Label-free visualization of type IV pili dynamics by interferometric scattering microscopy

Type IV pili (TFP) are slender objects that assemble by polymerization and secretion of protein subunits from bacterial cell surfaces. The mechanisms by which these surface structures of microscopic length and molecular diameter modulate the physical interaction of bacteria with their environment, however, remains poorly understood largely due to limitations in our ability to monitor and characterize the dynamics of individual TFP. Here, we demonstrate that interferometric scattering microscopy (iSCAT) enables label-free and dynamic visualization of TFP in intact cells of the opportunistic pathogen Pseudomonas aeruginosa. As a result, we can directly monitor extension, attachment and retraction events on millisecond timescale and nanometer length scale in three dimensions. These capabilities allow us to observe that P. aeruginosa is able to crawl against the direction of flow using short TFP filaments. Also, careful observation show that TFP retract rapidly after surface attachment, suggesting that P. aeruginosa senses contact of the pilus tip with the solid substrate. These results illustrate the power of iSCAT for the label-free visualization of small, dynamic microbial extracellular structures.