Identification and characterization of the cell-cell communication system of a multicellular cyanobacterium

Abstract

Around 2.5 billion years ago, cyanobacteria built the basis for life on Earth by raising the atmospheric oxygen level. Multicellularity among Cyanobacteria evolved very early and increased their fitness due to improved motility, economies of scale and faster adaption to changing environmental conditions. The importance of multicellularity for evolution of life and complex life styles gives reason for performing research on these organisms. Of particular interest is the cell-cell communication system, which allows molecular exchange between cells of the organism via direct cell contact. Without such a system, a multicellular lifestyle with division of labor between specialized cells would be impossible. The cell-cell communication system of the model organism Anabaena sp. PCC 7120 comprises a functional unit of a nanopore array in the septal peptidoglycan and of proteinaceous septal junctions (SJs). The SJs traverse the nanopores to connect the cytoplasm of adjacent cells. Little was known about the mode of action of this communication system and the structural protein components were obscure. This work revealed the in situ architecture of SJs as three-modular complexes involving a cytoplasmic cap and a membrane-embedded plug module at both sites of a length-variable tube module. Furthermore, it could be demonstrated that SJs are gated channels whose caps undergo a structural rearrangement to switch from an open into a closed state, which does not allow cell-to-cell diffusion. Regulation of intercellular diffusion was observed as a response on various intracellular stress conditions. Identification of the septum-localized membrane protein FraD as the first structural SJ component allowed the development of a co-immunoprecipitation using FraD as bait with the aim to discover further proteins of these complexes. By this, a so far hypothetical protein was identified as potential interaction partner of FraD and termed SepN. SJs of a mutant in this septal membrane protein lacked the plug module and exhibited a cap reminiscent to the closed state. Therefore, the function of the SJ plug was assumed to be important for holding the cap structure in its opened position as well as for SJ closure. A mutant in another identified protein, FraI, showed a severely reduced communication, which was linked to a drastic reduction in the number of nanopores. A relation to amidases that drill the nanopore array was therefore suggested. In this work, the complex network of proteins involved in cell-cell communication and the understanding of regulation of intercellular exchange were greatly extended. This includes the identification of two proteins that are essential to form the SJ complex, as well as the establishment of a microscopic assay to screen future mutants for their ability to regulate their communication, and the development of a workflow to identify further (membranous) SJ proteins.