The orchestration of intercellular communication is essential for multicellular organisms. One mechanism by which cells communicate is through long, actin-rich membranous protrusions called tunneling nanotubes (TNTs), which allow the intercellular transport of various cargoes, between the cytoplasm of distant cells in vitro and in vivo. With most studies failing to establish their structural identity and examine whether they are truly open-ended organelles, there is a need to study the anatomy of TNTs at the nanometer resolution. Here, we use correlative FIB-SEM, light- and cryo-electron microscopy approaches to elucidate the structural organization of neuronal TNTs. Our data indicate that they are composed of a bundle of open-ended individual tunneling nanotubes (iTNTs) that are held together by threads labeled with anti-N-Cadherin antibodies. iTNTs are filled with parallel actin bundles on which different membrane-bound compartments and mitochondria appear to transfer. These results provide evidence that neuronal TNTs have distinct structural features compared to other cell protrusions.

Highlights d Intestine-Chip reveals that Shigella infects enterocytes efficiently from the gut lumen d The crypt-like structure of the intestine is critical for Shigella adhesion d Peristaltic motion (mechanical forces) enhances Shigella invasion d Shigella exploits intestine architecture and mechanical forces to maximize infectivity

Recent data support the hypothesis that Gram-positive bacteria (monoderms) arose from Gram-negatives (diderms) through loss of the outer membrane (OM), but how this happened remains unknown. As tethering of the OM is essential for cell envelope stability in diderm bacteria, its destabilization may have been involved in this transition. Here, we present an in-depth analysis of the four known main OM tethering systems across the Tree of Bacteria (ToB). We show that the presence of such systems follows the ToB with a bimodal distribution matching the deepest phylogenetic divergence between Terrabacteria and Gracilicutes. Whereas the lipoprotein Pal is restricted to the Gracilicutes along with a more sporadic occurrence of OmpA, and Braun's lipoprotein Lpp is present only in a subclade of Gammaproteobacteria, diderm Terrabacteria display as the main system the OmpM protein. We propose an evolutionary scenario whereby OmpM represents a simple, ancestral OM tethering system that was later replaced by one based on Pal following the emergence of the Lol machinery to deliver lipoproteins to the OM, with OmpA as a possible transition state.We speculate that the existence of only one main OM tethering system in the Terrabacteria would have allowed the multiple OM losses specifically inferred in this clade through OmpM perturbation, and we provide experimental support for this hypothesis by inactivating all four ompM gene copies in the genetically tractable diderm Firmicute Veillonella parvula. High-resolution imaging and tomogram reconstructions reveal a non-lethal phenotype in which vast portions of the OM detach from the cells, forming huge vesicles with an inflated periplasm shared by multiple dividing cells. Together, our results highlight an ancient shift of OM tethering systems in bacterial evolution and suggest a possible mechanism for OM loss and the multiple emergences of the monoderm phenotype from diderm ancestors.

Most archaea divide by binary fission using an FtsZ-based system similar to that of bacteria, but they lack many of the divisome components described in model bacterial organisms. Notably, among the multiple factors that tether FtsZ to the membrane during bacterial cell constriction, archaea only possess SepF-like homologs. Here, we combine structural, cellular, and evolutionary analyses to demonstrate that SepF is the FtsZ anchor in the human-associated archaeon Methanobrevibacter smithii. 3D super-resolution microscopy and quantitative analysis of immunolabeled cells show that SepF transiently co-localizes with FtsZ at the septum and possibly primes the future division plane. M. smithii SepF binds to membranes and to FtsZ, inducing filament bundling. High-resolution crystal structures of archaeal SepF alone and in complex with the FtsZ C-terminal domain (FtsZCTD) reveal that SepF forms a dimer with a homodimerization interface driving a binding mode that is different from that previously reported in bacteria. Phylogenetic analyses of SepF and FtsZ from bacteria and archaea indicate that the two proteins may date back to the Last Universal Common Ancestor (LUCA), and we speculate that the archaeal mode of SepF/FtsZ interaction might reflect an ancestral feature. Our results provide insights into the mechanisms of archaeal cell division and pave the way for a better understanding of the processes underlying the divide between the two prokaryotic domains.

As a result of an author oversight in the originally published version of this article, the last name of author Samy Gobaa was misspelled as ''Gobba.'' This error has now been corrected in the article online. The authors apologize for the error and any inconvenience that may have resulted.