Identification, Characterization, and Intracellular Distribution of Cofilin in Dictyostelium discoideum

Hiroyuki Aizawa,Kazuo Sutoh,Satoshi Tsubuki,Seiichi Kawashima,Ai Ishii,Ichiro Yahara

doi:10.1074/jbc.270.18.10923

Abstract

We identified and purified an actin monomer-binding protein of apparent molecular weight of 15,000 from Dictyostelium discoideum. The 15-kDa protein depolymerized actin filaments in a pH-dependent manner. The protein also had an activity to decrease apparent viscosity of actin solutions in a dose-dependent manner. This activity was inhibited by phosphatidyl inositides. Molecular cloning of genes encoding this protein revealed that the protein is 42% identical in its primary sequence to yeast cofilin. We concluded that the 15-kDa protein is cofilin of this organism. D. discoideum cells contain two cofilin genes (DCOF1 and DCOF2) whose nucleotide sequences were entirely identical in their exsons while the promoter and intron regions were different. Promoter assay experiments revealed that DCOF1 is expressed both in vegetative and differentiating cells and that DCOF2 is not expressed under any conditions examined. Gene disruption experiments suggested that DCOF1 might be essential for the proliferation of D. discoideum cells whereas the disruption of DCOF2 was proven not to alter any phenotypes. Indirect immunofluorescence microscopic observations showed that cofilin is distributed diffusely throughout cytoplasm in vegetative cells. In flattened cells under starvation stress, cofilin localized at dramatically reorganizing actin-cytoskeletons in ruffling membranes of the leading edge, but not at rigid actin meshwork in focal adhesion plaques. These results suggest that cofilin may be involved in dynamic reorganization of membranous actin cytoskeletons.

Highlights

II To whom correspondence should be addressed: Dept. of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo 113, Japan
Identification and Purification ofan Actin Monomer-binding Protein with M; 15,000-8ince the cofilin family was first identified biochemically as an actin monomer-binding protein using DNase I affinity chromatography, we sought for actin monomer binding proteins in cell extracts of D. discoideum as described under "Experimental Procedures" (Fig. lA)
We described identification, biochemical characterization, primary structure, gene structure, and intracellular localization of D. discoideum cofilin

Summary

Introduction

The expression of mammalian cofilin or destrin rescued yeast cells from the lethality (Iida et al, 1993). DictyosteZium discoideum exhibits a number of cellular processes depending upon dynamic movements of actin filaments in various stages of spore formation induced under starvation stress. Those include cytokinesis, phagocytosis, intracellular vesicular transport, ruffling membrane formation, substrate adhesion, pseudopod formation, chemotactic migration toward cyclic AMP, and cell-cell adhesion to form a multicellular slug (Loomis, 1982). This organism is a powerful laboratory system for investigation on the actin-based cellular processes. We have investigated dynamic behavior of cofilin during processes leading to spore formation

Methods

Results

Conclusion