Caldesmon exhibits a clustered distribution along individual chicken gizzard native thin filaments.
Our earlier immuno-gold electron microscopic study indicated that the distribution of caldesmon (CaD) on actin filaments is not uniform and is restricted to the vicinity of the myosin filaments (Mabuchi K, Li Y, Tao T, Wang CLA (1996) J Muscle Res Cell Motil 17: 243). This suggested that CaD could effectively inhibit muscle contraction, if those actin filaments in the vicinity of myosin filaments were saturated with CaD. In the present study we further examined the distribution of CaD along isolated, crude and purified native thin filaments (NTF). Individual CaD molecules on purified NTF were visualized with the aid of a chemical crosslinker, 5,5'-dithiobis(2-nitrobenzoic acid), which efficiently crosslinks CaD to actin (Graceffa P, Adam LP, Lehman W (1993) Biochem J294: 63), and of a monoclonal anti-CaD antibody. The results indicated that individual NTF had alternating CaD-rich and CaD-deficient regions. Moreover, we found that the N-termini of all CaD molecules in a given cluster appeared on the same side of an actin filament. Electron microscopic images of crude NTF immunoprecipitated by a polyclonal antibody clearly indicated that the spacing between the CaD clusters is wide enough for myosin heads to interact with actin subunits. Similar clustering of CaD was also observed in plastic embedded tissue sections. These observations raise the possibility that CaD is not acting as a simple on/off switch, but more likely as a modulator, of smooth muscle contraction.
Mabuchi
K
K
Muscle and Motility Group, Boston Biomedical Research Institute, Watertown, MA 02472, USA.
Li
Y
Y
Carlos
A
A
Wang
C L
CL
Graceffa
P
P
D008841:Actin Cytoskeleton / Q000737:chemistry* / Q000648:ultrastructure; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D002148:Calmodulin-Binding Proteins / Q000032:analysis* / Q000276:immunology / Q000502:physiology; D002645:Chickens; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003553:Cystine / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian / Q000737:chemistry* / Q000648:ultrastructure; D006801:Humans; D007150:Immunohistochemistry; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D009119:Muscle Contraction; D016624:Negative Staining; D025941:Protein Interaction Mapping; D011817:Rabbits; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D002148:Calmodulin-Binding Proteins; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D013439:Sulfhydryl Reagents; D003553:Cystine; D004228:Dithionitrobenzoic Acid