Cell permeant polyphosphoinositide-binding peptides that block cell motility and actin assembly.
Polyphosphoinositides (PPIs) affect the localization and activities of many cellular constituents, including actin-modulating proteins. Several classes of polypeptide sequences, including pleckstrin homology domains, FYVE domains, and short linear sequences containing predominantly hydrophobic and cationic residues account for phosphoinositide binding by most such proteins. We report that a ten-residue peptide derived from the phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding region in segment 2 of gelsolin, when coupled to rhodamine B has potent PIP(2) binding activity in vitro; crosses the cell membrane of fibroblasts, platelets, melanoma cells, and neutrophils by a process not involving endocytosis; and blocks cell motility. This peptide derivative transiently disassembles actin filament structures in GFP-actin-expressing NIH3T3 fibroblasts and prevents thrombin- or chemotactic peptide-stimulated actin assembly in platelets and neutrophils, respectively, but does not block the initial [Ca(2+)] increase caused by these agonists. The blockage of actin assembly and motility is transient, and cells recover motility within an hour after their immobilization by 5-20 microm peptide. This class of reagents confirms the critical relation between inositol lipids and cytoskeletal structure and may be useful to probe the location and function of polyphosphoinositides in vivo.
Cunningham
C C
CC
Hematology Division, Brigham & Women's Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.
Vegners
R
R
Bucki
R
R
Funaki
M
M
Korde
N
N
Hartwig
J H
JH
Stossel
T P
TP
Janmey
P A
PA
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002465:Cell Movement; D003599:Cytoskeleton / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008856:Microscopy, Fluorescence; D009504:Neutrophils / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D011485:Protein Binding; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000199:Actins; D003902:Detergents; D010455:Peptides; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D017830:Octoxynol; D002118:Calcium