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Visualization of spatially and temporally regulated N-WASP activity during cytoskeletal reorganization in living cells.

ABSTRACT: 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.

SUBMITTER: Ward ME

PROVIDER: S-EPMC327126 | biostudies-literature | 2004 Jan

REPOSITORIES: biostudies-literature

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Visualization of spatially and temporally regulated N-WASP activity during cytoskeletal reorganization in living cells.

Ward Michael E ME Wu Jane Y JY Rao Yi Y

Proceedings of the National Academy of Sciences of the United States of America 20040119 4

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 ne ...[more]

PMID: 14732696

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Project description:BackgroundMorphogenetic events that shape the Drosophila melanogaster embryo are tightly controlled by a genetic program in which specific sets of genes are up-regulated. We used a suppressive subtractive hybridization procedure to identify a group of developmentally regulated genes during early stages of D. melanogaster embryogenesis. We studied the spatiotemporal activity of these genes in five different intervals covering 12 stages of embryogenesis.ResultsMicroarrays were constructed to confirm induction of expression and to determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes whose expression levels increased significantly in at least one developmental interval compared with a reference interval. Of these genes, 53% had a phenotype and/or molecular function reported in the literature, whereas 47% were essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. In situ hybridization assays were carried out on 23 uncharacterized genes, 15 of which proved to have spatiotemporally restricted expression patterns. Among these 15 uncharacterized genes, 13 were found to encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in Drosophila S2R+ cells and analyzed the subcellular distribution of recombinant proteins. We then focused on the functional characterization of the gene CG6234. Inhibition of CG6234 by RNA interference resulted in morphological defects in embryos, suggesting the involvement of this gene in germ band retraction.ConclusionOur data have yielded a list of developmentally regulated D. melanogaster genes and their expression profiles during embryogenesis and provide new information on the spatiotemporal expression patterns of several uncharacterized genes. In particular, we recovered a substantial number of unknown genes encoding putative secreted and transmembrane proteins, suggesting new components of signaling pathways that might be incorporated within the existing regulatory networks controlling D. melanogaster embryogenesis. These genes are also good candidates for additional targeted functional analyses similar to those we conducted for CG6234.See related minireview by Vichas and Zallen: http://www.jbiol.com/content/8/8/76.

Dataset Information

Visualization of spatially and temporally regulated N-WASP activity during cytoskeletal reorganization in living cells.

Publications

Visualization of spatially and temporally regulated N-WASP activity during cytoskeletal reorganization in living cells.

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