Acidic Compartment Size, Positioning, and Function during Myogenesis and Their Modulation by the Wnt/Beta-Catenin Pathway

Abstract

Lysosomes and acidic compartments are involved in breaking down of macromolecules, membrane recycling, and regulation of signaling pathways. Here, we analyzed the role of acidic compartments during muscle differentiation and the involvement of the Wnt/beta-catenin pathway in lysosomal function during myogenesis. Acridine orange was used to localize and quantify acidic cellular compartments in primary cultures of embryonic muscle cells from Gallus gallus. Our results show an increase in acidic compartment size and area, as well as changes in their positioning during the initial steps of myogenesis. The inhibition of lysosomal function by either the chloroquine Lys05 or the downregulation of LAMP-2 with siRNA impaired chick myogenesis, by inhibiting myoblast fusion. Two activators of the Wnt/beta-catenin pathway, BIO and Wnt3a, were able to rescue the inhibitory effects of Lys05 in myogenesis. These results suggest a new role for the Wnt/beta-catenin pathway in the regulation of acidic compartment size, positioning, and function in muscle cells.

The Role of Embryonic Chick Muscle Cell Culture in the Study of Skeletal Myogenesis

“…The meaning of these nuclear movements is still elusive during skeletal myogenesis, but the perinuclear region is becoming a focus of studies because of its highly dynamic and specialized concentration of molecules. The juxtanuclear area of muscle cells preferentially harbor specific organelles, such as centrosomes and lysosomes, as well as a number of structural and signaling proteins, such as desmin, Gli-1, and Lmo7 (Figure 1; Mermelstein et al, 2006;Possidonio et al, 2016;Teixeira et al, 2018;Bagri et al, 2020). Interestingly, at the end of skeletal muscle differentiation in primary chick muscle cell cultures, a single myotube may exhibit hundreds of nuclei within its cytoplasm (Figure 3), whereas myotubes formed in cultures of widely used skeletal muscle cell lines, such as mouse C2C12, rat L6 and L8 cells, and human CL25, are usually composed by only a few (2-20) nuclei.…”

“…The characterization and quantification of different aspects of skeletal myogenesis is relatively simple to achieve in culture of chick myoblasts ( Abrunhosa et al, 2014 ; Bastos et al, 2019 ; Bagri et al, 2020 ). Among them, information regarding the number of mononucleated myoblasts and multinucleated myotubes, as well as fibroblasts, the number of nuclei within each myotube, area of muscle cells, and size of myotubes can be acquired from image processing tools, such as ImageJ ( Schindelin et al, 2012 ).…”

“…The meaning of these nuclear movements is still elusive during skeletal myogenesis, but the perinuclear region is becoming a focus of studies because of its highly dynamic and specialized concentration of molecules. The juxtanuclear area of muscle cells preferentially harbor specific organelles, such as centrosomes and lysosomes, as well as a number of structural and signaling proteins, such as desmin, Gli-1, and Lmo7 ( Figure 1 ; Mermelstein et al, 2006 ; Possidonio et al, 2016 ; Teixeira et al, 2018 ; Bagri et al, 2020 ).…”

“…Importantly, chick skeletal muscle cell cultures have some disadvantages, such as the lack of widespread genetic manipulations, which are cutting edge approaches in other animal models of myogenesis, such as the fruit fly Drosophila ( Bryantsev et al, 2019 ). Nevertheless, the genetic approach weakness of the chick muscle culture model has been, in part, surpassed by some recent advances in the transfection of chick cultured cells with siRNA targeted to specific proteins and fluorescent/luminescent protein reporter systems ( Mermelstein et al, 2007 ; Possidonio et al, 2016 ; Yamashita et al, 2017 ; Bagri et al, 2020 ; de Andrade Rosa et al, 2020 ).…”

“…This trend is probably related to the tweaking of new molecular and cellular biology techniques to be used in chick cell cultures. Among them, we can cite tools to study gene function through cell transfection, such as plasmids for reporter-based assays (such as TOP-Flash assay for the Wnt/beta-catenin pathway), gene overexpression, siRNA-dependent knockdown, or CRISPR-based gene editing ( Mermelstein et al, 2007 ; Possidonio et al, 2016 ; Yamashita et al, 2017 ; Bagri et al, 2020 ; de Andrade Rosa et al, 2020 ).…”