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

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

Opposing Effects of PKCθ and WASp on Symmetry Breaking and Relocation of the Immunological Synapse

SummaryThe immunological synapse (IS) is a junction between the T cell and antigen-presenting cell and is composed of supramolecular activation clusters (SMACs). No studies have been published on naive T cell IS dynamics. Here, we find that IS formation during antigen recognition comprises cycles of stable IS formation and autonomous naive T cell migration. The migration phase is driven by PKCθ, which is localized to the F-actin-dependent peripheral (p)SMAC. PKCθ−/− T cells formed hyperstable IS in vitro and in vivo and, like WT cells, displayed fast oscillations in the distal SMAC, but they showed reduced slow oscillations in pSMAC integrity. IS reformation is driven by the Wiscott Aldrich Syndrome protein (WASp). WASp−/− T cells displayed normal IS formation but were unable to reform IS after migration unless PKCθ was inhibited. Thus, opposing effects of PKCθ and WASp control IS stability through pSMAC symmetry breaking and reformation