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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们用孔雀石绿色染色的小鼠乳腺和肝脏的电子断层扫描来研究细胞质脂滴和脂筏的生物发生。 孔雀石绿色（4-[（4-二甲氨基苯基）-苯基-甲基]-N，N-二甲基-苯胺）是一种亲锇染色剂，可与大多数细胞和组织中的脂质结合。 初步EM和断层扫描研究表明，孔雀石绿色染色的磷脂单层和双层，似乎对应于化学固定的大鼠和小鼠肝脏和乳腺上皮细胞中的高浓度胆固醇的特定区域。 在高尔基体池内的质膜和结构域中观察到斑片状染色。我们假设质膜和高尔基体结构域分别是成熟的脂筏和新生的脂筏。在细胞质脂滴（CLD）内ER与CLD表面并列的区域也观察到密集染色。 我们正致力于通过将孔雀石绿色染色引入冷冻置换方案来优化我们的制备，以便该方法可用于快速冷冻的细胞和组织。 这种方法将允许通过细胞的胆固醇运动的高分辨率调查，以及在高尔基体和质膜内的脂筏的3D结构。 此外，我们将应用该技术研究已知产生CLD的3种细胞类型（肝细胞、乳腺上皮细胞和脂肪细胞）中细胞质脂滴中的胆固醇密集定位。 这种染色方法，加上快速冷冻，冷冻置换和EM断层扫描，将有助于了解CLD的生物起源，并回答以下问题： 1)胆固醇集中的局部区域是否与特定的细胞膜并列？ 如果是，它们是什么？ 2)这些局部区域是否与CLD生物发生过程中的任何时间点相关？

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?