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Updated: May 15, 2026

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System
Published on: March 10, 2020
Fluorescence microscopy and proteomics to investigate subcellular localization, assembly, and function of the type II
Tanya L Johnson1, Aleksandra E Sikora, Ryszard A Zielke
1Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA.
Abstract:
Investigation of secretion systems is often critical to understanding the virulence mechanisms of bacterial pathogens. With estimates as high as 30-40% of proteins secreted or localized to the cell envelope, information about the subcellular localization and organization of secretion complexes and identification and functional characterization of their substrates are key steps toward understanding these intricate systems. Here we describe a protocol using fluorescent live-cell imaging of fusion proteins that can provide a powerful tool to potentially examine the localization, assembly, and role of each component in the secretion complex. In addition, we describe protocols for the identification of secreted substrates using 1D SDS-PAGE coupled with nano-liquid chromatography (LC) and tandem mass spectrometry (MS/MS), and isobaric tagging for absolute quantification (iTRAQ) coupled with two-dimensional LC and MS/MS. Both experimental approaches are applicable to any similar study of membrane transport systems.
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