PO-173 Critical requirement of the Sos1 and Sos2 RasGEFs for maintenance of mitochondrial homeostasis

Abstract

IntroductionThe homologous, ubiquitously expressed, members (Sos1 and Sos2) of the Sos family of RasGEFs participate in multiple signalling pathways but their specific cellular functions are not clearly defined yet.Material and methodsUsing a tamoxifen (4OHT)-inducible, conditional Sos1 null mutation, here we generated and analysed wild type (WT), single Sos1-KO, constitutive Sos2-KO and double Sos1/2-DKO primary mouse embryonic fibroblasts (MEFs) in an effort to ascertain the functional specificity or redundancy of Sos1 and Sos2 at the cellular level.Results and discussionsSos1-KO and Sos1/2-DKO MEFs exhibited distinct flat morphology, enlarged cell perimeter and altered cytoskeletal organisation that were not observed in WT and Sos2 KO counterparts. Sos1-KO and Sos1/2-DKO MEFs also displayed significant accumulation of cytoplasmic bodies identified as autophagosomes containing degraded mitochondria by means of electron microscopy and specific markers. Consistent with a mitophagic phenotype, in vivolabelling using specific fluorophores revealed increased levels of mitochondrial oxidative stress in the Sos1-KO and the Sos1/2-DKO cells as compared to Sos2-KO or WT MEFs. Treatment of the MEF cultures with antioxidants such as GSH and NAC corrected the altered perimeter size and proliferative rate of Sos1-KO and Sos1/2-DKO MEFs to levels similar to those of WT and Sos2-KO, but not recover oxidative stress. Furthermore, treatment with the specific mitochondrial superoxide scavenger mitoTEMPO recovered endogenous redox-homeostasis in Sos1-KO and Sos1/2-DKO to normal levels. Analysis of MEFs concomitantly loaded with MitoTracker Green (Δψ-independent) and MitoTracker Red (Δψ-dependent) showed a significantly increased of dysfunctional mitochondria in the Sos1/2-DKO MEFs in comparison to all other genotypes. Finally, Seahorse-based measurements of mitochondrial parameters showed that basal and maximal mitochondrial respiration, spare respiratory capacity, and ATP production, were also significantly decreased in Sos1/2-DKO cells as compared to MEFs of all the other genotypes.ConclusionOur data uncover a direct mechanistic link between Sos RasGEF proteins and the control of mitochondrial oxidative stress and respiration. Dysfunctional mitochondria are a key element in a variety of serious diseases, including cancer, and thus a promising therapeutic target.This work was supported by grants PI16/02137 (FIS) and CIBERONC (ONCG19//2017) from ISCIII; SA043U16 from FEDER-JCyL; FS/35–2017 from FSB and AECC, Spain.