Abstract
Desmin is the main intermediate filament (IF) protein of muscle cells. In skeletal muscle, desmin IFs form a scaffold that interconnects the entire contractile apparatus with the subsarcolemmal cytoskeleton and cytoplasmic organelles. The interaction between desmin and the sarcolemma is mediated by a number of membrane proteins, many of which are Ca2+-sensitive. In the present study, we analyzed the effects of the Ca2+ chelator EGTA (1.75 mM) on the expression and distribution of desmin in C2C12 myoblasts grown in culture. We used indirect immunofluorescence microscopy and reverse transcription polymerase chain reaction (RT-PCR) to analyze desmin distribution and expression in C2C12 cells grown in the presence or absence of EGTA. Control C2C12 myoblasts showed a well-spread morphology after a few hours in culture and became bipolar when grown for 24 h in the presence of EGTA. Control C2C12 cells showed a dense network of desmin from the perinuclear region to the cell periphery, whereas EGTA-treated cells showed desmin aggregates in the cytoplasm. RT-PCR analysis revealed a down-regulation of desmin expression in EGTA-treated C2C12 cells compared to untreated cells. The present results suggest that extracellular Ca2+ availability plays a role in the regulation of desmin expression and in the spatial distribution of desmin IFs in myoblasts, and is involved in the generation and maintenance of myoblast cell shape.
Highlights
Desmin (53 kDa) is the main intermediate filament protein of striated muscle cells
To better understand the role of extracellular Ca2+ in the expression and distribution of the muscle-specific protein desmin, skeletal muscle C2C12 cells were grown in the presence or in the absence of the Ca2+chelator EGTA (1.75 mM) and observed by phase contrast microscopy
C2C12 cells grown in normal medium without EGTA were well spread (Figure 1A), whereas the majority of C2C12 cells grown in medium with EGTA for 24 h drastically changed to a bipolar morphology (Figure 1B)
Summary
Desmin (53 kDa) is the main intermediate filament protein of striated muscle cells. This abundant protein polymerizes to form an extensive network of 10-nm intermediate filaments that span the nuclear envelope to the sarcolemma and link myofibrils together in a three-dimensional scaffold around Z discs, forming an extrasarcomeric component of the muscle cytoskeleton [1]. The association of desmin with the sarcolemma is supposed to be dependent on the versatile cytoskeletal linker protein plectin [5], which is a very large size polypeptide (>500 kDa) abundant in the subsarcolemmal region. Variations in extracellular Ca2+ concentration are one of the regulatory mechanisms involved in the activation of these membrane complexes through conformational changes [6,7]. One of the evolutionary advantages of Ca2+ ions as regulators of protein structure and function is the possibility of rapid changes in their concentration [8]
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