Organelle ion channel control of directional mesenchymal cell migration

Abstract

Endothelial cells (ECs) play a critical role in the maintenance of vascular function. They line blood and lymphatic vessels, forming a selectively permeable barrier essential for the exchange of nutrients, immune cells and waste products. In pathologies such as obesity and other metabolic disorders, circulating signals negatively affect vascular function and directly alter EC states, triggering cellular stress responses. These stress responses are particularly evident in obese individuals during pregnancy, where maternal stress can be transferred to the fetus through the umbilical cord in a process called fetal programming. In the offspring, this cellular stress contributes to insulin resistance and endothelial dysfunction. Although the mechanisms underlying endothelial dysfunction remain poorly understood, previous research has identified a disproportionate activation of the endoplasmic reticulum (ER) stress response, characterized by a dysregulated unfolded protein response (UPR). Because EC migration is critical for angiogenesis and vessel sprouting, this thesis investigates how ER stress affects EC behaviour during migration and identifies which UPR pathways drive these effects. Human umbilical vein endothelial cells (HUVECs) were treated with tunicamycin (TN), which in vitro mimics maternal obesity-induced ER stress. Using live-cell imaging, confocal microscopy, holotomography, ultrastructural electron microscopy, and advanced image analysis, the impact of TN-induced ER stress on EC migration was assessed during collective mesenchymal cell migration. ER stress induction led to reduced migration speed and directionality in both collective and single-cell migration on two-dimensional and one-dimensional substrates. This impaired migration correlated with cytoskeletal distortion: F-actin fibers and microtubules became misaligned relative to the migratory axis. The misalignment of F-actin also altered membrane dynamics. Although TN-induced ER stress increased membrane ruffling, these dynamics were non-productive for motility. Consistent with these cytoskeletal changes, organelle morphology and polarity were disrupted. Both the ER and mitochondria exhibited structural alterations and misalignments to the migratory axis. Pharmacological inhibition of the UPR identified RNA-dependent protein kinase (PKR)-like ER kinase (PERK) as the primary signalling branch regulating HUVEC migration. PERK inhibition prevented the ER stress-induced reduction in migration speed and directionality. Structurally, PERK inhibition also preserved cytoskeletal alignment and restored organelle organization to patterns similar to control conditions. Given the strong association between ER and mitochondria, confirmed through analysis of mitochondria-ER contacts (MERCs), the effects of ER stress on mitochondrial dynamics and function were further examined. Live-cell imaging revealed that mitochondria moved into protrusions that ultimately determined migration direction. Because mitochondrial function depends on mitochondrial membrane potential (∆y), changes in ∆y were monitored during directional migration and after ER stress induction. Directionally migrating HUVECs exhibited more frequent depolarization events in mitochondria positioned towards the cell front. ER stress prolonged the duration of these depolarization events, an effect that was prevented by PERK inhibition. Since ∆y relies on balanced ion transport, several ion channels were tested for their involvement in directional migration. While the inhibition of transient receptor potential channel 6 (TRPC6) or voltage-dependent anion channel 1 (VDAC1) did not prevent the ER stress-induced reduction in migration speed and directionality, VDAC1 was revealed as a key regulator of directional migration during collective mesenchymal migration under resting conditions. These findings suggest the involvement of a cryptic protein that regulates directional migration through PERK signalling and mitochondrial membrane potential. Overall, this thesis demonstrates a central role for PERK signalling in directional mesenchymal cell migration. PERK-dependent ER stress reduces migration speed and directionality, disrupts cytoskeletal and organelle organization, alters membrane dynamics, and modifies mitochondrial membrane potential behaviour. These results position PERK as a promising candidate for further investigation in the context of endothelial dysfunction.