Abstract
BackgroundIn specialized cells, such as mast cells, macrophages, T lymphocytes and Natural Killer cells in the immune system and for instance melanocytes in the skin, secretory lysosomes (SL) have evolved as bifunctional organelles that combine degradative and secretory properties. Mutations in lysosomal storage, transport or sorting molecules are associated with severe immunodeficiencies, autoimmunity and (partial) albinism. In order to analyze the function and content of secretory lysosomes in different cell populations, an efficient enrichment of these organelles is mandatory.ResultsBased on a combination of differential and density gradient centrifugation steps, we provide a protocol to enrich intact SL from expanded hematopoietic cells, here T lymphocytes and Natural Killer cells. Individual fractions were initially characterized by Western blotting using antibodies against an array of marker proteins for intracellular compartments. As indicated by the presence of LAMP-3 (CD63) and FasL (CD178), we obtained a selective enrichment of SL in one of the resulting organelle fractions. The robustness and reproducibility of the applied separation protocol was examined by a high-resolution proteome analysis of individual SL preparations of different donors by 2D difference gel electrophoresis (2D-DIGE).ConclusionThe provided protocol is readily applicable to enrich and isolate intact secretory vesicles from individual cell populations. It can be used to compare SL of normal and transformed cell lines or primary cell populations from healthy donors and patients with lysosomal storage or transport diseases, or from corresponding mutant mice. A subsequent proteome analysis allows the characterization of molecules involved in lysosomal maturation and cytotoxic effector function at high-resolution.
Highlights
In specialized cells, such as mast cells, macrophages, T lymphocytes and Natural Killer cells in the immune system and for instance melanocytes in the skin, secretory lysosomes (SL) have evolved as bifunctional organelles that combine degradative and secretory properties
In various hematopoietic cell lineages associated with secretory effector functions (CTL, Natural Killer (NK), mast cells and macrophages), a subset of lysosomes gained a second function as a storage and transport vehicle for effector molecules [2,3]
We provided a detailed analysis of the proteome of enriched SL from different NK cell lines and primary NK cells [32]
Summary
In specialized cells, such as mast cells, macrophages, T lymphocytes and Natural Killer cells in the immune system and for instance melanocytes in the skin, secretory lysosomes (SL) have evolved as bifunctional organelles that combine degradative and secretory properties. Cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells deliver their cytolytic effector molecules via so-called secretory lysosomes (SL). These organelles contain a number of cytotoxic proteins and undergo regulated secretion in response to external stimuli, for example to (page number not for citation purposes). In various hematopoietic cell lineages associated with secretory effector functions (CTL, NK, mast cells and macrophages), a subset of lysosomes gained a second function as a storage and transport vehicle for effector molecules [2,3]. FasL might participate in connecting SL to the actin-cytoskeleton via its interaction with Nck and associated actin-regulatory proteins [10,11,12]
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