Super Resolution Fluorescence Microscopy and Tracking of Bacterial Flotillin (Reggie) Paralogs Provide Evidence for Defined-Sized Protein Microdomains within the Bacterial Membrane but Absence of Clusters Containing Detergent-Resistant Proteins

Abstract

Biological membranes have been proposed to contain microdomains of a specific lipid composition, in which distinct groups of proteins are clustered. Flotillin-like proteins are conserved between pro—and eukaryotes, play an important function in several eukaryotic and bacterial cells, and define in vertebrates a type of so-called detergent-resistant microdomains. Using STED microscopy, we show that two bacterial flotillins, FloA and FloT, form defined assemblies with an average diameter of 85 to 110 nm in the model bacterium Bacillus subtilis. Interestingly, flotillin microdomains are of similar size in eukaryotic cells. The soluble domains of FloA form higher order oligomers of up to several hundred kDa in vitro, showing that like eukaryotic flotillins, bacterial assemblies are based in part on their ability to self-oligomerize. However, B. subtilis paralogs show significantly different diffusion rates, and consequently do not colocalize into a common microdomain. Dual colour time lapse experiments of flotillins together with other detergent-resistant proteins in bacteria show that proteins colocalize for no longer than a few hundred milliseconds, and do not move together. Our data reveal that the bacterial membrane contains defined-sized protein domains rather than functional microdomains dependent on flotillins. Based on their distinct dynamics, FloA and FloT confer spatially distinguishable activities, but do not serve as molecular scaffolds.

Author Summary

Many membrane proteins are not uniformly distributed within biological membranes, and may prefer specific lipid environments to function optimally. Using super resolution fluorescence microscopy, we show that several Bacillus subtilis membrane proteins indeed cluster into structures of 60 to 110 nm, verifying the existence of defined-size protein microdomains. Biochemical co-isolation of specific membrane proteins and flotillins, a family of proteins highly conserved between eukaryotic and bacterial cells, suggested that common “functional” microdomains exist, containing so-called “detergent-resistant” membrane proteins, that are centered by flotillins. Through high speed tracking of Bacillus subtilis FloA and FloT we show that both proteins are not present in the same microdomain, but move through the membrane with different velocities. Dual colour time lapse microscopy showed that contrarily to vertebrate flotillins, bacterial flotillins do not move together with detergent-resistant proteins, ruling out the existence of coclusters. The lack of both flotillins, but not of a single one, leads to striking defects in cell shape and in cell growth, indicating important overlapping functions of flotillin paralogs. Our data show that FloA and FloT perform spatially distinct functions, possibly in the insertion of membrane proteins that require a specific lipid environment, based on a close connection between FloA and FloT with the Sec membrane insertion machinery, but do not act as scaffolds for detergent resistant proteins. Our tracking analyses provide an important basis for the understanding of interactions between membrane proteins in living cells.