Abstract
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.
Highlights
Acto-myosin-based cell motility plays a central role in diverse cellular processes such as immune response [1,2], wound healing [3], development [4,5,6], and cancer metastasis [7,8]
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
Transitions between phases, defined by changes in the rate of area change, were distinguishable at up to 100 nM of cytochalasin D (CD), increased CD concentration disrupted the spatiotemporal organization of motility modules and decreased the final spread area of cells. These results indicated that the mechanism of transition between phases was relatively insensitive to barbed end inhibition by CD, similar to the above finding that altering the motility module of P0 with Rho kinase inhibitor did not affect the P0-P1 transition
Summary
Acto-myosin-based cell motility plays a central role in diverse cellular processes such as immune response [1,2], wound healing [3], development [4,5,6], and cancer metastasis [7,8]. While cytoskeletal motility depends on cellular context, the essential cytoskeletal proteins are conserved across eukaryotes [9] This similarity may explain why similar motility phenotypes such as blebbing (membrane protrusion following dissociation with the cytoskeleton), ruffling, filopodia (long, thin actin bundles), and lamellipodia (broad, thin membrane extensions) are observed across a broad range of cells such as mouse fibroblasts, endothelial cells, T-cells, neuronal cells, mammalian and amphibian epithelial cells, and drosophila wing-disk cells [10,11,12,13,14]. This concept can simplify biophysical modeling of cell motility by viewing the process as a sum of currently active motility modules
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