Department of Biochemistry University of Wisconsin-Madison, Madison, WI.
Department of Biochemistry University of Wisconsin-Madison, Madison, WI
J Cell Biol. 2018 Aug 6;217(8):2877-2890. doi: 10.1083/jcb.201710132. Epub 2018 Jun 21.
Cancer cells secrete copious amounts of exosomes, and elevated intracellular Ca is critical for tumor progression and metastasis, but the underlying cellular mechanisms are unknown. Munc13-4 is a Ca-dependent SNAP receptor- and Rab-binding protein required for Ca-dependent membrane fusion. Here we show that acute elevation of Ca in cancer cells stimulated a fivefold increase in CD63, CD9, and ALIX exosome release that was eliminated by Munc13-4 knockdown and not restored by Ca binding-deficient Munc13-4 mutants. Direct imaging of CD63-pHluorin exosome release confirmed its Munc13-4 dependence. Depletion of Munc13-4 in highly aggressive breast carcinoma MDA-MB-231 cells reduced the size of CD63 multivesicular bodies (MVBs), indicating a role for Munc13-4 in MVB maturation. Munc13-4 used a Rab11-dependent trafficking pathway to generate MVBs competent for exosome release. Membrane type 1 matrix metalloproteinase trafficking to MVBs by a Rab11-dependent pathway was also Munc13-4 dependent, and Munc13-4 depletion reduced extracellular matrix degradation. These studies identify a novel Ca- and Munc13-4-dependent pathway that underlies increased exosome release by cancer cells.
癌细胞大量分泌外泌体，细胞内 Ca 升高对于肿瘤的进展和转移至关重要，但潜在的细胞机制尚不清楚。Munc13-4 是一种 Ca 依赖性 SNAP 受体和 Rab 结合蛋白，对于 Ca 依赖性膜融合是必需的。在这里，我们发现癌细胞中 Ca 的急性升高刺激了 CD63、CD9 和 ALIX 外泌体释放增加了五倍，这一过程被 Munc13-4 敲低所消除，而不能被 Ca 结合缺陷型 Munc13-4 突变体所恢复。CD63-pHluorin 外泌体释放的直接成像证实了其对 Munc13-4 的依赖性。在高度侵袭性的乳腺癌 MDA-MB-231 细胞中耗尽 Munc13-4 减少了 CD63 多泡体 (MVB) 的大小，表明 Munc13-4 在 MVB 成熟中起作用。Munc13-4 使用 Rab11 依赖性转运途径来产生有能力释放外泌体的 MVB。通过 Rab11 依赖性途径转运到 MVB 的膜型 1 基质金属蛋白酶也依赖于 Munc13-4，Munc13-4 耗竭减少了细胞外基质的降解。这些研究确定了一种新的 Ca 和 Munc13-4 依赖性途径，该途径是癌细胞中外泌体释放增加的基础。
