Domain analysis of alpha-actinin reveals new aspects of its association with F-actin during cytokinesis.
alpha-Actinin is a rod-shaped actin cross-linking protein composed of actin binding domain, spectrin-like repeats of the central rod domain and the EF-hand domain. Cytokinesis in mammalian cells involves remodeling of equatorial actin filaments (F-actin) mediated by alpha-actinin. However, it remains unknown how alpha-actinin interacts with F-actin at the cleavage furrow. To address this question, we have conducted functional analysis of the mutant that either lacks the ability to cross-link F-actin (ABD) or to bind to F-actin (DeltaABD). We found that equatorial localization of alpha-actinin requires both its F-actin binding and cross-linking activities. Unexpectedly, we also found that overexpression of DeltaABD-GFP but not ABD-GFP frequently caused accelerated cytokinesis and ectopic furrowing similar to those observed in cells depleted of alpha-actinin. Immunofluorescence revealed that overexpression of DeltaABD-GFP caused displacement of endogenous alpha-actinin and a decrease in the density of F-actin throughout the entire cortex. Biochemical experiments showed that DeltaABD was able to form heterodimers with endogenous alpha-actinin. These results suggest that the central rod spectrin-like repeats of alpha-actinin is sufficient for its dimerization in vivo. Our findings uncover previously unappreciated functions of the alpha-actinin domains in a cell.
Low
Shyan Huey
SH
Temasek Life Sciences Laboratory, 1 Research Link, Singapore.
Mukhina
Svetlana
S
Srinivas
Vinayaka
V
Ng
Chang Zhi
CZ
Murata-Hori
Maki
M
D000185:Actinin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D048749:Cytokinesis / Q000502:physiology*; D003599:Cytoskeleton / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats
D000199:Actins; D000185:Actinin; D049452:Green Fluorescent Proteins