An Optogenetic Tool to Raise Intracellular pH in Single Cells and Drive Localized Membrane Dynamics.
An Optogenetic Tool to Raise Intracellular pH in Single Cells and Drive Localized Membrane Dynamics.
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DOI：
10.1021/jacs.1c02156
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发表时间：
2021-11-17
影响因子：
15
通讯作者：
White KA
中科院分区：
文献类型：
--
作者：
Donahue CET;Siroky MD;White KA
细胞内pH（pHi）动态对于调节正常细胞生理学是至关重要的。例如，pHi（7.2-7.6）的瞬时增加调节细胞行为，如细胞极化、肌动蛋白细胞骨架重塑和细胞迁移。大多数关于pH依赖性细胞行为的研究都是在群体水平上进行的，并使用非特异性方法来操纵pHi。缺乏在单细胞水平特异性操纵pHi的工具阻碍了对pHi动力学在驱动单细胞行为中的作用的研究。在这项工作中，我们表明，古视紫红质（ArchT），光驱动的外向质子泵，可用于引起强大的和生理的pHi增加超过分钟的时间尺度。我们发现，ArchT的激活是可重复的，能够在单细胞中维持高pHi长达45分钟。我们应用这种时空pHi操纵工具来确定增加的pHi是否是单细胞中膜皱褶的足够驱动因素。使用ArchT工具，我们表明，与未受刺激的ArchT细胞以及对照细胞相比，单细胞中增加的pHi可以在几秒钟内驱动局部膜皱褶反应，并增加膜动力学（突出和收缩事件）。总的来说，这个工具使我们能够直接研究增加的pHi和单细胞行为之间的关系，如膜皱褶。该工具将在促进实验方面具有变革性，这些实验是确定pHi增加在驱动单细胞行为中的作用所必需的。
Intracellular pH (pHi) dynamics are critical for regulating normal cell physiology. For example, transient increases in pHi (7.2–7.6) regulate cell behaviors like cell polarization, actin cytoskeleton remodeling, and cell migration. Most studies on pH-dependent cell behaviors have been performed at the population level and use nonspecific methods to manipulate pHi. The lack of tools to specifically manipulate pHi at the single-cell level has hindered investigation of the role of pHi dynamics in driving single cell behaviors. In this work, we show that Archaerhodopsin (ArchT), a light-driven outward proton pump, can be used to elicit robust and physiological pHi increases over the minutes time scale. We show that activation of ArchT is repeatable, enabling the maintenance of high pHi in single cells for up to 45 minutes. We apply this spatiotemporal pHi manipulation tool to determine whether increased pHi is a sufficient driver of membrane ruffling in single cells. Using the ArchT tool, we show that increased pHi in single cells can drive localized membrane ruffling responses within seconds and increased membrane dynamics (both protrusion and retraction events) compared to unstimulated ArchT cells as well as control cells. Overall, this tool allows us to directly investigate the relationship between increased pHi and single cell behaviors such as membrane ruffling. This tool will be transformative in facilitating experiments that are required to determine roles for increased pHi in driving single cell behaviors.
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影响因子：
5.2
作者：
Czowski BJ;Romero-Moreno R;Trull KJ;White KA
通讯作者：
White KA
影响因子：
8.8
作者：
El-Gaby M;Zhang Y;Wolf K;Schwiening CJ;Paulsen O;Shipton OA
通讯作者：
Shipton OA
影响因子：
--
作者：
Nin, Veronica;Hernandez, Julio A.;Chifflet, Silvia
通讯作者：
Chifflet, Silvia
影响因子：
5
作者：
Hulikova, Alzbeta;Swietach, Pawel
通讯作者：
Swietach, Pawel
DOI：
10.1152/ajpcell.1994.267.4.c1152
发表时间：
1994-10-01
影响因子：
5.5
作者：
GRINSTEIN, S;ROMANEK, R;ROTSTEIN, OD
通讯作者：
ROTSTEIN, OD