Isolation of mast cell secretory lysosomes using flow cytometry.
BACKGROUND
Mast cells are specialized secretory cells of the immune system. Through exocytosis of their secretory lysosomes and secretory granules, mast cells release biologically active substances such as histamine and proteases. Mast cell secretory granules have been studied extensively but much less attention has been given to secretory lysosomes. Studies on mast cell secretory lysosomes are limited by the lack of selective markers and the difficulty to isolate this organelle from conventional lysosomes. Our goal was to develop better tools to study secretory lysosomes.


METHODS
We engineered a rat mast cell line over expressing a rat mast cell protease (RMCP) tagged with a red fluorescent protein (RMCP-DsRed). We used single organelle flow analysis (SOFA) to detect fluorescently labeled secretory lysosomes. The labeled organelles were then sorted using the fluorescence-assisted organelle sorting (FAOS) method.


RESULTS
We show that the RMCP-DsRed fusion protein selectively localizes to the lysosomal compartment and is exocytosed upon activation, confirming its localization in secretory lysosomes. Lysosomal fractions from cells expressing the RMCP-DsRed fusion were analyzed by SOFA and a specific population of secretory lysosome was identified. Finally, we sorted secretory lysosomes and showed that the sorted material had a higher specific activity for the compartment marker hexosaminidase than a sample obtained by conventional methods.


CONCLUSIONS
Our work further demonstrates the usefulness of flow cytometry to study cellular organelles, and provides new tools to better understand the physiology of secretory lysosomes.
D000818:Animals; D045744:Cell Line, Tumor; D053818:Chymases; D003594:Cytoplasmic Granules; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006596:Hexosaminidases / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism*; D008407:Mast Cells / Q000201:enzymology / Q000378:metabolism*; D018613:Microscopy, Confocal; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors; D000097573:Red Fluorescent Protein
D053818:Chymases; D006596:Hexosaminidases; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D000097573:Red Fluorescent Protein