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Free keywords:
GREEN FLUORESCENT PROTEIN; IN-VIVO ANALYSIS; FUSION PROTEIN;
CYTOSKELETON DYNAMICS; SELF-ORGANIZATION; IMAGE-ANALYSIS; MOTILE CELLS;
PIP3 WAVES; F-ACTIN; DICTYOSTELIUMBiochemistry & Molecular Biology; Actin waves; Arp2/3 complex; Cell polarity; Myosin; Phosphoinositides;
PI3-kinase; PTEN; Ras; Self-organization;
Abstract:
When cells of Dictyostelium discoideum orientate in a gradient of chemoattractant, they are polarized into a protruding front pointing toward the source of attractant, and into a retracting tail. Under the control of chemotactic signal inputs, Ras is activated and PIP 3 is synthesized at the front, while the PIP3 - degrading phosphatase PTEN decorates the tail region. As a result of signal transduction, actin filaments assemble at the front into dendritic structures associated with the Arp2/3 complex, in contrast to the tail region where a loose actin meshwork is associated with myosin-II and cortexillin, an antiparallel actin-bundling protein. In axenically growing strains of D. discoideum, wave patterns built by the same components evolve in the absence of any external signal input. Since these autonomously generated patterns are constrained to the plane of the substrate-attached cell surface, they are optimally suited to the optical analysis of state transitions between front-like and tail-like states of the membrane and the actin cortex. Here, we describe imaging techniques using fl uorescent proteins to probe for the state of the membrane, the reorganization of the actin network, and the dynamics of wave patterns.
