Skinkle Allison D, Levental Kandice R, Levental Ilya
Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas; Biological and Biomedical Sciences Program, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas.
Biophys J. 2020 Mar 24;118(6):1292-1300. doi: 10.1016/j.bpj.2019.12.040. Epub 2020 Jan 28.
Giant plasma membrane vesicles (GPMVs) are a widely used experimental platform for biochemical and biophysical analysis of isolated mammalian plasma membranes (PMs). A core advantage of these vesicles is that they maintain the native lipid and protein diversity of the PM while affording the experimental flexibility of synthetic giant vesicles. In addition to fundamental investigations of PM structure and composition, GPMVs have been used to evaluate the binding of proteins and small molecules to cell-derived membranes and the permeation of drug-like molecules through them. An important assumption of such experiments is that GPMVs are sealed, i.e., that permeation occurs by diffusion through the hydrophobic core rather than through hydrophilic pores. Here, we demonstrate that this assumption is often incorrect. We find that most GPMVs isolated using standard preparations are passively permeable to various hydrophilic solutes as large as 40 kDa, in contrast to synthetic giant unilamellar vesicles. We attribute this leakiness to stable, relatively large, and heterogeneous pores formed by rupture of vesicles from cells. Finally, we identify preparation conditions that minimize poration and allow evaluation of sealed GPMVs. These unexpected observations of GPMV poration are important for interpreting experiments utilizing GPMVs as PM models, particularly for drug permeation and membrane asymmetry.
巨质膜囊泡（GPMV）是用于对分离的哺乳动物质膜（PM）进行生化和生物物理分析的广泛应用的实验平台。这些囊泡的一个核心优势在于，它们在保持质膜天然脂质和蛋白质多样性的同时，还具备合成巨囊泡所具有的实验灵活性。除了对质膜结构和组成的基础研究外，GPMV还被用于评估蛋白质和小分子与细胞衍生膜的结合以及类药物分子通过这些膜的渗透情况。此类实验的一个重要假设是GPMV是密封的，即渗透是通过疏水性核心扩散而非通过亲水性孔道发生的。在此，我们证明这一假设往往是错误的。我们发现，与合成的巨型单层囊泡相比，使用标准制备方法分离得到的大多数GPMV对高达40 kDa的各种亲水性溶质具有被动渗透性。我们将这种渗漏归因于细胞来源的囊泡破裂形成的稳定、相对较大且异质性的孔道。最后，我们确定了能将成孔最小化并允许对密封GPMV进行评估的制备条件。这些关于GPMV成孔的意外发现对于解释将GPMV用作质膜模型的实验，特别是对于药物渗透和膜不对称性的实验，具有重要意义。
