Dependence of Fibroblast Migration on Actin Severing Activity of Gelsolin

doi:10.1074/jbc.271.34.20516

Dependence of Fibroblast Migration on Actin Severing Activity of Gelsolin*

From the Medical Research Council Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

^‡ To whom correspondence should be addressed:
Rm. 4384, Medical Sciences Bldg., 8 Taddle Creek Rd., University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Tel.: 416-978-1258; Fax: 416-978-5956; E-mail: christopher.mcculloch{at}utoronto.ca.

Abstract

Gelsolin nucleates actin filament assembly, blocks the fast-exchanging ends of actin filaments, and severs filaments, processes that may play an important role in cell motility. We studied the relationship between cell migration, gelsolin content, and actin severing activity in human gingival fibroblasts. These cells were keratin negative and desmin negative but expressed vimentin and myosin II. Cells were separated by their ability to migrate in response to a chemoattractant stimulus. Northern analysis of mRNA, [³⁵S]methionine incorporation into immunoprecipitated gelsolin, immunoblots of cell lysates, and quantitative confocal microscopy showed 1.4-2-fold higher levels of gelsolin in nonmigrant compared with migrant cells. Because the concentration of intracellular gelsolin did not appear to be a central determinant of cell migration, we assessed its requirement for motility. Cells that were electroinjected with a gelsolin antibody that inhibits actin severing by gelsolin in vitro showed a 72% reduction of the number of migrant cells compared with controls treated with an irrelevant antibody. Cells that were electroinjected with free gelsolin exhibited a 33% increase in migration compared with cells electroinjected with bovine serum albumin. Compared with nonmigrant cells, migrant cells contained abundant free gelsolin and exhibited gelsolin-dependent F-actin severing activity, which required Ca²⁺. Serum stimulation of cell migration required increases in [Ca²⁺]_i because incubation with 3 μM 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)-ester blocked calcium flux and cell migration. Serum also stimulated the recruitment of gelsolin into the supernatants of lysates from migrant but not from nonmigrant cells. In fibroblasts, gelsolin concentration alone does not apparently determine migratory capacity. Instead, the Ca²⁺-dependent actin severing activity of free gelsolin appears to be a major determinant of cell migration.

Footnotes

↵* This work was supported in part by a Group Grant from the Medical Research Council of Canada (to C. A. G. M.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviation used are:

PBS

phosphate-buffered saline

BAPTA/AM

1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)-ester

FBS

fetal bovine serum

BSA

bovine serum albumin

SMA

smooth muscle actin.
- Received May 16, 1996.
- Revision received June 11, 1996.