Gelsolin binds to polyphosphoinositide-free lipid vesicles and simultaneously to actin microfilaments

Abstract

Gelsolin is a calcium-, pH- and lipid-dependent actin filament severing/capping protein whose main function is to regulate the assembly state of the actin cytoskeleton. Gelsolin is associated with membranes in cells, and it is generally assumed that this interaction is mediated by PPIs (polyphosphoinositides), since an interaction with these lipids has been characterized in vitro. We demonstrate that non-PPI lipids also bind gelsolin, especially at low pH. The data suggest further that gelsolin becomes partially buried in the lipid bilayer under mildly acidic conditions, in a manner that is not dependent of the presence of PPIs. Our data also suggest that lipid binding involves a number of sites that are spread throughout the gelsolin molecule. Linker regions between gelsolin domains have been implicated by other work, notably the linker between G1 and G2 (gelsolin domains 1 and 2 respectively), and we postulate that the linker region between the N-terminal and C-terminal halves of gelsolin (between G3 and G4) is also involved in the interaction with lipids. This region is compatible with other studies in which additional binding sites have been located within G4-6. The lipid-gelsolin interactions reported in the present paper are not calcium-dependent, and are likely to involve significant conformational changes to the gelsolin molecule, as the chymotryptic digest pattern is altered by the presence of lipids under our conditions. We also report that vesicle-bound gelsolin is capable of binding to actin filaments, presumably through barbed end capping. Gelsolin bound to vesicles can nucleate actin assembly, but is less active in severing microfilaments.

Force Spectroscopy Measurements Show That Cortical Neurons Exposed to Excitotoxic Agonists Stiffen before Showing Evidence of Bleb Damage

“…In experimental models, inhibitors of Nav and NMDA channels attenuate, postpone or prevent anoxic depolarization, and hypoxia/ischemia-induced demise of neurons; likewise for mechanically-traumatized neurons [1–11]. Dysregulation of excitatory channels engenders [Ca ++ ] int levels deemed “excitotoxic” because they overactivate Ca ++ -proteases [12] and Ca ++ -lipases [13], with calpain-cleaved spectrin products [14] being an unequivocal correlate of irreversible excitotoxic membrane damage. In erythrocytes, it is well recognized that plasma membrane integrity and functionality of integral membrane proteins depends on robust and extensive adhesive contacts between spectrin-actin membrane skeleton components and the bilayer.…”

Calcium‐induced conformational changes in the amino‐terminal half of gelsolin

“…A type I site is that which is coordinated both by gelsolin and actin residues while type II sites are solely coordinated by those of gelsolin. as previously reported [22]. G1-2 was produced recombinantly in E. coli.…”

“…Gelsolin was produced in E. coli (BL21 de3) carrying a cDNA encoding human gelsolin in the pMW172 plasmid as previously described [21]. We have defined the limits of the various gelsolin domains as previously reported [22]. G1–2 was produced recombinantly in E. coli .…”

“…Fluorescence of dansylated proteins was measured at an excitation of 340 nm. The collisional quenching of tryptophan fluorescence with acrylamide was carried out as previously described [22] using the Stern–Volmer relationship. F 0 and F are the fluorescence measured in the presence and the absence respectively of the quencher.…”

A direct interaction with calponin inhibits the actin-nucleating activity of gelsolin

“…Gelsolin's actin-binding activity is regulated by Ca 2+ ions [2], pH [3,4] phosphorylation [5,6] and phospholipids [7,8]. It is widely expressed in vertebrate cells [9] and is secreted from muscle [10] into the bloodstream.…”