Isolation of functional lysosomes from skeletal muscle
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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 Noteworthy
Lysosomes 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
Article activity feed
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Supplementary Fig. 1)
This data isn't linked anywhere.
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TRPML1 activity
This is a really nice result! Do you have a way to measure 'lysosome concentration' or do you just base everything off of normalized protein concentration? I'm wondering if there is a way to be quantitative about experiments like these from one batch to another.
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protein composition
It's really nice that this protocol is pretty agnostic to the source and doesn't require any modification of the natural biology!
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functional alterations.
How do you control for lysosomal quality here, or intactness from your isolation protocol?
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activity assay
For all of these assays, is the assumption that most of your lysosomes are intact? How do you then measure enzyme activity from the intact lysosomes? Do you assume that the substrates can all pass through the membranes?
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entrifuged at a higher speed to
Do you have details on what speed and for how long?
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This step was repeated two more times
This is a little unclear. Which step was repeated two more times? Just homogenization?
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