Enrichment and analysis of secretory lysosomes from lymphocyte populations

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.

The adapter protein Nck: Role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes

“…Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coat preparations of healthy donors by Ficoll density gradient centrifugation and PHA blasts were generated and cultivated as described previously. 33 The human CD4 þ T cell clone 12603 was established in our laboratory and maintained as described before. 34 293T cells (human embryonic kidney, SV40 transformed) were used for transient protein expression and cultured in DMEM medium (Invitrogen) supplemented with 1% (v/v) L-glutamine (Biochrom) and antibiotics.…”

“…33 Gel pieces were washed twice with HPLC grade water (Roth, Karlsruhe, Germany) and dehydrated in a first step with 25 mM ammonium bicarbonate (ABC) in 50% acetonitrile (ACN) and shrunk in pure ACN as a second step. The dry gel pieces were then rehydrated with 100 ng sequencing-grade trypsin (Serva, Heidelberg, Germany) in 10 lL of 5 mM ABC.…”

Growth of Murine Splenic Tissue Is Suppressed by Lymphotoxin β-Receptor Signaling (LTβR) Originating from Splenic and Non-Splenic Tissues

“…The tryptic digestion and mass spectrometric analysis was performed as described before [41]. In brief, after washing gel pieces in 25 mM ammonium bicarbonate (ABC) and shrinking them in pure ACN, they were rehydrated with 100 ng porcine trypsin (Serva, Heidelberg, Germany) in 10 μl of 5 mM ABC.…”

“…Western blotting was performed on 4–12% gradient Bis-Tris gels (Invitrogen, Karlsruhe, Germany) using 10 μg of splenic stroma lysates [41]. After electrophoresis proteins were transferred to 0.2 μm nitrocellulose membranes (Biometra, Goettingen, Germany) and subsequently blocked with bovine serum albumin in TBST (TBS + 0.05% Tween).…”

Control of death receptor ligand activity by posttranslational modifications

“…However, later work by Kassahn et al showed that ectopically-expressed FasL in Jurkat cells was rarely found in the same granules as CD63 and CD107a, both markers of secretory lysosomes [57], and the same pattern was also described for a murine CTL clone [49]. Indeed, a recent paper from Schmidt et al [58] revealed that, upon extensive fractioning of the cytolytic granules, FasL was enriched in a different fraction than perforin, granzymes, and other conventional secretory lysosomes markers. They speculated that distinct secretory lysosomes subtypes may form from an initial multivesicular complex, resulting in two independent compartments.…”