MINFLUX imaging of a bacterial molecular machine at nanometer resolution

Abstract

The resolution achievable with the established super-resolution fluorescence nanoscopy methods, such as STORM or STED, is in general not sufficient to resolve protein complexes or even individual proteins. Recently, minimal photon flux (MINFLUX) nanoscopy has been introduced that combines the strengths of STED and STORM nanoscopy and can achieve a localization precision of less than 5 nm. We established a generally applicable workflow for MINFLUX imaging and applied it for the first time to a bacterial molecular machine in situ, i.e., the injectisome of the enteropathogen Y. enterocolitica. We demonstrate with a pore protein of the injectisome that MINFLUX can achieve a resolution down to the single molecule level in situ. By imaging a sorting platform protein using 3D-MINFLUX, insights into the precise localization and distribution of an injectisome component in a bacterial cell could be accomplished. MINFLUX nanoscopy has the potential to revolutionize super-resolution imaging of dynamic molecular processes in bacteria and eukaryotes.

Super‐resolution fluorescence microscopy for investigating bacterial cell biology

“…STED microscopy and SIM were used to visualize the Yersinia enterocolitica T3SS pore complex proteins YopB and YopD (uni- With an isotropic localization precision of ~5 nm, these experiments could reproduce the size of the YscL structure determined by Cryo ET to be ~16 nm in diameter (Carsten et al, 2022;Berger et al, 2021). 3D MINFLUX experiments performed in whole bacteria showed that the YscL complexes localized almost exclusively at the plasma membrane and at very low distances to each other (down to ~10 nm apart; Figure 2d; Carsten et al, 2022).…”

“…The z‐ axis is color coded. The white boxed area has a depth of 200 nm along the x‐ axis (Carsten et al., 2022). (e) Trajectories of 3D super‐resolution SMT of eYFP‐labeled Y. enterocolitica T3SS component YscL.…”

“…For example, a combination of primary and fluorescently labeled secondary antibodies can already offset the fluorescent label by up to 20 nm relative to the molecule of interest (Fruh et al., 2021). To minimize this label error, fluorescent proteins, self‐labeling enzyme (SLE) tags (SNAP, Halo, CLIP) and nanobodies of around 3 nm in size have successfully been employed (Banaz et al., 2019; Carsten et al., 2022; Liss et al., 2015; Ries et al., 2012; Figure 1b). The label error in the visualization of proteins can also be reduced to the subnanometer scale by introducing noncanonical amino acids with bioorthogonal (“clickable”) side chains (Mihaila et al., 2022).…”

“…While the focus of this review is on the visualization of molecular processes in bacteria, all of the described microscopy approaches are also applicable in other cell types. A widely applicable workflow guiding researchers toward in cellulo MINFLUX imaging at molecular scale has been described previously (Carsten et al., 2022).…”

Cell-Based Assay to Determine Type 3 Secretion System Translocon Assembly in Pseudomonas aeruginosa Using Split Luciferase

“…The secretion apparatus is comprised of a cytosolic platform, an export apparatus, a multi-ring basal body that spans the inner membrane, peptidoglycan layer and outer membrane of bacteria, a hollow needle that is anchored in the membrane through the basal body and extends into the extracellular space, and an oligomeric tip . Proteins required for effector translocation are secreted through the needle and insert into the host membrane to assemble a hetero-oligomeric transmembrane protein complex or translocon. − Translocation of effectors into the host cell disrupts cell function and results in a localized disruption of neutrophils. ,− Inhibition of translocon assembly will prevent effector translocation, protect host cells (mainly neutrophils) from the cytotoxic effect of bacterial effector proteins, and reduce the virulence of the infecting P. aeruginosa strain. Furthermore, since the translocon is assembled and located outside the bacterial cell, , it serves as an ideal target for small-molecule inhibitors.…”