Dynamic Phase Transitions in Cell Spreading

We monitored isotropic spreading of mouse embryonic fibroblasts on fibronectin-coated substrates. Cell adhesion area versus time was measured via total internal reflection fluorescence microscopy. Spreading proceeds in well-defined phases. We found a power-law area growth with distinct exponents a_i in three sequential phases, which we denote basal (a_1=0.4+-0.2), continous (a_2=1.6+-0.9) and contractile (a_3=0.3+-0.2) spreading. High resolution differential interference contrast microscopy was used to characterize local membrane dynamics at the spreading front. Fourier power spectra of membrane velocity reveal the sudden development of periodic membrane retractions at the transition from continous to contractile spreading. We propose that the classification of cell spreading into phases with distinct functional characteristics and protein activity patterns serves as a paradigm for a general program of a phase classification of cellular phenotype. Biological variability is drastically reduced when only the corresponding phases are used for comparison across species/different cell lines.Comment: 4 pages, 5 figure

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

Periodic Lamellipodial Contractions Correlate with Rearward Actin Waves

AbstractCellular lamellipodia bind to the matrix and probe its rigidity through forces generated by rearward F-actin transport. Cells respond to matrix rigidity by moving toward more rigid matrices using an unknown mechanism. In spreading and migrating cells we find local periodic contractions of lamellipodia that depend on matrix rigidity, fibronectin binding and myosin light chain kinase (MLCK). These contractions leave periodic rows of matrix bound β3-integrin and paxillin while generating waves of rearward moving actin bound α-actinin and MLCK. The period between contractions corresponds to the time for F-actin to move across the lamellipodia. Shortening lamellipodial width by activating cofilin decreased this period proportionally. Increasing lamellipodial width by Rac signaling activation increased this period. We propose that an actin bound, contraction-activated signaling complex is transported locally from the tip to the base of the lamellipodium, activating the next contraction/extension cycle

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

Evidence for hierarchical control of conserved, discrete motility types in crawling motility

The 20th century saw a remarkable explosion of knowledge regarding the mechanisms of cell motility, with the recognition that different classes of motility are involved in almost every cell function. One class, crawling motility, is typical of metazoan derived tissue culture cells. We have developed a quantitative cell-spreading assay for describing crawling motility and have identified a small number of discrete, cytoskeletal organizations, or motility types. Each motility type is characterized by a specific organization of actin polymerization, myosin activity, and adhesion formation, and all have been observed during mouse fibroblast cell spreading as well as across a range of eukaryotic phyla and cell types. During spreading, cells exhibit a series of functional phases, including early adhesion and fast spreading, each with a different combination of motility types. The final phase of spreading is characterized by periodic lamellipodial contractions, a motility type that coordinates adhesion formation and edge protrusion, allowing for eventual polarization and migration. We propose that this hierarchy of functional phases and conserved, discrete motility types represents a general organizational principle in motility regulation

A three-dimensional stochastic spatio-temporal model of cell spreading

Cell motility is important for many physiological processes and the underlying
biochemical reactions motility have been well characterized. Mathematical models, using
the biochemical reactions and focused on different types of spreading behavior have been
constructed and analyzed. In this study, we build on these previous models to develop a
three-dimensional stochastic model of isotropic spreading of mammalian fibroblasts. The model is composed of three actin remodeling reactions that occur stochastically in space and time and are regulated by membrane resistance forces. Numerical simulations indicate that the model qualitatively captures the experimentally observed isotropic cell spreading behavior. We analyzed the effects of varying branching reaction rates, membrane resistance forces and capping protein concentrations on the dynamics of isotropic spreading. The simulations allowed us to identify the range within which branching reaction rates and membrane force values cooperate to yield isotropic spreading behavior. The model predicts increasing capping protein concentration would lead to a linear decrease in average peripheral velocity. We tested this prediction experimentally using varying concentrations of a pharmacologic agent (Cytochalasin D) that caps growing actin filaments. We find that the experimental results agree with the numerical simulations. Thus, a spatio-temporally complex model made up of a simple set of stochastic reactions near the cell surface, when constrained by membrane forces, can yield deterministic behavior as characterized by isotropic cell spreading