Fluorescent phosphoinositide derivatives reveal specific binding of gelsolin and other actin regulatory proteins to mixed lipid bilayers.
Fluorescent derivatives of phosphatidyl inositol (PtdIns)-(4,5)-P2 were synthesized and used to test the effects of the PtdIns-(4, 5)-P2-regulated proteins gelsolin, tau, cofilin, and profilin on labeled PtdIns-(4,5)-P2 that was either in micellar form or mixed with phosphatidylcholine (PtdCho) in bilayer vesicles. Gelsolin increased the fluorescence of 7-nitrobenz-2-oxa-1,3-diazole (NBD)- or pyrene-labeled PtdIns-(4,5)-P2 and NBD-PtdIns-(3,4,5)-P3. Cofilin and profilin produced no detectable change at equimolar ratios to PtdIns-(4,5)-P2, while tau decreased NBD-PtdIns-(4,5)-P2 fluorescence. Fluorescence enhancement by gelsolin of NBD-PtdIns-(4, 5)-P2 in mixed lipid vesicles depended on the mole fraction of PtdIns-(4,5)-P2 in the bilayer. Specific enhancement of 3% NBD-PtdIns-(4,5)-P2 : 97% PtdCho was much lower than that of 10% PtdIns-(4,5)-P2 : 90% PtdCho, but the enhancement of 3% NBD-PtdIns-(4,5)-P2 could be increased by addition of 7% unlabeled PtdIns-(4,5)-P2. The gelsolin-dependent increase in NBD-PtdIns-(4, 5)-P2 fluorescence was reversed by addition of Ca2+ or G-actin. Significant, but weaker, fluorescence enhancement was observed with the gelsolin N-terminal domain (residues 1-160) and a peptide comprised of gelsolin residues 150-169. Fluorescence energy transfer from gelsolin to pyrene-PtdIns-(4,5)-P2 was much stronger with intact gelsolin than the N-terminal region of gelsolin containing the PtdIns-(4,5)-P2 binding sites, suggesting that PtdIns-(4,5)-P2 may bind near a site formed by the juxtaposition of the N- and C-terminal domains of gelsolin.
Tuominen
E K
EK
Lipid Research Laboratory, Department of Medical Chemistry, Institute of Biomedicine, University of Helsinki, Finland.
Holopainen
J M
JM
Chen
J
J
Prestwich
G D
GD
Bachiller
P R
PR
Kinnunen
P K
PK
Janmey
P A
PA
D009327:4-Chloro-7-nitrobenzofurazan; D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003285:Contractile Proteins; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D018260:Gelsolin / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes; D008823:Micelles; D008840:Microfilament Proteins / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D010716:Phosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D051304:Profilins; D011485:Protein Binding; D011721:Pyrenes; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D016875:tau Proteins / Q000378:metabolism
D051339:Actin Depolymerizing Factors; D000199:Actins; D003285:Contractile Proteins; D005456:Fluorescent Dyes; D018260:Gelsolin; D008051:Lipid Bilayers; D008081:Liposomes; D008823:Micelles; D008840:Microfilament Proteins; C495738:PFN1 protein, human; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D051304:Profilins; D011721:Pyrenes; D011994:Recombinant Proteins; D016875:tau Proteins; D015544:Inositol 1,4,5-Trisphosphate; C030984:pyrene; D009327:4-Chloro-7-nitrobenzofurazan