A Makam Aishwarya, Wahlund Jonathan, Gandasi Nikhil R, Hatamie Amir
Cell Metabolism Lab (GA-08), Department of Developmental Biology and Genetics (DBG), Indian Institute of Science (IISc), Bengaluru 560012, India.
Institution of Health Sciences, University of Skövde, Högskolevägen 1, Skövde 541 28, Sweden.
ACS Omega. 2025 May 1;10(18):18889-18898. doi: 10.1021/acsomega.5c00864. eCollection 2025 May 13.
Cellular communication is a critical process that relies on exocytosis, during which cells release stored chemical messengers contained within intracellular nanoscale vesicles (50-500 nm in diameter). Before this occurs, the vesicle membrane must open and form a fusion pore, allowing its contents to be released into the extracellular space. This subcellular process involves various biomolecules, such as lipids and proteins, within the membrane, and any changes in their levels can impact dynamic pore formation and, consequently, the exocytosis process. Due to their small size, intracellular location, and sensitivity, direct studies of vesicles are challenging yet highly valuable. One of these crucial biomolecules is phosphatidylinositol-4,5-bisphosphate (PIP2), a lipid involved in membrane dynamics and related processes including exocytosis. In this study, we employed a combination of sensitive confocal microscopy and vesicle impact electrochemical cytometry (VIEC)-a novel amperometric technique using microelectrodes (D, 33 μm)-to test the hypothesis that elevated PIP2 levels regulate vesicle membrane properties and indirectly influence the exocytosis process. To investigate this, we used nanoscale vesicles isolated from neural cells as a biological model system. First, imaging analysis revealed that high PIP2 levels led to its accumulation in both cell and vesicle membranes, where it also participates in exocytosis. Next, direct analysis of PIP2-treated and untreated single nanoscale vesicles using VIEC demonstrated that while the vesicle content (i.e., the number of stored catecholamines) remained unchanged after PIP2 treatment, the vesicle opening dynamics were altered compared to the control. Specifically, our results showed that the vesicle opening rate increased by 1 ms, and the duration of vesicle opening extended from 5.7 to 6.9 ms in PIP2-treated vesicles compared to the control. In addition to the recognized roles of PIP2, these findings indicate that an extra level of PIP2 modulates the vesicle opening rate and suggest that PIP2 enhances membrane stability while delaying the vesicle opening process. Interestingly, this observation aligns with previous experimental and computational studies, which reported that abnormally high levels of PIP2 or other lipids can modify membrane properties and then exocytosis too. In our study, we observed this effect for PIP2 at abnormal levels through single vesicle electroanalysis. Furthermore, these results open a new way of investigating similar membrane components and their roles in disease mechanisms and cellular processes.
细胞通讯是一个依赖胞吐作用的关键过程，在此过程中，细胞释放包含在细胞内纳米级囊泡（直径50 - 500纳米）中的储存化学信使。在此之前，囊泡膜必须打开并形成融合孔，使其内容物释放到细胞外空间。这个亚细胞过程涉及膜内的各种生物分子，如脂质和蛋白质，它们水平的任何变化都会影响动态孔的形成，进而影响胞吐过程。由于其体积小、位于细胞内且敏感性高，对囊泡进行直接研究具有挑战性但非常有价值。这些关键生物分子之一是磷脂酰肌醇 - 4,5 - 二磷酸（PIP2），一种参与膜动态及包括胞吐作用在内的相关过程的脂质。在本研究中，我们采用了灵敏的共聚焦显微镜和囊泡撞击电化学细胞术（VIEC）——一种使用微电极（直径33μm）的新型安培技术——来检验PIP2水平升高调节囊泡膜特性并间接影响胞吐过程这一假设。为了研究这一点，我们使用从神经细胞分离的纳米级囊泡作为生物模型系统。首先，成像分析表明高PIP2水平导致其在细胞膜和囊泡膜中积累，它也参与胞吐作用。接下来，使用VIEC对经PIP2处理和未处理的单个纳米级囊泡进行直接分析表明，虽然PIP2处理后囊泡内容物（即储存的儿茶酚胺数量）保持不变，但与对照相比，囊泡打开动力学发生了改变。具体而言，我们的结果表明，与对照相比，经PIP2处理的囊泡打开速率增加了1毫秒，囊泡打开持续时间从5.7毫秒延长至6.9毫秒。除了PIP2已被认可的作用外，这些发现表明额外水平的PIP2调节囊泡打开速率，并表明PIP2增强膜稳定性同时延迟囊泡打开过程。有趣的是，这一观察结果与之前的实验和计算研究一致，这些研究报告称异常高水平的PIP2或其他脂质可改变膜特性进而影响胞吐作用。在我们的研究中，我们通过单个囊泡电分析观察到了异常水平的PIP2产生的这种效应。此外，这些结果为研究类似的膜成分及其在疾病机制和细胞过程中的作用开辟了一条新途径。
