Abstract
Phospholipase D3 (PLD3) is a non-classical, poorly characterized member of the PLD superfamily of signaling enzymes. PLD3 is a type II glycoprotein associated with the endoplasmic reticulum, is expressed in a wide range of tissues and cells, and undergoes dramatic upregulation in neurons and muscle cells during differentiation. Using an in vitro skeletal muscle differentiation system, we define the ER-tethering mechanism and report that increased PLD3 expression enhances myotube formation, whereas a putatively dominant-negative PLD3 mutant isoform reduces myotube formation. ER stress, which also enhances myotube formation, is shown here to increase PLD3 expression levels. PLD3 protein was observed to localize to a restricted set of subcellular membrane sites in myotubes that may derive from or constitute a subdomain of the endoplasmic reticulum. These findings suggest that PLD3 plays a role in myogenesis during myotube formation, potentially in the events surrounding ER reorganization.
Highlights
Myogenesis is a highly complex, multi-tiered, cell-cell fusion event
A prior microarray study had reported that Phospholipase D3 (PLD3) mRNA expression increases in C2C12 myoblasts during differentiation [7], which we confirmed in preliminary studies by performing qRT-PCR on RNA collected from differentiating C2C12 myoblasts (Fig. 1A)
Whole cell lysates were collected from C2C12 myoblasts and analyzed by western blot analysis, which revealed that PLD3 protein expression levels increased along with mRNA expression during C2C12 myoblast differentiation (Fig. 1B)
Summary
The immature muscle cells, proliferate until they receive intra- and extra-cellular cues to withdraw from the cell cycle, initiate muscle-specific gene expression and fuse together to form nascent myotubes. These nascent myotubes continue to increase in size and nuclei number by fusing to more myoblasts until they eventually form mature myofibers [1]. Myoblast/myotubes make significant changes to their membrane systems to form muscle-specific organelles. The plasma membrane, called the sarcolemma, forms specialized invaginations called transverse (T) tubules, an extensive membrane system with lipid and protein compositions distinct from the sarcolemma [6]. The T-tubules function to transfer action potentials to the SR, eventually leading to muscle contraction
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