of groups. Here we present an additional model which specifically includes an evolution equation for the lubricant excreted by the bacteria. We show that when the diffusion of the fluid is governed by nonlinear diffusion coefficient branching patterns evolves. We study the effect of the rates of emission and decomposition of the lubricant fluid on the observed patterns. The results are compared with experimental observations. We also include fields of chemotactic agents and food chemotaxis and conclude that these features are needed in order to explain the observations.Comment: 1 latex file, 16 jpeg files, submitted to Phys. Rev.

Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal.

The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient