Quantification of Cell Movement Reveals Distinct Edge Motility Types During Cell Spreading

Actin-based motility is central to cellular processes such as migration, bacterial engulfment, and cancer metastasis, and requires precise spatial and temporal regulation of the cytoskeleton. We studied one such process, fibroblast spreading, which involves three temporal phases: early, middle, and late spreading, distinguished by differences in cell area growth. In these studies, aided by improved algorithms for analyzing edge movement, we observed that each phase was dominated by a single, kinematically and biochemically distinct cytoskeletal organization, or motility type. Specifically, early spreading was dominated by periodic blebbing; continuous protrusion occurred predominantly during middle spreading; and periodic contractions were prevalent in late spreading. Further characterization revealed that each motility type exhibited a distinct distribution of the actin-related protein VASP, while inhibition of actin polymerization by cytochalasin D treatment revealed different dependences on barbed-end polymerization. Through this detailed characterization and graded perturbation of the system, we observed that although each temporal phase of spreading was dominated by a single motility type, in general cells exhibited a variety of motility types in neighboring spatial domains of the plasma membrane edge. These observations support a model in which global signals bias local cytoskeletal biochemistry in favor of a particular motility type

Spheres and Prolate and Oblate Ellipsoids from an Analytical Solution of Spontaneous Curvature Fluid Membrane Model

An analytic solution for Helfrich spontaneous curvature membrane model (H. Naito, M.Okuda and Ou-Yang Zhong-Can, Phys. Rev. E {\bf 48}, 2304 (1993); {\bf 54}, 2816 (1996)), which has a conspicuous feature of representing the circular biconcave shape, is studied. Results show that the solution in fact describes a family of shapes, which can be classified as: i) the flat plane (trivial case), ii) the sphere, iii) the prolate ellipsoid, iv) the capped cylinder, v) the oblate ellipsoid, vi) the circular biconcave shape, vii) the self-intersecting inverted circular biconcave shape, and viii) the self-intersecting nodoidlike cylinder. Among the closed shapes (ii)-(vii), a circular biconcave shape is the one with the minimum of local curvature energy.Comment: 11 pages, 11 figures. Phys. Rev. E (to appear in Sept. 1999

Shape transformation transitions of a tethered surface model

A surface model of Nambu and Goto is studied statistical mechanically by using the canonical Monte Carlo simulation technique on a spherical meshwork. The model is defined by the area energy term and a one-dimensional bending energy term in the Hamiltonian. We find that the model has a large variety of phases; the spherical phase, the planar phase, the long linear phase, the short linear phase, the wormlike phase, and the collapsed phase. Almost all two neighboring phases are separated by discontinuous transitions. It is also remarkable that no surface fluctuation can be seen in the surfaces both in the spherical phase and in the planar phase.Comment: 7 pages with 8 figure

Spontaneous streptomycin resistance and efficiency of Rhizobium strains native in Brazilian cerrados associated with Stylosanthes

Foram efetuados isolamentos de Rhizobium sp nativos nos Cerrados, associados a Stylosanthes spp, com o objetivo de estudar a natureza dos fatores que interferem no estabelecimento das estirpes de Rhizobium inoculadas, e de avaliar o potencial de fixação de N2 das estirpes nativas. Dentre 122 estirpes isoladas em solo sob Cerrado cultivado, 35% foram resistentes a concentrações iguais ou superiores a 40 μg/ml, enquanto que apenas 10% dentre 92 estirpes isoladas em solo sob Cerrado virgem apresentaram esse nível de resistência. Esses dados sugerem que modificações ecológicas promovidas pelo cultivo dos Cerrados podem induzir a vantagens competitivas para estirpes de Rhizobium sp. resistentes à estreptomicina. Experimentos conduzidos em vasos Leonard com areia e solução nutritiva esterilizada indicaram que cerca de 61% das estirpes isoladas em S. guianensis var. Canescens e S. grandifolia eram pouco ou moderadamente efetivas no hospedeiro homólogo, enquanto que apenas 14% das estirpes apresentaram um nível elevado de fixação do N2. Esse baixo índice de estirpes eficientes sugere a necessidade de se iniciar um trabalho de seleção de Rhizobium sp. adaptado às condições dos Cerrados, visando a produção de inoculantes de alta qualidade para a região.Rhizobium strains were isolated from nodules of Stylosanthes spp. in undisturbed or cultivated Cerrado soils that have never been inoculated, in order to study the nature of the factors that affect stablishment of Rhizobium strains introduced in these soils, as well as to evaluate the N2 fixing capacity of the native strains. Streptomycin resistance level of the Rhizobium strains were tested in yeast-mannitol-agar plates. Among 122 strains isolated in cultivated Cerrado, 35%  were resistant to concentrations equal or above 40 μg/ml, while only 10% within 92 strains isolated in undisturbed Cerrados were resistant to that level. These data suggest that the ecological changes caused by cultivation of virgin savanna land result in competitive advantages for Streptomycin resistent Rhizobium strains. Experiments carried out in Leonard jar assemblies with san and sterilized solution indicated that 61% of the strains isolated in S. guianensis var. Canescens and S. grandifolia were low or moderately effective in the homologous host, while only 14% of the strains had a high level of N2 fixation. This low percentage of efficient strains suggests the necessity of a Rhizobium strain selection program adapted to the Cerrado conditions, aiming to obtain high quality inoculant for the Cerrados region

Lateral Membrane Waves Constitute a Universal Dynamic Pattern of Motile Cells

We have monitored active movements of the cell circumference on specifically coated substrates for a variety of cells including mouse embryonic fibroblasts and T cells, as well as wing disk cells from fruit flies. Despite having different functions and being from multiple phyla, these cell types share a common spatiotemporal pattern in their normal membrane velocity; we show that protrusion and retraction events are organized in lateral waves along the cell membrane. These wave patterns indicate both spatial and temporal long-range periodic correlations of the actomyosin gel

Coupled Growth and Division of Model Protocell Membranes

The generation of synthetic forms of cellular life requires solutions to the problem of how biological processes such as cyclic growth and division could emerge from purely physical and chemical systems. Small unilamellar fatty acid vesicles grow when fed with fatty acid micelles and can be forced to divide by extrusion, but this artificial division process results in significant loss of protocell contents during each division cycle. Here we describe a simple and efficient pathway for model protocell membrane growth and division. The growth of large multilamellar fatty acid vesicles fed with fatty acid micelles, in a solution where solute permeation across the membranes is slow, results in the transformation of initially spherical vesicles into long thread-like vesicles, a process driven by the transient imbalance between surface area and volume growth. Modest shear forces are then sufficient to cause the thread-like vesicles to divide into multiple daughter vesicles without loss of internal contents. In an environment of gentle shear, protocell growth and division are thus coupled processes. We show that model protocells can proceed through multiple cycles of reproduction. Encapsulated RNA molecules, representing a primitive genome, are distributed to the daughter vesicles. Our observations bring us closer to the laboratory synthesis of a complete protocell consisting of a self-replicating genome and a self-replicating membrane compartment. In addition, the robustness and simplicity of this pathway suggests that similar processes might have occurred under the prebiotic conditions of the early Earth.Exobiology Program (U.S.) (Grant EXB02- 0031-0018)United States. National Aeronautics and Space Administration (Exobiology Program) (Grant EXB02-0031-0018)Howard Hughes Medical Institute (Investigator

An Adhesion-Dependent Switch between Mechanisms That Determine Motile Cell Shape

Keratocytes are fast-moving cells in which adhesion dynamics are tightly coupled to the actin polymerization motor that drives migration, resulting in highly coordinated cell movement. We have found that modifying the adhesive properties of the underlying substrate has a dramatic effect on keratocyte morphology. Cells crawling at intermediate adhesion strengths resembled stereotypical keratocytes, characterized by a broad, fan-shaped lamellipodium, clearly defined leading and trailing edges, and persistent rates of protrusion and retraction. Cells at low adhesion strength were small and round with highly variable protrusion and retraction rates, and cells at high adhesion strength were large and asymmetrical and, strikingly, exhibited traveling waves of protrusion. To elucidate the mechanisms by which adhesion strength determines cell behavior, we examined the organization of adhesions, myosin II, and the actin network in keratocytes migrating on substrates with different adhesion strengths. On the whole, our results are consistent with a quantitative physical model in which keratocyte shape and migratory behavior emerge from the self-organization of actin, adhesions, and myosin, and quantitative changes in either adhesion strength or myosin contraction can switch keratocytes among qualitatively distinct migration regimes

Cell–Matrix De-Adhesion Dynamics Reflect Contractile Mechanics

Measurement of the mechanical properties of single cells is of increasing interest both from a fundamental cell biological perspective and in the context of disease diagnostics. In this study, we show that tracking cell shape dynamics during trypsin-induced de-adhesion can serve as a simple but extremely useful tool for probing the contractility of adherent cells. When treated with trypsin, both SW13−/− epithelial cells and U373 MG glioma cells exhibit a brief lag period followed by a concerted retraction to a rounded shape. The time–response of the normalized cell area can be fit to a sigmoidal curve with two characteristic time constants that rise and fall when cells are treated with blebbistatin and nocodazole, respectively. These differences can be attributed to actomyosin-based cytoskeletal remodeling, as evidenced by the prominent buildup of stress fibers in nocodazole-treated SW13−/− cells, which are also two-fold stiffer than untreated cells. Similar results observed in U373 MG cells highlights the direct association between cell stiffness and the de-adhesion response. Faster de-adhesion is obtained with higher trypsin concentration, with nocodazole treatment further expediting the process and blebbistatin treatment blunting the response. A simple finite element model confirms that faster contraction is achieved with increased stiffness