Quantifying and localizing actin-free barbed ends in neutrophils.
We describe here a permeablization method that retains coupling between N-formylmethionyl-leucyl-phenylalanine (fMLP) receptor stimulation and barbed-end actin nucleation in neutrophils. Using fluorescently-tagged actin monomers, we are able to quantify and localize actin-free barbed ends generated downstream of chemoattractant receptors. Partial permeabilization of the neutrophils with the mild detergent n-octyl-beta-glucopyranoside maintains signaling from membrane receptor to the actin cytoskeleton while allowing for the introduction of inhibitors and activators of signal transduction pathways implicated in regulating actin cytoskeleton dynamics. This is a useful assay for studying signal transduction to the actin cytoskeleton in neutrophils.
Glogauer
Michael
M
Canadian Institutes of Health Research Group in Matrix Dynamics, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada.
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002463:Cell Membrane Permeability / Q000187:drug effects; D003599:Cytoskeleton / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D018375:Neutrophil Activation; D009504:Neutrophils / Q000737:chemistry* / Q000378:metabolism*; D012235:Rhodamines / Q000493:pharmacokinetics / Q000494:pharmacology; D013194:Staining and Labeling
D000199:Actins; D012235:Rhodamines