Gated junctions in multicellular cyanobacteria

细胞间的通讯是多细胞生物功能的先决条件。最具组织性的多细胞细菌是蓝藻门，其中的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，异囊以半规则的模式从营养细胞分化出来，并专门用于固氮。这两种细胞类型完成特定的代谢任务，并在整个多细胞丝的稳态网络中相互连接。分子的运输和细胞间的通讯是通过多聚体蛋白结构，即间隔连接，通过间隔细胞壁进行的。在以前的研究中，我们已经在间隔肽聚糖中发现了纳米孔阵列，最近我们通过离子束磨细丝的电子冷冻断层扫描（ECT）解决了间隔连接的原位结构。这些多蛋白复合物通过纳米孔连接细胞间的细丝的所有细胞。间隔连接由一根管子穿过纳米孔内的间隔肽聚糖组成。每个管端在细胞质膜内包含一个塞，其中含有FraD蛋白。在细胞质一侧，塞被一个具有5倍对称的帽状结构所覆盖。光漂白后的荧光恢复表明，细胞间的通信在胁迫下以可逆的方式被阻断。在这个项目中，我们希望与苏黎世联邦理工学院的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，它们的功能类似后生动物的间隙连接。为此，我们将使用不同的方法识别仍然未知的间隔连接成分，并通过诱变，蛋白质定位和生化研究来研究它们的个体功能。此外，我们希望阐明这些蓝藻的多细胞生活方式及其适应不断变化的环境条件和捕食的生理意义。

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.