Exosome secretion kinetics are controlled by temperature
Exosome secretion kinetics are controlled by temperature
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DOI：
10.1016/j.bpj.2023.02.025
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发表时间：
2023-04-04
影响因子：
3.4
通讯作者：
Knowles，Michelle K.
中科院分区：
文献类型：
--
作者：
Mahmood，Anarkali;Otruba，Zdenek;Knowles，Michelle K.
当多泡内体（MVE）与质膜融合时，外泌体被释放到细胞外空间，在那里它们可以影响其他细胞。外泌体调节附近或远处细胞的能力取决于它们是否保持附着在分泌细胞膜上。外泌体分泌的调节和动力学尚未得到很好的表征，但用于直接成像单个MVE融合事件的探针已经允许融合和释放过程的可视化。特别地，在四跨膜蛋白CD 63中间具有pH敏感性染料的外泌体标记物的设计促进了个体MVE融合事件的研究。使用TIRF显微术，在保持在23-37°C的A549细胞中测量单个融合事件，并使用自动化检测算法鉴定事件。稳定对接融合之前几乎总是和温度的降低伴随着内容物损失率和融合事件的频率降低。在融合位点处测量CD 63-pHluorin荧光的损失，并与单或双指数衰减拟合，其中大多数事件需要两个组分和平台，因为荧光的损失通常是不完全的。为了解释动力学，融合事件被模拟为与CD 63的膜扩散偶联的系留/未系留外泌体的局部释放。实验观察到的衰变在模拟中需要三个组分：1）游离的外泌体，2）从内体膜到质膜中的CD 63膜扩散，和3）栓系的外泌体。用系留的外泌体的缓慢扩散（0.0015-0.004μm2/s）建模准确地拟合了所有温度的实验数据。然而，使用固定的系绳或没有系绳的模拟无法复制数据。我们的模型表明，外泌体从融合位点的释放是不完全的，由于融合后，膜附着。
When multivesicular endosomes (MVEs) fuse with the plasma membrane, exosomes are released into the extracellular space where they can affect other cells. The ability of exosomes to regulate cells nearby or further away depends on whether they remain attached to the secreting cell membrane. The regulation and kinetics of exosome secretion are not well characterized, but probes for directly imaging single MVE fusion events have allowed for visualization of the fusion and release process. In particular, the design of an exosome marker with a pH-sensitive dye in the middle of the tetraspanin protein CD63 has facilitated studies of individual MVE fusion events. Using TIRF microscopy, single fusion events were measured in A549 cells held at 23–37°C and events were identified using an automated detection algorithm. Stable docking precedes fusion almost always and a decrease in temperature was accompanied by decrease in the rate of content loss and in the frequency of fusion events. The loss of CD63-pHluorin fluorescence was measured at fusion sites and fit with a single or double exponential decay, with most events requiring two components and a plateau because the loss of fluorescence was typically incomplete. To interpret the kinetics, fusion events were simulated as a localized release of tethered/untethered exosomes coupled with the membrane diffusion of CD63. The experimentally observed decay required three components in the simulation: 1) free exosomes, 2) CD63 membrane diffusion from the endosomal membrane into the plasma membrane, and 3) tethered exosomes. Modeling with slow diffusion of the tethered exosomes (0.0015–0.004μm2/s) accurately fits the experimental data for all temperatures. However, simulating with immobile tethers or the absence of tethers fails to replicate the data. Our model suggests that exosome release from the fusion site is incomplete due to postfusion, membrane attachment.