Gated junctions in multicellular cyanobacteria

细胞与细胞之间的通讯是多细胞生物体功能的先决条件。最高度组织化的多细胞细菌是在蓝藻门中发现的，其中细丝可能由数百个紧密相连的细胞组成。在丝状蓝藻Anabaena sp. PCC 7120中，异形胞以半规则模式从营养细胞分化，并专门从事固氮。这两种细胞类型都完成特定的代谢任务，并在整个多细胞细丝的稳态网络中相互连接。分子的运输和细胞间的通讯沿着丝通过隔细胞壁由多聚体蛋白结构，隔连接发生。在以前的研究中，我们已经确定了一个纳米孔阵列中的间隔肽聚糖，最近我们解决了原位结构的间隔连接的电子冷冻断层扫描（ECT）的离子束铣削细丝。这些多蛋白质复合物通过纳米孔连接从细胞到细胞的细丝的所有细胞。隔膜连接由穿过纳米孔内的隔膜肽聚糖的管组成。每个管端包括细胞质膜内的塞子，其含有FraD蛋白。塞被在胞质侧具有5重对称性的帽结构覆盖。光漂白后的荧光恢复表明，细胞-细胞通讯被阻断后，压力在一个可逆的方式。这种门控伴随着帽中的可逆构象变化。在这个项目中，我们希望与苏黎世ETH的Martin Pilhofer密切合作，继续研究这些细菌间隙连接类似物，他将进行ECT分析。我们想知道这些门控通道的组成和机制，这些通道的功能类似于后生动物的缝隙连接。为此，我们将确定仍然未知的隔连接组件使用不同的方法，并通过诱变，蛋白质定位和生化研究调查他们的个人功能。此外，我们希望阐明门控的生理意义，这些蓝藻的多细胞生活方式和适应不断变化的环境条件和捕食。

Cell-to-cell communication is a prerequisite for the functioning of a multicellular organism. The most highly organized multicellular bacteria are found in the phylum cyanobacteria, where filaments may consist of hundreds of tightly interconnected cells. In the filamentous cyanobacterium Anabaena sp. PCC 7120, heterocysts differentiate from vegetative cells in a semi-regular pattern and are specialized in nitrogen fixation. Both cell types fulfill specific metabolic tasks and are interconnected in a homeostatic network of the entire multicellular filament. Transport of molecules and cell-cell communication along the filament occurs through the septal cell wall by multimeric protein structures, the septal junctions. In previous studies we have identified a nanopore array in the septal peptidoglycan and recently we solved the in situ architecture of septal junctions by electron cryo tomography (ECT) of ion beam milled filaments. These multiprotein complexes connect all cells of the filaments reaching from cell to cell thru the nanopores. The septal junctions consist of a tube traversing the septal peptidoglycan inside the nanopores. Each tube end comprises a plug inside the cytoplasmic membrane, which contains the FraD protein. The plug is covered by a cap structure with 5-fold symmetry on the cytosolic side. Fluorescence recovery after photobleaching showed that cell-cell communication was blocked upon stress in a reversible manner. This gating was accompanied by a reversible conformational change in the cap. In this project we want to continue our research on these bacterial gap junction analogs in close cooperation with Martin Pilhofer at ETH, Zürich, who will perform the ECT analysis. We want to know the composition and mechanism of these gated channels, which functional resemble metazoan gap junctions. For this we will identify the still unknown septal junction components using different approaches and investigate their individual function by mutagenesis, protein localization and biochemical studies. In addition we want to elucidate the physiological significance of the gating for the multicellular life style of these cyanobacteria and their adaptation to changing environmental conditions and predation.