Abstract
The myotendinous junction is a specialized structure of the muscle fibre enriched in mechanosensing complexes, including costameric proteins and core elements of the z-disc. Here, laser capture microdissection was applied to purify membrane regions from the myotendinous junctions of mouse skeletal muscles, which were then processed for proteomic analysis. Sarcolemma sections from the longitudinal axis of the muscle fibre were used as control for the specificity of the junctional preparation. Gene ontology term analysis of the combined lists indicated a statistically significant enrichment in membrane-associated proteins. The myotendinous junction preparation contained previously uncharacterized proteins, a number of z-disc costameric ligands (e.g., actinins, capZ, αB cristallin, filamin C, cypher, calsarcin, desmin, FHL1, telethonin, nebulin, titin and an enigma-like protein) and other proposed players of sarcomeric stretch sensing and signalling, such as myotilin and the three myomesin homologs. A subset were confirmed by immunofluorescence analysis as enriched at the myotendinous junction, suggesting that laser capture microdissection from muscle sections is a valid approach to identify novel myotendinous junction players potentially involved in mechanotransduction pathways.
Highlights
Laser-assisted cell microdissection in combination with laser-pressure catapulting has been exploited for over a decade to isolate pure population of cells, specific regions from tissue sections or even single chromosomes [1,2,3,4]
The sarcomeric z-disc has emerged as a plausible structure that mediates adaptive responses to mechanical stresses
800 cuts were collected from the myotendinous junction and a similar amount from the extra junctional membrane regions, hereafter referred to as samples MTJ and M, respectively
Summary
Laser-assisted cell microdissection in combination with laser-pressure catapulting (commonly referred to as laser capture microdissection or LCM) has been exploited for over a decade to isolate pure population of cells, specific regions from tissue sections or even single chromosomes [1,2,3,4]. LCM has been applied to identify the unique expression profiles of specialized regions within complex cells. In an attempt to identify candidate proteins for mechanotransduction processes, we decided to focus on the myotendinous junction (MTJ). Actin filaments bundled with alphaactinin and desmin project from the electrodense terminal sarcomeric z-discs [15] towards the sarcolemma. At the sarcolemma, they interact with the dystrophin-associated and the α7β1 integrin protein complexes, which in turn connect with the extracellular matrix through the basal lamina protein laminin, following a similar arrangement to the costamere [16,17,18,19,20]. The myotendinous junction is enriched in costameric proteins and core elements of the z-disc
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