Abstract
<jats:p>Bacterial nanotubes are widespread intercellular membranous bridges mediating molecular exchange among neighboring cells, yet their formation and dynamics remain largely unexplored. By combining live-cell imaging with in situ cryo-electron tomography, we describe nanotube biogenesis in the Gram-positive Bacillus subtilis. Nanotubes form elaborate, highly dynamic networks, with extending nanotubes exhibiting multidirectional movement toward nearby cells, repeatedly scanning their surface. Ultimately, nanotubes penetrate through the recipient cell wall to establish an intercellular bridge. Strikingly, nanotubes emerge within milliseconds from lipid-enriched sites representing a previously unrecognized membranous organelle, termed tubeosome, located in an unusual pseudo-periplasmic space beneath the cell wall. We present evidence that tubeosomes comprise proto-nanotube reservoirs that unfold to project nanotubes. Our study uncovers the tubeosome as a novel bacterial organelle and provides the first real-time visualization of intercellular bridge formation. As connecting nanotubes are found across bacteria, archaea, and eukaryotes, this mechanism may reflect a broadly conserved strategy for cellular communication. </jats:p>