Monitoring Autophagy in Muscle Stem Cells

Methods Mol Biol. 2017;1556:255-280. doi: 10.1007/978-1-4939-6771-1_14.

Abstract

Autophagy is critical not only for the cell's adaptive response to starvation but also for cellular homeostasis, by acting as quality-control machinery for cytoplasmic components. This basal autophagic activity is particularly needed in postmitotic cells for survival maintenance. Recently, basal autophagic activity was reported in skeletal muscle stem cells (satellite cells) in their dormant quiescent state. Satellite cells are responsible for growth as well as for regeneration of muscle in response to stresses such as injury or disease. In the absence of stress, quiescence is the stem cell state of these cells throughout life, although which mechanisms maintain long-life quiescence remains largely unknown. Our recent findings showed that autophagy is necessary for quiescence maintenance in satellite cells and for retention of their regenerative functions. Importantly, damaged organelles and proteins accumulated in these cells with aging and this was connected to age-associated defective autophagy. Refueling of autophagy through genetic and pharmacological strategies restored aged satellite cell functions, and these finding have biomedical implications. In this chapter, we describe different experimental strategies to evaluate autophagic activity in satellite cells in resting muscle of mice. They should facilitate our competence to investigate stem cell functions both during tissue homeostasis as in pathological conditions.

Keywords: Aging; Autophagy; Quiescence; Satellite cell; Skeletal muscle; Stem cell.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagy* / drug effects
  • Autophagy* / genetics
  • Biological Assay / methods*
  • Cell Separation / methods
  • Cellular Senescence
  • Enzyme Inhibitors / pharmacology
  • Flow Cytometry / methods
  • Fluorescent Antibody Technique
  • Gene Expression
  • Gene Expression Profiling
  • Genes, Reporter
  • Macrolides / pharmacology
  • Mice
  • Mice, Transgenic
  • Microscopy, Fluorescence
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism
  • Muscle, Skeletal / cytology*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Resting Phase, Cell Cycle / genetics
  • Satellite Cells, Skeletal Muscle / cytology
  • Satellite Cells, Skeletal Muscle / metabolism
  • Stem Cells / cytology*
  • Stem Cells / drug effects
  • Stem Cells / metabolism*
  • Stem Cells / ultrastructure
  • Transcriptome

Substances

  • Enzyme Inhibitors
  • Macrolides
  • Microtubule-Associated Proteins
  • Recombinant Fusion Proteins
  • bafilomycin A