Enrichment and analysis of secretory lysosomes from lymphocyte populations.
BACKGROUND
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. 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.


RESULTS
Based 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).


CONCLUSIONS
The 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.
Schmidt
Hendrik
H
Molecular Immunology, Institute of Immunology, University Hospital Schleswig-Holstein Campus Kiel, Kiel, Germany. schmidt@immunologie.uni-kiel.de
Gelhaus
Christoph
C
Lucius
Ralph
R
Nebendahl
Melanie
M
Leippe
Matthias
M
Janssen
Ottmar
O
D002458:Cell Fractionation / Q000379:methods*; D053222:Fas Ligand Protein / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008214:Lymphocytes / Q000276:immunology* / Q000648:ultrastructure; D051907:Lysosomal Membrane Proteins / Q000276:immunology / Q000378:metabolism; D008247:Lysosomes / Q000276:immunology* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D009363:Neoplasm Proteins / Q000276:immunology / Q000378:metabolism; D013347:Subcellular Fractions / Q000276:immunology / Q000648:ultrastructure; D059666:Lysosomal-Associated Membrane Protein 3
D053222:Fas Ligand Protein; D051907:Lysosomal Membrane Proteins; D009363:Neoplasm Proteins; C505661:FASLG protein, human; C114233:LAMP3 protein, human; D059666:Lysosomal-Associated Membrane Protein 3