Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) export pathway by live cell imaging methods

Lorna MacLean (Lead / Corresponding author), Helen Price, Peter O'Toole

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review

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.

Original languageEnglish
Title of host publicationUnconventional protein secretion
Subtitle of host publicationmethods and protocols
EditorsAndrea Pompa , Francesca De Marchis
Place of PublicationNew York
PublisherSpringer
Pages191-203
Number of pages13
Volume1459
ISBN (Electronic)9781493938049
ISBN (Print)9781493938025
DOIs
Publication statusPublished - 2016

Publication series

NameMethods in Molecular Biology
Volume1459
ISSN (Print)1064-3745

Keywords

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

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