Disruption of the actin cytoskeleton in living nonmuscle cells by microinjection of antibodies to domain-3 of caldesmon.
Two classes of affinity-purified polyclonal antibodies directed against the four different domains of gizzard caldesmon were prepared and their specificity was verified by immunochemical assays. One set of antibodies recognized exclusively domain-3 spanning residues 483-578 and the other one included IgG reactive toward the remaining domains-1, -2 and -4 corresponding to residues 1-482 and 581-756. Microinjection into cultured fibroblasts of each antibody preparation was employed to probe the functional importance of the interaction between caldesmon and tropomyosin within living nonmuscle cells. Low concentrations of the antibody to domain-3 caused a rapid, severe and reversible disassembly of the microfilament network. In contrast, the antibodies to domains-1, -2 and -4 were ineffective. The effects of the anti-domain-3 IgG were observed not only with the purified antibody but also when present in the whole caldesmon antiserum serving for its isolation. The microfilament disintegrating activity of this antibody was completely abolished upon preincubation with native caldesmon or a proteolytic caldesmon fragment encompassing amino acids 483-578. In enzyme-linked immunosorbent assay competition experiments, tropomyosin, but not F-actin, significantly decreased the binding of the domain-3 antibody to caldesmon. Consistent with recent mutational studies pinpointing to the contribution of domain-3 in the in vitro binding of cellular caldesmon to tropomyosin, the findings suggest that the microinjected domain-3 antibody selectively disrupts the association of tropomyosin with caldesmon domain-3, thereby destabilizing the protein complex and alleviating its known protective action against F-actin severing in the cell. Thus, our data further highlight the in vivo involvement of this particular domain in the attachment of non-muscle caldesmon to tropomyosin as well as the direct participation of the caldesmon-tropomyosin complex in the cytoskeletal organization of the microfilaments.
Lamb
N J
NJ
Centre de Recherches de Biochimie Macromoléculaire du CNRS, INSERM U 249, Université de Montpellier I, France.
Fernandez
A
A
Mezgueldi
M
M
Labbé
J P
JP
Kassab
R
R
Fattoum
A
A
D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000199:Actins / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D000070559:Gizzard, Non-avian / Q000737:chemistry; D008845:Microinjections; D011817:Rabbits; D051381:Rats; D014335:Tropomyosin / Q000187:drug effects / Q000378:metabolism
D000199:Actins; D000906:Antibodies; D002148:Calmodulin-Binding Proteins; D014335:Tropomyosin