Department of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.
Department of Applied Physics, California Institute of Technology, Pasadena, California, USA.
J Bacteriol. 2018 Nov 6;200(23). doi: 10.1128/JB.00460-18. Print 2018 Dec 1.
Rapid changes in extracellular osmolarity are one of many insults microbial cells face on a daily basis. To protect against such shocks, and other microbes express several types of transmembrane channels that open and close in response to changes in membrane tension. In , one of the most abundant channels is the mechanosensitive channel of large conductance (MscL). While this channel has been heavily characterized through structural methods, electrophysiology, and theoretical modeling, our understanding of its physiological role in preventing cell death by alleviating high membrane tension remains tenuous. In this work, we examine the contribution of MscL alone to cell survival after osmotic shock at single-cell resolution using quantitative fluorescence microscopy. We conducted these experiments in an strain which is lacking all mechanosensitive channel genes save for MscL, whose expression was tuned across 3 orders of magnitude through modifications of the Shine-Dalgarno sequence. While theoretical models suggest that only a few MscL channels would be needed to alleviate even large changes in osmotic pressure, we find that between 500 and 700 channels per cell are needed to convey upwards of 80% survival. This number agrees with the average MscL copy number measured in wild-type cells through proteomic studies and quantitative Western blotting. Furthermore, we observed zero survival events in cells with fewer than ∼100 channels per cell. This work opens new questions concerning the contribution of other mechanosensitive channels to survival, as well as regulation of their activity. Mechanosensitive (MS) channels are transmembrane protein complexes which open and close in response to changes in membrane tension as a result of osmotic shock. Despite extensive biophysical characterization, the contribution of these channels to cell survival remains largely unknown. In this work, we used quantitative video microscopy to measure the abundance of a single species of MS channel in single cells, followed by their survival after a large osmotic shock. We observed total death of the population with fewer than ∼100 channels per cell and determined that approximately 500 to 700 channels were needed for 80% survival. The number of channels we found to confer nearly full survival is consistent with the counts of the numbers of channels in wild-type cells in several earlier studies. These results prompt further studies to dissect the contribution of other channel species to survival.
细胞外渗透压的快速变化是微生物细胞每天面临的众多刺激之一。为了防止这种冲击， 和其他微生物表达几种跨膜通道，这些通道会根据膜张力的变化而打开和关闭。 在 中，最丰富的通道之一是机械敏感的大电导通道（MscL）。虽然这个通道已经通过结构方法、电生理学和理论建模进行了大量的研究，但我们对它在缓解高膜张力以防止细胞死亡方面的生理作用的理解仍然很薄弱。在这项工作中，我们使用定量荧光显微镜在单细胞分辨率下检查了 MscL 单独对渗透压冲击后细胞存活的贡献。我们在一个 菌株中进行了这些实验，该菌株除了 MscL 之外，还缺乏所有的机械敏感通道基因，MscL 的表达通过对 Shine-Dalgarno 序列的修饰在 3 个数量级上进行了调整。虽然理论模型表明，即使是渗透压的巨大变化，只需要几个 MscL 通道就可以缓解，但我们发现，每个细胞需要 500 到 700 个通道才能传递 80%以上的存活率。这个数字与通过蛋白质组学研究和定量 Western blot 测量的野生型 细胞中 MscL 的平均拷贝数一致。此外，我们观察到每个细胞中少于约 100 个通道的细胞中没有任何存活事件。这项工作提出了关于其他机械敏感通道对存活的贡献以及它们活性调节的新问题。机械敏感（MS）通道是跨膜蛋白复合物，由于渗透压冲击导致膜张力变化，它们会打开和关闭。尽管进行了广泛的生物物理特性表征，但这些通道对细胞存活的贡献在很大程度上仍然未知。在这项工作中，我们使用定量视频显微镜测量单个细胞中单一 MS 通道的丰度，然后在经历大渗透压冲击后测量它们的存活率。我们观察到每个细胞中少于约 100 个通道的细胞群体完全死亡，并确定大约需要 500 到 700 个通道才能获得 80%的存活率。我们发现的赋予几乎完全存活的通道数量与之前几项研究中野生型细胞的通道数量计数一致。这些结果促使进一步的研究来剖析其他通道物种对存活的贡献。
