BIOGENESIS OF CYTOPLASMIC LIPID DROPLETS AND LIPID RAFTS USING EM TOMOGRAPHY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们正在使用孔雀石绿染色的小鼠乳腺和肝脏的电子断层扫描，以研究细胞质脂滴和脂筏的生物发生。孔雀石绿(4-[(4-二甲氨基苯基)-苯基-甲基]-N，N-二甲基苯胺)是一种嗜奥斯染料，在大多数细胞和组织中与脂类结合。初步的EM和断层扫描研究表明，孔雀石绿在化学固定的大鼠和小鼠肝脏和乳腺上皮中染色特定区域的磷脂单分子层和双分子层，似乎对应于高浓度的胆固醇。高尔基体池内的质膜和结构域可见斑片状染色。我们假设质膜和高尔基结构域分别是成熟的脂筏和新生的脂筏。在ER与CLD表面并列的区域，细胞质脂滴(CLDs)内也可观察到浓染。我们正在努力通过将孔雀绿染色引入冷冻替代方案来优化我们的准备工作，以便该方法可以用于快速冷冻的细胞和组织。这种方法将允许高分辨率研究胆固醇在细胞中的移动，以及高尔基体内和质膜上的脂筏的3D结构。此外，我们将应用这项技术来研究3种细胞类型(肝细胞、乳腺上皮细胞和脂肪细胞)中细胞质脂滴中胆固醇致密的定位。这种染色方法与快速冷冻、冷冻替代和EM断层扫描相结合，将有助于理解CLDs的生物发生，并回答以下问题： 1)局部的胆固醇浓缩区域是否并列在特定的细胞膜上？如果是，它们是什么？2)这些局部区域与慢性腰椎病生物发生过程中的任何时间点相关吗？

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are using electron tomography of mouse mammary glands and liver, stained with malachite green, to study the biogenesis of cytoplasmic lipid droplets and lipid rafts. Malachite Green (4-[(4-dimethylaminophenyl)-phenyl-methyl]-N,N-dimethyl-aniline) is an osmiophilic stain that binds to lipids in most cells and tissues. Preliminary EM and tomographic studies suggest that malachite green stains specific areas of phospholipid monolayers and bilayers that seem to correspond with high concentrations of cholesterol in chemically-fixed rat and mouse liver and mammary epithelia. Patch-like staining is observed in the plasma membrane and domains within Golgi cisternae. We hypothesize that the plasma membrane and Golgi domains are mature lipid rafts and nascent lipid rafts, respectively. Dense staining is also observed within cytoplasmic lipid droplets (CLDs) at regions where ER is juxtaposed to the CLD surface. We are working to optimize our preparations by introducing malachite green stain to freeze-substitution protocols so that the method can be used with rapidly-frozen cells and tissues. This approach will allow for high-resolution investigations of cholesterol movement through the cell, as well as the 3D architecture of lipid rafts within the Golgi and at the plasma membrane. Additionally, we will apply this technology to the investigation of cholesterol-dense localizations in cytoplasmic lipid droplets in 3 cell types known to produce CLDs (hepatocytes, mammary epithelial cells, and adipocytes). This staining method, coupled with rapid-freezing, freeze-substitution and EM tomography, will facilitate the understanding of the biogenesis of CLDs and answer the following questions: 1) Are localized areas of concentrated cholesterol juxtaposed to specific cellular membranes? If so, what are they? 2) Do these localized areas correlate with any time point during the biogenesis of the CLDs?