A MULTIORGANELLE PLATFORM REGULATES EGFR ENDOCYTOSIS AND SIGNALLING

Endocytosis plays a crucial role in regulating the signalling of the epidermal growth factor receptor (EGFR). The internalization of the EGFR can occur through different mechanisms. Clathrin-mediated endocytosis (CME) operates at all concentrations of EGF and in all cell contexts, directing the majority of receptors towards recycling. In contrast, non-clathrin endocytosis (NCE) is activated only at high (but physiological) EGF concentrations, alongside CME, and leads to the degradation of EGFR. Thus, the NCE pathway can serve as an intrinsic cellular system to suppress EGFR levels and signalling, which could be particularly relevant in situations where the receptor or its ligands are excessively upregulated, such as in cancer. Through a proteomics investigation of EGFR-loaded NCE vesicles coupled with RNA interference screening, several functional regulators of NCE have been discovered, including the endoplasmic reticulum (ER)-shaping factor reticulon-3 (RTN3) and various mitochondrial proteins. RTN3 plays a critical role in EGFR internalization via NCE by promoting the formation of contact sites between the ER and the plasma membrane (PM). These contact sites are essential for the formation and elongation of NCE intermediates known as tubular invaginations (TIs). Calcium, released from the ER at these sites through the inositol trisphosphate receptor (IP3R), is required, along with the dynamin, for the fission of TIs and the completion of EGFR-NCE. Preliminary data from our laboratory suggested the possible involvement of mitochondria in EGFR-NCE: a significant enrichment in mitochondrial proteins was observed in isolated NCE vesicles. Electron microscopy (EM) analysis of PM-ER contact sites also revealed the presence of mitochondria adjacent to these structures. Hence, the main objectives of this thesis were to investigate the precise role of mitochondria in EGFR-NCE and to gain an understanding of the potential crosstalk between EGFR signalling and the specific subset of mitochondria at NCE contact sites. Specifically, we aimed to determine whether EGFR signalling can directly influence mitochondrial metabolism and, if so, what the impacts of this would be on broader cellular responses. Importantly, using EM, we established that EGFR activation enhances the physical association between internalizing EGFR-NCE structures, ER stacks, and mitochondria, forming a tripartite interorganelle platform. At the molecular level, using intracellular calcium sensors targeted to the PM or mitochondria, we discovered that calcium signalling at these sites occurs through oscillatory waves that are sensed by mitochondria, leading to an increase in mitochondrial energetics. Furthermore, the activation of EGFR-NCE results in a localized burst of ATP, as measured by an ATP-dependent enzyme (Luciferase) targeted to the PM. This accumulation of ATP in the subplasmalemmal region depends on EGFR-NCE activation. The interplay between active EGFR, calcium signalling, and mitochondrial metabolism has a dual impact on cell physiology. It is crucial for the recruitment of the actin machinery, which acts in concert with dynamin in the fission of NCE TIs. Additionally, it regulates the remodelling of cortical actin, thereby promoting cell migration, as evidenced in wound healing assays performed on human immortalized keratinocytes (HaCaT cells). In conclusion, the tripartite structure involving the PM, ER, and mitochondria not only plays a role in the endocytosis of EGFR but also serves as a signalling platform that mediates cellular responses to high-dose EGF stimulation. This work reveals an additional layer of complexity in the regulation of EGFR signalling, involving a new player (NCE multiorganelle platform) that connects EGFR endocytosis to cellular metabolism and, ultimately, to the wider cellular response, e.g., cell motility. It is worth noting that while NCE acts as a negative regulator of EGFR signalling by promoting receptor degradation in the presence of excess ligand, it also serves as a positive regulator of EGF-related cellular responses in specific cell contexts, such as the migration of normal keratinocytes. Thus, considering the crucial role of NCE in the regulation of EGFR signalling and the fact that EGFR signalling is frequently altered in pathological conditions, this research could improve our understanding of how EGF-induced biological responses are regulated in the physiological contexts. Additionally, it may shed light on novel mechanisms responsible for the dysregulation of EGFR signalling in diseases like cancer, offering valuable insights for the development of innovative therapeutic strategies that combine EGFR- and mitochondria-targeting therapies.