Abstract
The enrichment of lysosomes is a useful way to study their structure and function. These dynamic vesicles can be enriched from cell cultures in a variety of ways including immunoprecipitation and fluorescence-activated organelle sorting. These methods are extremely precise but often require the transfection and expression of an affinity or fluorophore-tagged lysosomal membrane protein. A simpler approach uses differential density of subcellular organelles, which are characteristic to a particular type of organelle. Separation of organelles along a density-gradient enables fractionation to enrich for specific organelles (such as lysosomes) in their native state. This protocol outlines an optimized method for enriching lysosomes from HeLa cells with a continuous density-gradient that contains Percoll. Gentle cell lysis and extraction conditions yield dense-fractions that are enriched with functional and intact lysosomes, which can be assayed in downstream analyses. This method is quick (conducted in less than 2 h after harvesting cells), and can be easily scaled and optimized for other cell types.
Keywords: Lysosome, Organelle enrichment, HeLa, Density gradient, Subcellular fractionation
Background
The lysosomal system is the cell’s recycling center and is responsible for breaking down macromolecules such as proteins, carbohydrates and lipids. This system is responsible for quality control and is one of the cell’s front-line defenses against aging. As such, researching lysosomal function has found application in age-related diseases such as Alzheimer’s and Parkinson’s disease, which are both genetically associated with the lysosomal system (Gao et al., 2018), and display profound lysosomal system dysfunction (Whyte et al., 2017). The enrichment or purification of cellular organelles that are a part of the lysosomal system is a powerful way to study their structure and function. Enrichment of subcellular structures such as lysosomes and related organelles from cell cultures can be conducted in a variety of ways including immunoprecipitation, fluorescence-activated organelle sorting, and magnetic separation of organelles that contain superparamagnetic iron nanoparticles (Gauthier et al., 2008; Abu-Remaileh et al., 2017; Lloyd-Lewis et al., 2018). Whilst being extremely precise these methods often require the transfection and expression of an affinity or fluorophore-tagged lysosomal membrane protein (e.g., LAMP1, LAMP2 or TMEM192). Density-gradients offer an alternative simple approach that takes advantage of the different densities of specific organelles. The use of Percoll for the creation of density gradients with which cells can be fractionated and organelles enriched is an easy-to-use and well-established method (Pertoft et al., 1978). Separation of cell lysates along a density-gradient enables fractionation to enrich for specific organelles (such as lysosomes) in their native state. Percoll media itself is made of polydisperse silica particles that are coated in polyvinylpyrrolidone that have an average size in water of 35 nm. One of the main advantages of using Percoll above other media is that it has a low osmolality. Osmolality does not change with respect to position in a Percoll gradient. This contrasts with sucrose gradients, and even Ficoll (sucrose polymer) gradients that change in osmolality with respect to position in the density gradient (Pertoft, 2000).This protocol outlines an optimized method for enriching lysosomes from HeLa cells with a density-gradient that contains Percoll (summarized in Figure 1A). Gentle extraction conditions yield dense-fractions that are enriched with functional and intact lysosomes. These fractions can be measured for lysosomal proteins and lysosomal enzyme activity (Cui et al., 2019). This method is quick (i.e., can be performed in < 2 h after harvesting cells), and can be easily scaled for size and optimized for other cell types.
Materials and Reagents
Equipment
Procedure
Recipes
Prepare the following solutions and store at 4 °C until required. Note: For all solutions, prepare more than is required to allow for pipetting errors.
Acknowledgments
This work was supported by the Hopwood Centre for Neurobiology, a Research Training Stipend and a Commonwealth Scholarship by the Australian Government (awarded to J.M.C). This protocol was adapted and modified from Cui et al. (2019). J.M.C and K.J.H optimized and performed key experiments. J.M.C wrote the manuscript, analysed data and prepared all figures. J.M.C, K.J.H, R.D.T, Y.C, Z.Y and T.J.S critically reviewed the manuscript.
Competing interests
T.J.S and co-authors declare no conflicts of interest.
References
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