Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods.
Leishmania major is a human-infective protozoan parasite transmitted by the bite of the female phlebotomine sand fly. The L. major hydrophilic acylated surface protein B (HASPB) is only expressed in infective parasite stages suggesting a role in parasite virulence. HASPB is a "nonclassically" secreted protein that lacks a conventional signal peptide, reaching the cell surface by an alternative route to the classical ER-Golgi pathway. Instead HASPB trafficking to and exposure on the parasite plasma membrane requires dual N-terminal acylation. Here, we use live cell imaging methods to further explore this pathway allowing visualization of key events in real time at the individual cell level. These methods include live cell imaging using fluorescent reporters to determine the subcellular localization of wild type and acylation site mutation HASPB18-GFP fusion proteins, fluorescence recovery after photobleaching (FRAP) to analyze the dynamics of HASPB in live cells, and live antibody staining to detect surface exposure of HASPB by confocal microscopy.
Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK. l.m.maclean@dundee.ac.uk.
Lorna
L
MacLean
Centre for Applied Entomology and Parasitology, School of Life Sciences, Keele University, Keele, Staffordshire, ST5 5BG, UK.
Helen
H
Price
Technology Facility, Department of Biology, University of York, York, YO10 5DD, UK.
Peter
P
O'Toole
D000215:Acylation; D000953:Antigens, Protozoan / Q000378:metabolism*; D017930:Genes, Reporter; D007891:Leishmania / Q000378:metabolism*; D018320:Leishmania major / Q000378:metabolism; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging; D021381:Protein Transport; D015800:Protozoan Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D055571:Secretory Pathway
D000953:Antigens, Protozoan; C401884:HASPB protein, Leishmania; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins
Acylation; FRAP (fluorescence recovery after photobleaching); HASPB (hydrophilic acylated surface protein B); Leishmania; Live cell imaging; Nonclassical protein secretion