Aberrant cellular cholesterol accumulation contributes to the pathophysiology of many diseases including neurodegenerative disorders such as Niemann-Pick Type C (NPC) and Alzheimer's Disease^1–4. Many aspects of cholesterol efflux from cells remain elusive. Here we describe the utility of cholesterol-rich giant plasma membrane vesicles (GPMVs) as a means to monitor cholesterol that is translocated to the plasma membrane for secretion. We demonstrate that small molecules known to enhance lipid efflux, including those in clinical trials for lipid storage disorders, enhance this GPMV formation. Conversely, pharmacological inhibition of cholesterol efflux blocks GPMV formation. We show that microtubule stabilization via paclitaxel treatment and increased tubulin acetylation via HDAC6 inhibition promotes the formation of GPMVs with concomitant reduction in cellular cholesterol in a cell model of NPC disease. The pan-deacetylase inhibitor panobinostat, which has been shown to reduce the severity of cholesterol storage in NPC, elicited a similar response. Further, the disruption of actin polymerization inhibits the formation of GPMVs, whereas the small GTP-binding protein Arl4c promotes actin remodeling at sites overlapping with GPMV formation. Thus, monitoring the formation of GPMVs provides a new avenue to better understand diseases whose pathology may be sensitive to alterations in cellular cholesterol.

异常的细胞胆固醇蓄积有助于许多疾病的病理生理，包括神经退行性疾病，如尼曼-匹克C型（NPC）和阿尔茨海默氏病^1-4。从细胞中胆固醇外排的许多方面仍然难以捉摸。在这里，我们描述了富含胆固醇的巨质膜囊泡（GPMVs）的实用程序，作为监测转运至质膜分泌的胆固醇的一种手段。我们证明，已知增强脂质外流的小分子，包括那些在脂质存储障碍临床试验中的分子，都会增强GPMV的形成。相反，药理抑制胆固醇外流可阻止GPMV的形成。我们显示通过紫杉醇治疗和通过HDAC6抑制增加微管蛋白乙酰化的微管稳定促进了NPC疾病的细胞模型中细胞胆固醇的同时降低GPMVs的形成。泛脱乙酰基酶抑制剂panobinostat已被证明可降低NPC中胆固醇存储的严重程度，引起了类似的反应。此外，肌动蛋白聚合反应的破坏抑制了GPMV的形成，而小的GTP结合蛋白Arl4c促进了与GPMV形成重叠的部位的肌动蛋白重塑。因此，监测GPMV的形成为更好地了解其病理可能对细胞胆固醇变化敏感的疾病提供了一条新途径。