CRISPR CAS9 ENGINEERED TETRASPANIN DEFICIENT CELL LINES FOR THE ANALYSIS OF EXTRACELLULAR VESICLES
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| Rok publikování | 2023 |
| Druh | Konferenční abstrakty |
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| Popis | Tetraspanins are essential proteins that play a crucial role in the biogenesis, secretion and uptake of extracellular vesicles (EVs). These transmembrane proteins can form tetraspanin-enriched microdomains (TEMs) on the plasma membrane by interacting with each other and with additional membrane proteins and lipids. These TEMs are critical for sorting proteins and lipids into EVs, regulating EV biogenesis and release, and facilitating EV uptake by target cells. Our study focuses on understanding the roles of individual tetraspanins in EV biology. Specifically, we are analysing EVs from four cell lines, including the wild type cell line DU145 and three tetraspanin-deficient cell lines prepared using CRISPR-Cas9 gene editing. After producing of conditioned media with EVs, we isolate them using ultracentrifugation coupled to sucrose cushion. We confirm the tetraspanin deficiency by western blotting and analyze the size and concentration of EVs using dynamic light scattering and cryo-electron microscopy. Next, we quantify their protein composition using tandem mass spectrometry. Each tetraspanin has a unique function in the process of EV biogenesis and secretion. CD9, for example, engages in the biogenesis and secretion of EVs in various cell types, while CD63 is implicated in the sorting of proteins and lipids into EVs. CD81 plays a role in the fusion of EVs with target cells, leading to the uptake of EVs by target cells. However, comprehensive analysis of the major tetraspanin knock-out (KO) cells, i.e. CD9 KO, CD63 KO and CD81 KO, in the same background has not been performed. Understanding the specific roles of individual tetraspanins in EV biology is essential for revealing the mechanisms underlying EV biogenesis, secretion and uptake. Our experimental approach will provide a comprehensive understanding of the roles of tetraspanins in EV biology, therefore contributing to the broader field of EV research. |