Localization of RalB signaling at endomembrane compartments and its modulation by autophagy

Abstract

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.

Autophagy Is Polarized toward Cell Front during Migration and Spatially Perturbed by Oncogenic Ras

“…Finally, to evaluate the impact of oncogenic Ras on autophagy topology in a genetically controlled cell model, we compared isogenic normal HEK-HT cells (human embryonic kidney cells, immortalized but not transformed) and transformed HEK-HT-H-RasV12 cells (tumorigenic, invasive, and metastatic, due to expression of constitutive active H-RasV12) [23,24]. Consistently with previous reports on Ras-mutated cancer cells [7][8][9], the transformation of HEK-HT cells by oncogenic H-RasV12 leads to increased autophagy [25].…”

RalA and PLD1 promote lipid droplet growth in response to nutrient withdrawal

“…Instead of total cellular RalA activity, it is possible that only a subset of RalA in specific cellular compartments is acting to promote LD accumulation. A recent study showed that RalB activity is increased specifically at autophagosomes upon starvation (Singh et al, 2019). Thus, we stably expressed either a GTPase-deficient RalA Q72L (GTP bound) or the dominant-negative RalA S28N mutant (GDP bound) in RalA KO MEFs (Figure S5C).…”

STK 38 kinase acts as XPO 1 gatekeeper regulating the nuclear export of autophagy proteins and other cargoes

“…HeLa [8], HEK293T (CRL-3216; ATCC), and A549 (CCL-185; ATCC) cell lines were grown in DMEM (Gibco) supplemented with 10% FBS (Biosera), 1% penicillin/streptomycin (Gibco), and with 1% Lglutamine (Gibco). HeLa cells stably expressing the GFP-LC3-RFP-LC3DG autophagic flux probe (pMRX-IP-GFP-LC3-RFP-LC3DG, 84572; Addgene) as well as HEK-HT-iRFP-LC3 cells [49] were cultured in the appropriate medium supplemented with 1 lg/ml puromycin. Cells stably expressing APEX-STK38 (pWZL-APEX2-STK38; this work) were cultured in appropriate medium supplemented with 10 lg/ml blasticidin.…”

“…Cells stably expressing APEX-STK38 (pWZL-APEX2-STK38; this work) were cultured in appropriate medium supplemented with 10 lg/ml blasticidin. HeLa cells stably expressing the GFP-LC3-RFP-LC3DG autophagic flux probe (pMRX-IP-GFP-LC3-RFP-LC3DG, 84572; Addgene) as well as HEK-HT-iRFP-LC3 cells [49] were cultured in the appropriate medium supplemented with 1 lg/ml puromycin. HAP1 cell lines (C631; Horizon Discovery) were grown in IMDM (Gibco) supplemented with 10% FBS (Biosera), 1% penicillin/streptomycin (Gibco), and with 1% Lglutamine (Gibco).…”