Department of Physics and Astronomy, Rice University, Houston, Texas, USA.
Biophys J. 2012 Oct 17;103(8):1627-36. doi: 10.1016/j.bpj.2012.07.052. Epub 2012 Oct 16.
Prestin is the membrane motor protein that drives outer hair cell (OHC) electromotility, a process that is essential for mammalian hearing. Prestin function is sensitive to membrane cholesterol levels, and numerous studies have suggested that prestin localizes in cholesterol-rich membrane microdomains. Previously, fluorescence recovery after photobleaching experiments were performed in HEK cells expressing prestin-GFP after cholesterol manipulations, and revealed evidence of transient confinement. To further characterize this apparent confined diffusion of prestin, we conjugated prestin to a photostable fluorophore (tetramethylrhodamine) and performed single-molecule fluorescence microscopy. Using single-particle tracking, we determined the microscopic diffusion coefficient from the full time course of the mean-squared deviation. Our results indicate that prestin undergoes diffusion in confinement regions, and that depletion of membrane cholesterol increases confinement size and decreases confinement strength. By interpreting the data in terms of a mathematical model of hop-diffusion, we quantified these cholesterol-induced changes in membrane organization. A complementary analysis of the distribution of squared displacements confirmed that cholesterol depletion reduces prestin confinement. These findings support the hypothesis that prestin function is intimately linked to membrane organization, and further promote a regulatory role for cholesterol in OHC and auditory function.
Prestin 是一种驱动外毛细胞（OHC）电活动的膜运动蛋白，该过程对于哺乳动物的听力至关重要。Prestin 的功能对膜胆固醇水平敏感，许多研究表明 Prestin 定位于富含胆固醇的膜微区。先前，在表达 prestin-GFP 的 HEK 细胞中进行了荧光恢复后光漂白实验，这些实验揭示了瞬态限制的证据。为了进一步描述 Prestin 这种明显的受限扩散，我们将 Prestin 与一种光稳定荧光团（四甲基罗丹明）缀合，并进行了单分子荧光显微镜检查。使用单粒子跟踪，我们从均方偏差的完整时间过程中确定了微观扩散系数。我们的结果表明 Prestin 在限制区域内进行扩散，并且膜胆固醇的耗竭会增加限制大小并降低限制强度。通过用 hop-diffusion 的数学模型来解释数据，我们量化了这些胆固醇诱导的膜组织变化。对平方位移分布的补充分析证实，胆固醇耗竭会降低 Prestin 的限制。这些发现支持 Prestin 功能与膜组织密切相关的假设，并进一步促进胆固醇在外毛细胞和听觉功能中的调节作用。
