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Novel methods to analyze localization of bacterial proteins with super-resolution microscopy

Emerging super-resolution microscopy techniques allow us to overcome the diffraction limit of light, so that taking images (often from live cells) in a resolution of less than 100 nm. They unveiled detailed structure of eukaryotic cells including organelles. Whilst bacterial cells are small (typically 2-3 µm long and 0.8-1 µm wide) and not compartmentised, it is now well appreciated that they are also well organized and far from « bag of enzymes ». In rod-shaped bacteria, cell poles play an important role for the systematic arrangement of multi-component cellular processes. In the cholera pathogen Vibrio cholerae, polar landmark protein HubP tethers at least three proteins involved in chromosome segregation, chemotaxis and flagella biosynthesis. However, how HubP links multiple cellular machineries and how these are precisely organized in such a small area at the cell pole are yet to be elucidated.
Two I2BC teams, the Yamaichi group in the Genome Biology department and Merrifield group in the Cell Biology Department collaborated to tackle these questions by using PALM super-resolution microscopy.

Major breakthrough includes :
(1) Development of MatLab-based software “Vibio” to analyze subcellular localization of proteins from PALM microscopy in high-throughput manner.
(2) Development of novel cell labelling techniques (see Figure) that overcome the big problem in outlining the cell (which is essential for precise analysis of subcellular localization).
Altogether, quantitative image analyses of single molecules with hundreds of cells revealed that HubP shows skewed localization at the cell pole closer to the inner curvature of the vibrioid cell. While its interaction partners showed rather loose polar localization.

Novel bacterial cell labelling for PALM
Novel bacterial cell labelling for PALM
Representative PALM image of V. cholerae cells simultaneously expressing DronPA-MTS and ssDsbA-PAmCherry. Consequently, inner membrane (A) and periplasm (B) were labelled so that cell periphery can be identified. Strikingly, when the two images were merged (C and D), it can be appreciated that periplasmic PAmCherry are slightly more external than those for inner membrane DronPA-MTS, and the difference can be as small as 30 nm which correlates well with the width of the periplasmic space.

Super-resolution microscopy experiments were carried out at the Imagerie-Gif imaging platform.

Read more : "Single molecule super-resolution imaging of bacterial cell pole proteins with high-throughput quantitative analysis pipeline"
by Ipek Altinoglu, Christien J. Merrifield and Yoshiharu Yamaichi
Scientific Reports 9 : 6680 (2019), doi : 10.1038/s41598-019-43051-7

par Communication - publié le