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 classe s of polypeptide sequences, including pleckstrin homology domains, FYVE dom ains, and short linear sequences containing predominantly hydrophobic and c ationic residues account for phosphoinositide binding by most such proteins . We report that a ten-residue peptide derived from the phosphatidylinosito l 4,5-bisphosphate (PIP2) binding region in segment 2 of gelsolin, when cou pled to rhodamine B has potent PIP2 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 peptid e derivative transiently disassembles actin filament structures in GFP-acti n-expressing NIH3T3 fibroblasts and prevents thrombin- or chemotactic pepti de-stimulated actin assembly in platelets and neutrophils, respectively, bu t does not block the initial [Ca2+] increase caused by these agonists. The blockage of actin assembly and motility is transient, and cells recover mot ility within an hour after their immobilization by 5-20 muM peptide. This c lass of reagents confirms the critical relation between inositol lipids and cytoskeletal structure and may be useful to probe the location and functio n of polyphosphoinositides in vivo.