In Dictyostelium, mechanical resistance induces bleb-driven chemotactic movement that is controlled through PI3-kinase.

Two motors can drive extension of the leading edge of motile cells: actin polymerization and myosin-driven contraction of the cortex, producing fluid pressure and the formation of blebs. Dictyostelium cells can move with both blebs and actin-driven pseudopods at the same time, and blebs, like pseudopods, can be orientated by chemotactic gradients. Here we ask how bleb sites are selected and how the two forms of projection cooperate. We show that membrane curvature is an important, yet overlooked, factor. Dictyostelium cells were observed moving under agarose, which efficiently induces blebbing, and the dynamics of membrane deformations were analyzed. Blebs preferentially originate from negatively curved regions, generated on the flanks of either extending pseudopods or blebs themselves. This is true of cells at different developmental stages, chemotaxing to either folate or cyclic AMP and moving with both blebs and pseudopods or with blebs only. A physical model of blebbing suggests that detachment of the cell membrane is facilitated in concave areas of the cell, where membrane tension produces an outward directed force, as opposed to pulling inward in convex regions. Our findings assign a role to membrane tension in spatially coupling blebs and pseudopods, thus contributing to clustering protrusions to the cell front.C rawling cells must restrict protrusions to a limited part of their periphery if they are to move efficiently, and when these cells chemotax, the location of projections must be further controlled by the chemotactic gradient (1-3). Cellular protrusions are of two main types: those driven by actin polymerization, such as pseudopods or lamellipods, and those driven by fluid pressure, which are usually called blebs. Blebs form when the cell membrane locally detaches from the underlying cortex and is driven outward by hydrostatic pressure, created by myosin-IIdriven contraction of the cortex (4, 5). When blebs form, the cortex is left behind as an "F-actin scar," which depolymerizes, while a new actin cortex forms at the freshly exposed membrane.Blebbing is important in cells migrating in three-dimensional environments, such as during tumor invasion (6, 7), zebrafish primordial germ cell migration (8, 9), or migration of the pathogen Entamoeba histolytica in the liver (10). Dictyostelium amoebae can also move with blebs (11). In standard conditions on a 2D surface under buffer, they move mainly with F-actin-driven pseudopods, but switch progressively to bleb-driven motility when faced with mechanical resistance to their movement (12). This can be conveniently applied by inducing the cells to migrate under an elastic overlay, such as agarose, which they must deform to progress (13). Blebbing is stimulated by acute treatment with the chemoattractant cyclic AMP (14), and blebs can be chemotactically orientated by cyclic-AMP gradients (11,12).Actin-driven pseudopods are preferentially formed up-gradient by chemotaxing cells, and they can be induced on the flanks of cells by applying a steep gradient of ch...

Abstract. Cell motility is an integral part of a diverse set of biological processes. The quest for mathematical models of cell motility has prompted the development of automated approaches for gathering quantitative data on cell morphology, and the distribution of molecular players involved in cell motility. Here we review recent approaches for quantifying cell motility, including automated cell segmentation and tracking. Secondly, we present our own novel method for tracking cell boundaries of moving cells, the Electrostatic Contour Migration Method (ECMM), as an alternative to the generally accepted level set method (LSM). ECMM smoothly tracks regions of the cell boundary over time to compute local membrane displacements using the simple underlying concept of electrostatics. It offers substantial speed increases and reduced computational overheads in comparison to the LSM. We conclude with general considerations regarding boundary tracking in the context of mathematical modelling.

SummaryDetection of substances tasting bitter to humans occurs in diverse organisms including the social amoeba Dictyostelium discoideum. To establish a molecular mechanism for bitter tastant detection in Dictyostelium, we screened a mutant library for resistance to a commonly used bitter standard, phenylthiourea. This approach identified a G-protein-coupled receptor mutant, grlJ 2 , which showed a significantly increased tolerance to phenylthiourea in growth, survival and movement. This mutant was not resistant to a structurally dissimilar potent bitter tastant, denatonium benzoate, suggesting it is not a target for at least one other bitter tastant. Analysis of the cell-signalling pathway involved in the detection of phenylthiourea showed dependence upon heterotrimeric G protein and phosphatidylinositol 3-kinase activity, suggesting that this signalling pathway is responsible for the cellular effects of phenylthiourea. This is further supported by a phenylthiourea-dependent block in the transient cAMP-induced production of phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) in wild-type but not grlJ 2 cells. Finally, we have identified an uncharacterized human protein c-aminobutyric acid (GABA) type B receptor subunit 1 isoform with weak homology to GrlJ that restored grlJ 2 sensitivity to phenylthiourea in cell movement and PIP 3 regulation. Our results thus identify a novel pathway for the detection of the standard bitter tastant phenylthiourea in Dictyostelium and implicate a poorly characterized human protein in phenylthiourea-dependent cell responses.

Increasing concern regarding the use of animals in research has triggered a growing need for non-animal research models in a range of fields. The development of 3Rs (replacement, refinement, and reduction) approaches in research, to reduce the reliance on the use of animal tissue and whole-animal experiments, has recently included the use of Dictyostelium. In addition to not feeling pain and thus being relatively free of ethical constraints, Dictyostelium provides a range of distinct methodological advantages for researchers that has led to a number of breakthroughs. These methodologies include using cell behavior (cell movement and shape) as a rapid indicator of sensitivity to poorly characterized medicines, natural products, and other chemicals to help understand the molecular mechanism of action of compounds. Here, we outline a general approach to employing Dictyostelium as a 3Rs research model, using cell behavior as a readout to better understand how compounds, such as the active ingredient in chilli peppers, capsaicin, function at a cellular level. This chapter helps scientists unfamiliar with Dictyostelium to rapidly employ it as an advantageous model system for research, to reduce the use of animals in research, and to make paradigm shift advances in our understanding of biological chemistry.