外泌體是細胞經由多囊泡體 (MVB) 與細胞膜融合而分泌的脂質雙層奈米顆粒。它們介導多種腫瘤促進功能，包括癌症轉移、腫瘤進展和免疫逃脫。通常情況下，大多數MVB都傾向於被溶酶體降解而不是外泌體分泌；然而，控制MVB命運的機制仍未完全被了解。在本研究中，我們發現E3連接酶接頭WDR4透過降解ESCRT相關的Bro1家族蛋白PTPN23來促進外泌體的生物合成。從機制上講，PTPN23及其旁系同源物ALIX競爭與syntenin的結合，從而促進不同MVB群體的形成，即PTPN23+MVB和ALIX+MVB。PTPN23+MVB主要位於核週，優先被溶酶體降解，而ALIX+MVB在細胞質中分散性較高，較容易與細胞膜融合而分泌外泌體。蛋白質體學分析表明，ALIX特異性結合蛋白參與肌動蛋白細胞骨架重塑。例如，ALIX會招募封閉蛋白 (如CAPZA1和CAPZB) 來防止分支F-肌動蛋白在MVB周圍積聚。這種重塑提供了一條無障礙的運輸路線，使ALIX+MVB能夠移動到細胞膜周圍與細胞膜融合，分泌外泌體。WDR4作為PTPN23的負調節劑，從而增強ALIX誘導的外泌體的生物合成。此外，我們也發現Rab7和Arl8b分別調節MVB的降解和分泌。功能上，WDR4/ALIX誘導的外泌體透過LAMP2A選擇性地包裹促腫瘤蛋白，包括MCAM、PTK2和NRP1，從而促進癌症轉移、腫瘤生長和免疫逃脫。總的來說，我們的研究結果發現了MVB生物合成及其命運之間的耦合關系，突顯了其在塑造外泌體內容物的作用。這些研究結果為大規模外泌體生產和標靶癌症治療的開發提供了重要的線索。

Exosomes are lipid bilayer nanoparticles secreted by cells through the fusion of multivesicular bodies (MVBs) with the plasma membrane. They mediate cell physiology and pathological functions, including cancer metastasis, tumor progression, and immune evasion. In general, MVBs are directed to lysosomal degradation, however, the mechanisms governing the distinct fates of MVBs remain elusive. In this study, we identified the E3 ligase adaptor WDR4 promotes exosome biogenesis by degrading the ESCRT-associated Bro1-family protein PTPN23. Mechanistically, PTPN23 and its paralog ALIX competitively bind syntenin, thereby facilitating the formation of different MVB populations, i.e. PTPN23+MVBs and ALIX+MVBs. PTPN23+MVBs are predominantly perinuclear and preferentially undergo lysosomal degradation, whereas ALIX+MVBs are more dispersed in the cytosol and are more likely to undergo exosomal secretion. Proteomic analysis revealed that ALIX-specific binding proteins are involved in actin cytoskeleton remodeling. For example, barbed-end capping proteins, such as CAPZA1 and CAPZB, are recruited by ALIX to prevent the accumulation of branched F-actin around MVBs. This remodeling provides a barrier-free trafficking route, enabling ALIX+MVBs to translocate to the cell periphery for secretion. WDR4 acts as a negative regulator of PTPN23, thereby enhancing the biogenesis of ALIX-dependent exosomes. Additionally, Rab7 and Arl8b were found to regulate MVB degradation and secretion, respectively. Functionally, WDR4/ALIX-dependent exosomes selectively incorporate pro-tumor proteins, including MCAM, PTK2, and NRP1, via LAMP2A, promoting cancer metastasis, tumor growth, and immune evasion. Collectively, our findings elucidate a novel coupling between exosome biogenesis and MVB fate determination, highlighting its role in shaping the cargo landscape of exosomes. These insights provide a foundation for large-scale exosome production and the development of targeted cancer therapeutics.