Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

This protocol is useful for characterizing the assembling functions of bacterial secretion complexes and can lead to a fundamental understanding of the molecular mechanisms that are required for host pathogen interactions. This technique integrates complementary approaches that preserve the native structure of the sample and allows and see to visualization of proteins complexes in intact bacterial cells. It increases our understanding of the structural function relationships of bacterial secretion systems.

This method can be adapted to study other types of secretion systems or other cellular complexes in a wide variety of bacterial species. Start by making agarose pads for live cell imaging. Prepare about 30 mL of 1%low melt agarose solution in water and microwave it in a glass flask for about 90 seconds swirling it occasionally until the agarose is completely dissolved.