Differential effects of gelsolins on tissue culture cells

Abstract

Gelsolins, prepared from a number of different sources, showed similar severing activity on F-actin in vitro or on stress fibers of detergent-extracted cells but differed in their effects on actin in stress fibers of microinjected cells. When human gelsolin isolated from plasma was injected into cells in a Ca(++)-containing buffer, stress fibers were degraded, the cellular morphology was changed, and numerous actin patches appeared. These effects were particularly striking when the Ca(++)-insensitive N-terminal proteolytic fragment of this gelsolin was injected. By contrast, Ca(++)-sensitive gelsolins isolated from human platelets, pig stomach smooth muscle and pig plasma showed no comparable activity. Furthermore, the Ca(++)-independent N-terminal proteolytic fragments prepared from these gelsolins also had no effect despite their in vitro actin severing activity. Most striking was the finding that human plasma gelsolin expressed in E. coli did not degrade stress fibers, in contrast to the same protein isolated from plasma; nor was there any stress fiber disruption observed with the N-terminal half of human gelsolin expressed in Escherichia coli. The different behavior of these gelsolins in cells cannot be explained by sequence diversity between plasma and cytoplasmic forms, nor by variability in the Ca++ sensitivity of the preparations. It suggests the presence of factors, as yet unidentified, that may regulate gelsolin activity in the cytoplasm of living cells and discriminate between gelsolins of different origin. Such discrimination could be achieved as a result of post-translational modification of the gelsolin; only in this way can differences between apparently identical proteins isolated from human plasma and expressed in E. coli be reconciled.

Sodium Channel Activity in Leukemia Cells Is Directly Controlled by Actin Polymerization

“…The disrupting effect of gelsolin on the cortical cytoskeleton remaining in the excised membrane fragment is in line with the results of gelsolin-produced disassembly of the actin cytoskeleton in permeabilized and microinjected cultured cells (28,29). This effect may be accounted for by changes that gelsolin generates in the cortical actin network by shortening actin filaments.…”

Disruption of the actin cytoskeleton after microinjection of proteolytic fragments of alpha-actinin.

“…solin from some sources had no affect on stress fibers in nonmuscle cells (Cooper et al ., 1987 ;Huckriede et al, 1990) . Stress fibers have also been disrupted by microinjection of vitamin D binding protein and DNase I, two other proteins that bind monomeric actin (Sanger et al ., 1990) .…”

Gelsolin overexpression enhances neurite outgrowth in PC12 cells

“…Because this truncation removes the calcium requirement for gelsolin activation, this negative effect on neurite outgrowth suggests that, without regulation, gelsolin can have a detrimental effect on actin‐based motility. In addition, cells microinjected with gelsolin showed a destructive loss of actin filaments with only certain preparations of gelsolin [13,14]. Therefore, it is not clear whether gelsolin is critical for actin‐based cell migration events, particularly for neurite extension.…”