Identification, characterization, and intracellular distribution of cofilin in Dictyostelium discoideum.
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.
Aizawa
H
H
Department of Cell Biology, Tokyo Metropolitan Institute of Medical Science, Japan.
Sutoh
K
K
Tsubuki
S
S
Kawashima
S
S
Ishii
A
A
Yahara
I
I
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004023:Dictyostelium / Q000737:chemistry / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005656:Fungal Proteins / Q000302:isolation & purification; D005800:Genes, Fungal; D008840:Microfilament Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013172:Spores, Fungal
D051339:Actin Depolymerizing Factors; D000199:Actins; D017931:DNA Primers; D005656:Fungal Proteins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D004121:Dimethyl Sulfoxide