The effects of proton motive force on thylakoid architecture and function

类囊体膜是光合作用复合体的骨架，光合作用复合体将电子传递耦合到跨膜的质子运动，从而产生质子动力(PMF，由Delta-pH和Delta-Psi组成)，驱动ATP的产生。类囊体结构与高效的光合作用密切相关。类囊体是扁平的而不是球形的，增加了每腔面积的光合作用复合体的浓度以驱动PMF，同时为可溶性电子载体叶绿体通过类囊体腔的扩散提供了方向性。然而，类囊体结构并不是一成不变的，它对光的质量和数量的变化做出反应。在蓝藻、绿藻和植物中的初步电子显微镜观察表明，随着光照强度的增加，类囊体腔膨胀。一个合乎逻辑的假设是，管腔酸化(Delta-pH)和离子通量驱动的膜电位变化(Delta-Psi)可能与扩张管腔的渗透力相耦合。然而，这种架构变化的确切动力和机制仍未确定。该项目的目标是了解光诱导类囊体胀大的机制基础和生理后果，并具体测试PMF的不同成分是如何调节这种肿胀的。这项研究将通过我们先进的低温电子断层扫描(CRYO-ET)工作流程实现，我们开发的工作流程可以以亚纳米精度测量天然类囊体结构(膜和管腔宽度)，并分析沿着这些膜的每个光合作用复合体的组织。首先，我们的目的是(I)表征绿藻(Chlamydomonas Rehardtii)和蓝藻(聚球藻)中类囊体膨胀的动力学。6803)通过在从黑暗过渡到不同光强度之后执行野生型细胞的详细低温时间序列。接下来，我们的目标是(Ii)通过将冷冻-ET与小分子离子载体以及ATP合成酶和KEA3离子逆向转运蛋白的遗传扰动相结合，来研究Delta-pH和Delta-Psi对类囊体腔扩张的贡献。同时，我们的目标是(Iii)测试细胞色素b6f对类囊体腔动力学的特异性PMF贡献。最后，我们的目标是(Iv)通过将我们的低温辐射数据与来自该联盟其他成员的PMF和光合作用功能测量相结合，创建依赖光的管腔扩张的集成模型。我们项目的重点是实现GoPMF的总体目标3(能量转换系统重构中的类囊体膜结构动力学)。我们的工作也将有助于实现总体目标1、2和4。

Thylakoid membranes scaffold photosynthetic complexes, which couple electron transfer to proton movement across the membrane, thereby generating the proton motive force (PMF, composed of Delta-pH and Delta-Psi) that drives the production of ATP. Thylakoid architecture is intimately linked to efficient photosynthesis. Thylakoids are flat instead of spherical, increasing the concentration of photosynthetic complexes per luminal area to drive the PMF, while providing directionality for the diffusion of the soluble electron carrier plastocyanin through the thylakoid lumen. However, thylakoid architecture is not static and responds to changes in light quality and quantity. Preliminary electron microscopy observations in cyanobacteria, green algae, and plants have shown that the thylakoid lumen swells under increased light intensity. A logical hypothesis would be that lumen acidification (Delta-pH) and ion flux-driven changes in membrane potential (Delta-Psi) may be coupled to osmotic forces that expand the lumen. However, the precise dynamics and mechanism of this architectural change remain uncharacterized. The goal of this project is to understand the mechanistic basis and physiological consequences of light-induced thylakoid swelling, and to specifically test how this swelling is modulated by different components of the PMF. This study will be enabled by our advanced cryo-electron tomography (cryo-ET) workflow, which we have developed to measure native thylakoid architecture (membrane and lumen width) with sub-nanometer precision and analyze the organization of each photosynthetic complex along these membranes. First, we aim (i) to characterize the kinetics of thylakoid swelling in green algae (Chlamydomonas reinhardtii) and cyanobacteria (Synechocystis sp. 6803) by performing a detailed cryo-ET time-series of wild-type cells after transition from darkness to different light intensities. Next, we aim (ii) to examine the contributions of Delta-pH and Delta-Psi to thylakoid lumen expansion by combining cryo-ET with small molecule ionophores and genetic perturbations of ATP synthase and KEA3 ion antiporters. In parallel, we aim (iii) to test the specific PMF contributions of cytochrome b6f to thylakoid lumen dynamics. Finally, we aim (iv) to create an integrated model of light-dependent lumen expansion by combining our cryo-ET data with functional measurements of PMF and photosynthetic function from other members of the consortium. The focus of our project is to deliver on the overall aim 3 of GoPMF (Structural dynamics of thylakoid membranes in remodeling of energy conversion systems). Our work will also contribute to the overall aims 1, 2, and 4.