Identification and characterization of the nano-sized vesicles released by muscle cells

Abstract

Several cell types secrete small membranous vesicles that contain cell-specific collections of proteins, lipids, and genetic material. The function of these vesicles is to allow cell-to-cell signaling and the horizontal transfer of their cargo molecules. Here, we demonstrate that muscle cells secrete nano-sized vesicles and that their release increases during muscle differentiation. Analysis of these nanovesicles allowed us to characterize them as exosome-like particles and to define the potential role of the multifunctional protein Alix in their biogenesis.

Figure 1. Muscle cells release nanovesicles

A) Electron microscope analyses of 3D cultures of differentiating C2C12 cells show the presence of outward budding intermediates (nascent vesicles) at the plasma membrane with an average diameter of 80 nm (scale bar=100 nm). B) Transmission electron microscopy shows immunogold labeling of Alix in the nanovesicles from 3D cultures of differentiated C2C12 myotubes (10 nm gold particles). Left panel shows a nascent nanovesicle, right panel shows a released vesicle (PM=plasma membrane, ECM=extra-cellular matrix) (scale bar=100 nm).

Figure 2. Characterization of nanovesicles released by differentiated muscle cells

A) Electron-microscope analysis of whole-mounted vesicles purified from conditioned-media (CM) of C2C12 myotubes (DIII) (scale bar=100 nm). B) Equal amounts of total proteins (5 µg) extracted from the nanovesicle preparations were immunoblotted for Alix. Alix is mainly present in the nanovesicle preparation from differentiated muscle cells (DIII). Controls of the procedure show the purity of the nanovesicle preparation. Note, the 10,000 × g pellets (10k×g), containing cell debris and large microvesicles, were almost negative for Alix. C) Assessment of myogenic differentiation of C2C12 cells. (Top) Myoblasts (D0) and myotubes after two (DII), three (DIII) and six days (DVI) of differentiation. (Bottom) Western blots showing the expression of MyHC, myogenin, and MyoD in lysates of C2C12 cells after the indicated days of differentiation. Ponceau staining is shown as a control of the total protein loaded per lane. D) Characterization of the protein content of muscle-released nanovesicles showed the presence of proteins, indicated by gene symbol, that are identified more often in exosomes of different cell types.

Figure 3. Alix-depletion affects release and protein content of muscle-derived nanovesicles

A) Light microscopy analysis (top panel) and transmission electron microscopy (bottom panel) of 3D cultures of differentiated C2C12 cells (DIII) silenced for Alix showed an altered phenotype. B) Cytoplasmic (top) and nuclear (bottom) fractions from Mock- and Alix-silenced myotubes were immunoblotted for the indicated apoptotic markers. Ponceau staining is shown as a control of the total protein loaded per lane. C) Treatment of C2C12 myoblast with Alix-specific double stranded siRNA pools led to a significant reduction (≈80%) in Alix expression compared to mock-transfected cells. Nanovesicles released by Alix-silenced C2C12 cells exhibited a significant accumulation of Hsc70, enolase and actin, and a reduction of CD63.