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Live‐Cell Fluorescent Visualization of T3SS Needle and Its Dynamics

The Type III Secretion System (T3SS) is a complex transmembrane apparatus used by many Gram‐negative pathogens for protein translocation and host invasion. An essential T3SS component is the needle, a hollow substructure extending from the bacterial membrane, spanning the space from the bacterium to the host cytosol. The needle is formed by helical polymerization of a single small protein, which is secreted by the T3SS and assembled outside the bacterium. Visualization of the needle previously depended on immunofluorescence, which requires cell fixation. We hereby describe a simple methodology for T3SS needle fluorescent tagging and visualization, which enables live‐cell microscopy and investigation of needle kinetics and dynamics.