Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, 75005, Paris, France.
ART group, Inserm U830, 75005, Paris, France.
Sci Rep. 2019 Jun 20;9(1):8910. doi: 10.1038/s41598-019-45443-1.
The monomeric GTPase RalB controls crucial physiological processes, including autophagy and invasion, but it still remains unclear how this multi-functionality is achieved. Previously, we reported that the RalGEF (Guanine nucleotide Exchange Factor) RGL2 binds and activates RalB to promote invasion. Here we show that RGL2, a major activator of RalB, is also required for autophagy. Using a novel automated image analysis method, Endomapper, we quantified the endogenous localization of the RGL2 activator and its substrate RalB at different endomembrane compartments, in an isogenic normal and Ras-transformed cell model. In both normal and Ras-transformed cells, we observed that RGL2 and RalB substantially localize at early and recycling endosomes, and to lesser extent at autophagosomes, but not at trans-Golgi. Interestingly the use of a FRET-based RalB biosensor indicated that RalB signaling is active at these endomembrane compartments at basal level in rich medium. Furthermore, induction of autophagy by nutrient starvation led to a considerable reduction of early and recycling endosomes, in contrast to the expected increase of autophagosomes, in both normal and Ras-transformed cells. However, autophagy mildly affected relative abundances of both RGL2 and RalB at early and recycling endosomes, and at autophagosomes. Interestingly, RalB activity increased at autophagosomes upon starvation in normal cells. These results suggest that the contribution of endosome membranes (carrying RGL2 and RalB molecules) increases total pool of RGL2-RalB at autophagosome forming compartments and might contribute to amplify RalB signaling to support autophagy.
单体 GTPase RalB 控制着包括自噬和入侵在内的关键生理过程，但尚不清楚这种多功能性是如何实现的。之前，我们报道了 RalGEF（鸟嘌呤核苷酸交换因子）RGL2 结合并激活 RalB 以促进入侵。在这里，我们表明 RGL2，RalB 的主要激活剂，也需要自噬。使用一种新的自动图像分析方法 Endomapper，我们在同基因正常和 Ras 转化细胞模型中，定量了 RGL2 激活剂及其底物 RalB 在不同内体膜区室中的内源性定位。在正常和 Ras 转化的细胞中，我们观察到 RGL2 和 RalB 大量定位于早期和再循环内体，并且在较小程度上定位于自噬体，但不在反式高尔基体上。有趣的是，使用基于 FRET 的 RalB 生物传感器表明，在富含营养的培养基中，RalB 信号在这些内体膜区室中处于基础活性状态。此外，营养饥饿诱导的自噬导致早期和再循环内体的数量明显减少，与自噬体数量的预期增加相反，这在正常和 Ras 转化的细胞中都是如此。然而，自噬对内体膜上 RGL2 和 RalB 的相对丰度的影响较小，无论是在早期和再循环内体上，还是在自噬体上。有趣的是，在正常细胞中，饥饿时 RalB 活性在自噬体上增加。这些结果表明，内体膜（携带 RGL2 和 RalB 分子）的贡献增加了自噬体形成区室中 RGL2-RalB 的总池，并可能有助于放大 RalB 信号以支持自噬。
