Caldesmon is necessary for maintaining the actin and intermediate filaments in cultured bladder smooth muscle cells

Abstract

Caldesmon (CaD), a component of microfilaments in all cells and thin filaments in smooth muscle cells, is known to bind to actin, tropomyosin, calmodulin, and myosin and to inhibit actin-activated ATP hydrolysis by smooth muscle myosin. Thus, it is believed to regulate smooth muscle contraction, cell motility and the cytoskeletal structure. Using bladder smooth muscle cell cultures and RNA interference (RNAi) technique, we show that the organization of actin into microfilaments in the cytoskeleton is diminished by siRNA-mediated CaD silencing. CaD silencing significantly decreased the amount of polymerized actin (F-actin), but the expression of actin was not altered. Additionally, we find that CaD is associated with 10 nm intermediate-sized filaments (IF) and in vitro binding assay reveals that it binds to vimentin and desmin proteins. Assembly of vimentin and desmin into IF is also affected by CaD silencing, although their expression is not significantly altered when CaD is silenced. Electronmicroscopic analyses of the siRNA-treated cells showed the presence of myosin filaments and a few surrounding actin filaments, but the distribution of microfilament bundles was sparse. Interestingly, the decrease in CaD expression had no effect on tubulin expression and distribution of microtubules in these cells. These results demonstrate that CaD is necessary for the maintenance of actin microfilaments and intermediate-sized filaments in the cytoskeletal structure. This finding raises the possibility that the cytoskeletal structure in smooth muscle is affected when CaD expression is altered, as in smooth muscle de-differentiation and hypertrophy seen in certain pathological conditions.

Amino acid mutations in the caldesmon COOH-terminal functional domain increase force generation in bladder smooth muscle

“…Lack of homozygous mice with the knockin constructs indirectly shows that the CaD COOH-terminal functional domain is critical for embryonic development. This is not surprising, considering the importance of CaD in actin filament assembly in test tube experiments (8) and the lack of actin-containing microfilaments and intermediate size filaments with down regulation of CaD expression via CaD small interfering RNA (10). Cytoplasmic intermediate size filaments form a dense filament network radiating from the nucleus and extending to the plasma membrane (11).…”

Actin‐dependent dynamics of keratin filament precursors

“…Litjens et al, 2003;Rezniczek et al, 2004], and certain plectin isoforms have been localized to the vicinity of focal adhesions [Rezniczek et al, 2003;Tian et al, 2006]. Another recently identified candidate to coordinate actin filaments and IFs is caldesmon [Deng et al, 2007]. Once KFPs approach the peripheral KF network they integrate end-on [ Fig.…”

The roles and regulation of the actin cytoskeleton, intermediate filaments and microtubules in smooth muscle cell migration

“…Second, caldesmon is a component of microfilaments in all cells and thin filaments in smooth muscle cells. Caldesmon is able to interact with intermediate filaments and polymerized actin, and is required for maintaining the intermediate filament network and actin filaments in smooth muscle cells [75]. Caldesmon phosphorylation by the serine/threonine protein kinase PFTAIRE1 promotes its binding to F-actin and stress fiber formation in motile cells [76].…”