Unconventional Protein Secretion pp 191-203

Part of the Methods in Molecular Biology book series (MIMB, volume 1459) | Cite as

Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods

Protocol

Abstract

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.

Key words

Live cell imaging Leishmania Nonclassical protein secretion FRAP (fluorescence recovery after photobleaching) HASPB (hydrophilic acylated surface protein B) Acylation 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, College of Life SciencesUniversity of DundeeDundeeUK
  2. 2.Centre for Applied Entomology and Parasitology, School of Life SciencesKeele UniversityKeele, StaffordshireUK
  3. 3.Technology Facility, Department of BiologyUniversity of YorkYorkUK

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