title: Dynamic Organization of Myristoylated Src in the Live Cell Plasma Membrane.
abstract: The spatial organization of lipid-anchored proteins in the plasma membrane directly influences cell signaling, but measuring such organization in situ is experimentally challenging. The canonical oncogene, c-Src, is a lipid anchored protein that plays a key role in integrin-mediated signal transduction within focal adhesions and cell-cell junctions. Because of its activity in specific plasma membrane regions, structural motifs within the protein have been hypothesized to play an important role in its subcellular localization. This study used a combination of time-resolved fluorescence fluctuation spectroscopy and super-resolution microscopy to quantify the dynamic organization of c-Src in live cell membranes. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) showed that a small fraction of c-Src transiently sorts into membrane clusters that are several times larger than the monomers. Photoactivated localization microscopy (PALM) confirmed that c-Src partitions into clusters with low probability and showed that the characteristic size of the clusters is 10-80 nm. Finally, time-resolved fluorescence anisotropy measurements were used to quantify the rotational mobility of c-Src to determine how it interacts with its local environment. Taken together, these results build a quantitative description of the mobility and clustering behavior of the c-Src nonreceptor tyrosine kinase in the live cell plasma membrane.
authors.affiliation: Department of Chemistry, University of California , Berkeley, California 94720, United States.|Department of Chemistry, University of Akron , Akron, Ohio 44303, United States.
authors.forename: Adam W
authors.initials: AW
authors.lastname: Smith
authors.affiliation: Department of Chemistry, University of California , Berkeley, California 94720, United States.|Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California , Berkeley, California 94720, United States.
authors.forename: Hector H
authors.initials: HH
authors.lastname: Huang
authors.affiliation: Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California , Berkeley, California 94720, United States.
authors.forename: Nicholas F
authors.initials: NF
authors.lastname: Endres
authors.affiliation: Department of Chemistry, University of California , Berkeley, California 94720, United States.
authors.forename: Christopher
authors.initials: C
authors.lastname: Rhodes
authors.affiliation: Department of Chemistry, University of California , Berkeley, California 94720, United States.
authors.forename: Jay T
authors.initials: JT
authors.lastname: Groves
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D019814:Myristic Acid / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*
chemical_list: D019814:Myristic Acid; D016392:Proto-Oncogene Proteins pp60(c-src)