Methods for Studying Membrane-Associated Bacterial Cytoskeleton Proteins In Vivo by TIRF Microscopy

Charlène Cornilleau (1) , Arnaud Chastanet (1) , Cyrille Billaudeau (1) , Rut Carballido-Lopez (1)

1 MICALIS - MICrobiologie de l'ALImentation au Service de la Santé

MreB proteins are actin homologs present in nonspherical bacteria. They assemble into membrane-associated discrete filamentous structures that exhibit different dynamic behaviors along the bacterial sidewalls. Total internal reflection fluorescence (TIRF) microscopy, a sensitive method for studying molecular events at cell surfaces with high contrast and temporal resolution, is a method of choice to characterize the localization and dynamics of cortical MreB assemblies in vivo. This chapter describes the methods for visualizing fluorescently tagged MreB proteins in live Bacillus subtilis cells. We detail how to (1) grow B. subtilis strains for reproducible TIRF observations, (2) immobilize cells on agarose pads and (3) in CellASIC ® microfluidic plates, and (4) acquire TIRF images and time lapses.

Mots clés: TIRF microscopy; Actin-like MreB proteins; Bacterial cytoskeleton; Agarose pad; CellASIC ® microfluidic system; Bacillus subtilis

Domaines: Bactériologie; Organisation et fonctions cellulaires [q-bio.SC]

Charlène Cornilleau, Arnaud Chastanet, Cyrille Billaudeau, Rut Carballido-Lopez. Methods for Studying Membrane-Associated Bacterial Cytoskeleton Proteins In Vivo by TIRF Microscopy. Cytoskeleton Dynamics: Methods and Protocols, Methods in Molecular Biology, pp.123-133, 2020, ⟨10.1007/978-1-0716-0219-5_8⟩. ⟨hal-02918510⟩