BIOGENESIS OF CYTOPLASMIC LIPID DROPLETS AND LIPID RAFTS USING EM TOMOGRAPHY

使用电子断层扫描技术研究细胞质脂滴和脂筏的生物发生

• 批准号: 7955063
• 负责人: MARK S LADINSKY
• 金额: $ 1.07万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955063
• 关键词: Adipocytes; Aniline; Architecture; Area; Binding; Biogenesis; Cell membrane; Cells; Cellular Membrane; Cellular Structures; Cholesterol; Computer Retrieval of Information on Scientific Projects Database; Coupled; Epithelial Cells; Freeze Substitution; Freezing; Funding; Golgi Apparatus; Grant; Hepatocyte; Institution; Investigation; Lipids; Liver; Mammary gland; Membrane Microdomains; Methods; Movement; Mus; Phospholipids; Preparation; Protocols documentation; Rattus; Research; Research Personnel; Resolution; Resources; Source; Staining method; Stains; Surface; Technology; Time; Tissues; United States National Institutes of Health; Work; cell type; electron tomography; mammary epithelium; monolayer; tomography

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?

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 我们使用的是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质液滴和脂质筏的生物发生。 孔雀石绿（4- [（（4-二甲基苯基） - 苯基甲基] -n，N-二甲基苯胺）是一种渗透性染色，在大多数细胞和组织中与脂质结合。 初步的EM和层析成像研究表明，孔雀石绿色染色的磷脂单层和双层的特定区域似乎与化学固定的大鼠和小鼠肝脏和乳腺上皮中的高浓度胆固醇相对应。 在质膜和高尔基水库内的结构域中观察到类似斑块的染色。我们假设质膜和高尔基体分别是成熟的脂质筏和新生的脂质筏。还观察到在ER并置至CLD表面的区域的细胞质脂质液滴（CLD）中观察到致密染色。 我们正在努力通过将孔雀石绿色染色引入冻结固定方案来优化我们的制剂，以便该方法可以与快速变形的细胞和组织一起使用。 这种方法将允许对通过细胞的胆固醇运动以及高尔基体和质膜内的脂质筏的3D结构进行高分辨率研究。 此外，我们将将这项技术应用于对已知产生CLDS（肝细胞，乳腺上皮细胞和脂肪细胞）的3种细胞类型的细胞质脂质液滴中胆固醇致密局部的研究。 这种染色方法，再加上快速冻结，冻结固定和EM层析成像，将促进对CLD的生物发生的理解，并回答以下问题： 1）是否与特定的细胞膜并列浓胆固醇的局部区域？ 如果是这样，他们是什么？ 2）这些局部区域是否与CLD的生物发生过程中的任何时间点相关？