Controlled exosome release from the retinal pigment epithelium in situ
Controlled exosome release from the retinal pigment epithelium in situ
复制标题
DOI：
10.1016/j.exer.2014.10.010
复制
发表时间：
2014-12-01
影响因子：
3.4
通讯作者：
McKay, Brian S.
中科院分区：
文献类型：
--
作者：
Locke, Christina J.;Congrove, Nicole R.;McKay, Brian S.
视网膜色素上皮细胞（RPE）表达GPR 143和myocilin，它们以信号转导依赖的方式相互作用。在异源系统中，GPR 143与配体的激活导致肌球蛋白瞬时募集到内化受体，这似乎是肌球蛋白进入内吞途径的入口点。在一些但不是所有细胞中，肌球蛋白也通过多泡体（MVB）运输，并以信号转导依赖性方式释放在外泌体表面。关于外泌体在RPE中的作用知之甚少，但它们可能作为RPE和外视网膜之间的通信模式。在这项研究中，我们使用从新鲜的人供体眼睛中取出视网膜的后极作为模型来测试内源性系统中GPR 143、肌球蛋白和外泌体之间的关系。我们使用由RPE调节的培养基的差速离心分离由RPE释放的外泌体25分钟，然后使用纳米颗粒追踪来表征外泌体以确定外泌体的数量和大小。接下来，我们测试了使用L-DOPA的GPR 143的配体刺激是否改变了RPE外泌体释放。最后，我们研究了肌钙蛋白是否存在于RPE释放的外泌体上，以及L-DOPA刺激GPR 143是否引起肌钙蛋白向内吞途径的募集，正如我们先前使用培养细胞观察到的那样。GPR 143的激活停止了RPE外泌体的释放，同时将肌钙蛋白募集到内吞区室。总之，我们的结果表明，GPR 143和myocilin在信号转导系统中起作用，该系统可以控制RPE的外泌体释放。(C)2014爱思唯尔有限公司版权所有。
Retinal Pigment Epithelial cells (RPE) express both GPR143 and myocilin, which interact in a signal transduction-dependent manner. In heterologous systems, activation of GPR143 with ligand causes transient recruitment of myocilin to internalized receptors, which appears to be the entry point of myocilin to the endocytic pathway. In some but not all cells, myocilin also traffics through the multivesicular body (MVB) and is released on the surface of exosomes in a signal transduction-dependent fashion. Little is known regarding the role of exosomes in RPE, but they likely serve as a mode of communication between the RPE and the outer retina. In this study, we used posterior poles with retina removed from fresh human donor eyes as a model to test the relationship between GPR143, myocilin, and exosomes in an endogenous system. We isolated exosomes released by RPE using differential centrifugation of media conditioned by the RPE for 25 min, and then characterized the exosomes using nanoparticle tracking to determine the number and size of the exosomes. Next, we tested whether ligand stimulation of GPR143 using L-DOPA altered RPE exosome release. Finally, we investigated whether myocilin was present on the exosomes released by RPE and whether L-DOPA stimulation of GPR143 caused recruitment of myocilin to the endocytic pathway, as we have previously observed using cultured cells. Activation of GPR143 halted RPE exosome release, while simultaneously recruiting myocilin to the endocytic compartment. Together, our results indicate that GPR143 and myocilin function in a signal transduction system that can control exosome release from RPE. (C) 2014 Elsevier Ltd. All rights reserved.