A new method for direct detection of the sites of actin polymerization in intact cells and its application to differentiated vascular smooth muscle.
Here we report and validate a new method, suitable broadly, for use in differentiated cells and tissues, for the direct visualization of actin polymerization under physiological conditions. We have designed and tested different versions of fluorescently labeled actin, reversibly attached to the protein transduction tag TAT, and have introduced this novel reagent into intact differentiated vascular smooth muscle cells (dVSMCs). A thiol-reactive version of the TAT peptide was synthesized by adding the amino acids glycine and cysteine to its NH(2)-terminus and forming a thionitrobenzoate adduct: viz. TAT-Cys-S-STNB. This peptide reacts readily with G-actin, and the complex is rapidly taken up by freshly enzymatically isolated dVSMC, as indicated by the fluorescence of a FITC tag on the TAT peptide. By comparing different versions of the construct, we determined that the optimal construct for biological applications is a nonfluorescently labeled TAT peptide conjugated to rhodamine-labeled actin. When TAT-Cys-S-STNB-tagged rhodamine actin (TSSAR) was added to live, freshly enzymatically isolated cells, we observed punctae of incorporated actin at the cortex of the cell. The punctae are indistinguishable from those we have previously reported to occur in the same cell type when rhodamine G-actin is added to permeabilized cells. Thus this new method allows the delivery of labeled G-actin into intact cells without disrupting the native state and will allow its further use to study the effect of physiological intracellular Ca(2+) concentration transients and signal transduction on actin dynamics in intact cells.
Kim
Hak Rim
HR
Department of Health Sciences, Boston University, Boston, MA 02215, USA.
Leavis
Paul C
PC
Graceffa
Philip
P
Gallant
Cynthia
C
Morgan
Kathleen G
KG
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D002454:Cell Differentiation; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D013194:Staining and Labeling / Q000379:methods*
D000199:Actins; D005456:Fluorescent Dyes; D015696:Gene Products, tat; D010455:Peptides