Visualization of spatially and temporally regulated N-WASP activity during cytoskeletal reorganization in living cells.
Members of the WASP/WAVE family of proteins are key regulators of cytoskeletal reorganization across a diverse range of cellular processes. Despite a wealth of biochemical data about WASP/WAVE regulation in vitro, our understanding of the in vivo regulation of these proteins is hampered by the inability to monitor subcellular regulation of their activities in living cells. Here we establish a fluorescence resonance energy transfer-based approach to visualize spatial and temporal regulation of neuronal Wiskott-Aldrich syndrome protein (N-WASP) activity in living cells. Using time-lapse microscopy, we characterize the activation of N-WASP in response to growth-factor stimulation, and we reveal that N-WASP is activated inside extending filopodia. Furthermore, we suggest a role of N-WASP in regulating membrane ruffling downstream of phosphatidylinositol 4,5-bisphosphate and Cdc42.
Ward
Michael E
ME
Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8108, St Louis, MO 63110, USA.
Wu
Jane Y
JY
Rao
Yi
Y
D000818:Animals; D002460:Cell Line; D003599:Cytoskeleton / Q000502:physiology*; D004735:Energy Transfer; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal
D009419:Nerve Tissue Proteins; C495771:WASL protein, human; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal