Department of Physics, ‡Center for Biosystems Science and Engineering, ¶Department of Chemical Engineering, and §Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science Bangalore 560 012, India.
Langmuir. 2016 Sep 20;32(37):9649-57. doi: 10.1021/acs.langmuir.6b01848. Epub 2016 Sep 8.
Membrane-protein interactions play a central role in membrane mediated cellular processes ranging from signaling, budding, and fusion, to transport across the cell membrane. Of particular significance is the process of efficient protein olgomerization and transmembrane pore formation on the membrane surface; the primary virulent pathway for the action of antimicrobial peptides and pore forming toxins (PFTs). The suggested nanoscopic length scales and dynamic nature of such membrane lipid-protein interactions makes their detection extremely challenging. Using a combination of super-resolution stimulated emission depletion nanoscopy with fluorescence correlation spectroscopy (STED-FCS) we unravel the emergence of nanoscale lateral heterogeneity in supported bilayer membranes made up of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol upon interaction with the PFT, listeriolysin O (LLO). A distinct length scale-dependent dynamical crossover (<200 nm) from a Brownian diffusive regime is observed at 33 and 50% cholesterol compositions, indicating the partitioning of lipids into domains with variable cholesterol content. At 25% cholesterol content, this dyamical crossover is observed only in bilayers incubated with LLO providing evidence for the existence of sub ∼100 nm dynamical lipid nanodomains bound to LLO pore assemblies. By introducing asymmetry in cholesterol composition across the bilayer leaflets we infer that this domain formation is driven largely due to active cholesterol sequestration and transient trapping of lipids to the membrane bound motifs present in the toxins, en route to LLO oligomerization and subsequent pore formation. Bilayers prepared with labeled lipids present in either the proximal or distal leaflet allow us to track the dynamical perturbation in a leaflet-dependent manner upon LLO incubation. From the differences in the extent and intensity of the dynamical crossover as observed with STED-FCS, these experiments reveal that the affinity for cholesterol in the membrane binding motifs of the LLO subdomains induce cholesterol and lipid reorganization to a greater extent in the distal (upper) leaflet when compared with the proximal (lower) leaflet. The observed length scale-dependent membrane reorganization that occurs due to invasion by LLO could be generalized to other cholesterol-dependent cytolysins and emphasizes the significant advantage of using super-resolution STED nanoscopy to unravel complex lipid-protein interactions in membrane and cellular biophysics.
膜蛋白相互作用在膜介导的细胞过程中起着核心作用，这些过程包括信号转导、出芽和融合，以及跨细胞膜的运输。特别重要的是在膜表面上有效进行蛋白质寡聚化和跨膜孔形成的过程；这是抗菌肽和孔形成毒素（PFT）作用的主要毒力途径。这种膜脂-蛋白相互作用的纳米级长度尺度和动态性质使得它们的检测极具挑战性。我们使用超分辨率受激发射损耗纳米显微镜与荧光相关光谱（STED-FCS）相结合，揭示了由 1,2-二油酰基-sn-甘油-3-磷酸胆碱（DOPC）和胆固醇组成的支撑双层膜在与 PFT 李斯特溶素 O（LLO）相互作用时出现纳米级横向异质性。在 33%和 50%胆固醇组成的情况下，观察到一个明显的依赖于长度尺度的动力学交叉（<200nm），从布朗扩散转变为一个扩散受限的状态，表明脂质分配到具有不同胆固醇含量的域中。在 25%胆固醇含量的情况下，这种动力学交叉仅在与 LLO 孵育的双层膜中观察到，这为存在与 LLO 孔组装结合的亚 100nm 动态脂质纳米域提供了证据。通过在双层叶片中引入胆固醇组成的不对称性，我们推断这种域形成主要是由于胆固醇的主动隔离和脂质的瞬时捕获到毒素中存在的膜结合基序，LLO 寡聚化和随后的孔形成。在近端或远端叶片中存在标记脂质的双层膜允许我们以依赖于叶层的方式跟踪LLO 孵育后的动力学干扰。从观察到的 STED-FCS 动力学交叉的程度和强度的差异中，这些实验表明，LLO 亚域的膜结合基序中胆固醇的亲和力导致胆固醇和脂质在远端（上）叶片中比在近端（下）叶片中更大程度地重组。LLO 入侵引起的依赖于长度尺度的膜重组可以推广到其他依赖胆固醇的细胞溶素，并强调了使用超分辨率 STED 纳米显微镜来揭示膜和细胞生物物理学中复杂的脂质-蛋白相互作用的重要优势。
