Department of Medicine, Northwestern University, Chicago, IL, USA.
Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA.
J Extracell Vesicles. 2014 Jun 11;3. doi: 10.3402/jev.v3.22653. eCollection 2014.
Cancer cell-derived micro-particles (MPs) play important regulatory roles on cellular and system levels. These activities are attributed in part to protein factors carried by MPs. However, recruitment strategies for sequestering certain protein factors in MPs are poorly understood. In the current study, using exogenous and endogenously expressed phospholipid-binding probes, we investigated the distribution of membrane phospholipids in MPs as a potential mechanism for electrostatically enriching cationic protein factors in MPs. We detected a significant level of externalised phosphatidylethanolamine (PE) at the outer surface of MPs. This was accompanied, in the inner leaflet of the MP membrane, by a greater density of negatively charged phospholipids, particularly phosphatidylserine (PS). The local enrichment of PS in the inner surface of MPs was correlated with an elevated presence of small GTPases in a polybasic region (PBR)-dependent fashion. By employing a series of RhoA derivatives, including constitutively active and RhoA derivatives lacking a PBR, we could demonstrate that the congregation of RhoA in MPs was dependent on the presence of the PBR. A chimer with the fusion of PBR sequence alone to GFP significantly enhanced GFP localisation in MPs, indicative of a positive contribution of electrostatic interactions in RhoA recruitment to MPs. Using in silico thermodynamic simulations, we characterised the electrostatic interactions between PBR and anionic lipid membrane surface. In summary, the redistribution of membrane phospholipids in MPs has an impact on the local ionic density, and is likely a contributing factor in the electrostatic recruitment of membrane-associated proteins to MPs in a PBR-dependent fashion.
癌细胞衍生的微颗粒 (MPs) 在细胞和系统水平上发挥着重要的调节作用。这些活动部分归因于 MPs 携带的蛋白质因子。然而，将特定蛋白质因子募集到 MPs 中的招募策略还了解甚少。在本研究中，我们使用外源性和内源性表达的磷脂结合探针，研究了 MPs 中膜磷脂的分布，作为一种通过静电作用将阳离子蛋白因子募集到 MPs 中的潜在机制。我们检测到 MPs 外表面有显著水平的外显化磷脂酰乙醇胺 (PE)。这伴随着 MPs 膜内层中带负电荷的磷脂，特别是磷脂酰丝氨酸 (PS) 的密度增加。PS 在 MPs 内表面的局部富集与多碱性区域 (PBR) 依赖性的小 GTPases 的高表达相关。通过使用一系列 RhoA 衍生物，包括组成性激活和缺乏 PBR 的 RhoA 衍生物，我们可以证明 RhoA 在 MPs 中的聚集依赖于 PBR 的存在。与 GFP 融合的 PBR 序列嵌合体显著增强了 GFP 在 MPs 中的定位，表明静电相互作用在 RhoA 募集到 MPs 中具有积极贡献。通过计算机热力学模拟，我们描述了 PBR 与阴离子脂质膜表面之间的静电相互作用。总之，MPs 中膜磷脂的重新分布会影响局部离子密度，并且可能是通过 PBR 依赖的静电作用将膜相关蛋白募集到 MPs 中的一个促成因素。
