Isolation of functional lysosomes from skeletal muscle

Abstract

Lysosomes are membrane-bound organelles responsible for the degradation of damaged or dysfunctional cellular components, including mitochondria. Their acidic internal environment and the presence of an array of hydrolytic enzymes facilitate the efficient breakdown of macromolecules such as proteins, lipids, and nucleic acids. Mitochondria play a critical role in maintaining skeletal muscle homeostasis to meet the energy demands under physiological and pathological conditions. Mitochondrial quality control within skeletal muscle during processes such as exercise, disuse, and injury is regulated by mitophagy, where dysfunctional mitochondria are targeted for lysosomal degradation. The limited understanding of quality control mechanisms in skeletal muscle necessitates the need for isolating intact lysosomes to assess organelle integrity and the degradative functions of hydrolytic enzymes. Although several methods exist for lysosome isolation, the complex structure of skeletal muscle makes it challenging to obtain relatively pure and functional lysosomes due to the high abundance of contractile proteins. Here we describe a method to isolate functional lysosomes from small amounts of mouse skeletal muscle tissue, preserving membrane integrity. We also describe functional assays that allow direct evaluation of lysosomal enzymatic activity and we provide data indicating reduced lysosomal degradative activity in lysosomes from aging muscle. We hope that this protocol provides a valuable tool to advance our understanding of lysosomal biology in skeletal muscle, supporting investigations into lysosome-related dysfunction in aging, disease, and exercise adaptations. New and NoteworthyLysosomes within skeletal muscle function to degrade dysfunctional debris and initiate retrograde signaling pathways. We developed a method to isolate purified lysosomal fractions using a small portion of skeletal muscle, eliminating the need for density gradients or lysosome-modifying agents, ensuring high lysosomal purity without compromising structure or function. By enabling functional analysis via acid phosphatase, cathepsin-B activity, and calcium release, this approach offers a powerful tool to study lysosomal roles in muscle physiology, disease, and exercise. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/675181v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@f41e09org.highwire.dtl.DTLVardef@e013a0org.highwire.dtl.DTLVardef@12871edorg.highwire.dtl.DTLVardef@4f96cb_HPS_FORMAT_FIGEXP M_FIG C_FIG