Nonspecific Binding Correction for Single-Cell Mass Cytometric Analysis of Autophagy and Myoblast Differentiation.

Abstract

Satellite cells provide regenerative capacity to the skeletal muscle after injury. In this process, termed myogenesis, satellite cells get activated, proliferate, and differentiate. Myogenesis is recapitulated in the tissue culture of myoblasts that differentiate by fusion and then by the formation of myotubes. Autophagy plays an important role in myogenesis, but the asynchronous and unique trajectory of differentiation of each myoblast along the myogenic lineage complicates teasing apart at what stages of differentiation autophagy plays a critical role. In this report, we describe a mass cytometric, multidimensional, individual cell analysis of differentiating myoblasts that characterizes autophagy flux (i.e., autophagy rate) at separate myogenesis stages. Because mass cytometry uses a set of lanthanide-tagged antibodies, each being specific for a desired molecular target, quantification of each molecular target could be exaggerated by nonspecific binding of its respective antibody to other nontarget cellular regions. In this report, we used lanthanide-tagged isotypes, which allowed for correction for nonspecific binding at the single-cell level. Using this approach, myoblasts were phenotypically identified by their position in the myogenic lineage, simultaneously with the quantification of autophagic flux in each identified subset. We found that generally autophagy flux is upregulated specifically during myoblast fusion and declines in myotubes. We also observed that mitophagy (i.e., selective autophagic degradation of mitochondria) is also active after myotube formation. The ability to track different types of autophagy is another feature of this methodology, which could be key to expand the current understanding of autophagy regulation in regenerating the skeletal muscle.