Arf6 is required for endocytosis and filamentous actin assembly during angiogenesis in vitro

Abstract

ObjectiveEndocytosis is a process vital to angiogenesis and vascular homeostasis. In pathologies where supraphysiological growth factor signaling underlies disease etiology, such as in diabetic retinopathy and solid tumors, strategies to limit chronic growth factor signaling by way of blunting endocytic processes have been shown to have tremendous clinical value. ADP ribosylation factor 6 (Arf6) is a small GTPase that promotes the assembly of actin necessary for clathrin‐mediated and clathrin‐independent endocytosis. In its absence, growth factor signaling is greatly diminished, which has been shown to ameliorate pathological signaling input in diseased vasculature. However, it is less clear if there are bystander effects related to loss of Arf6 on angiogenic behaviors. Our goal was to provide an analysis of Arf6's function in angiogenic endothelium, focusing on its role in actin and endocytosis as well as sprouting morphogenesis.MethodsPrimary endothelial cells were cultured in both 2D and 3D environments. Here, endothelial cells were fixed and stained for various proteins or transfected with fluorescently‐tagged constructs for live‐cell imaging.ResultsWe found that Arf6 localized to both filamentous actin and sites of endocytosis in two‐dimensional culture. Loss of Arf6 distorted both apicobasal polarity and reduced the total cellular filamentous actin content, which may be the primary driver underlying gross sprouting dysmorphogenesis in its absence.ConclusionsOur findings highlight that endothelial Arf6 is a potent mediator of both actin regulation and endocytosis and is required for proper sprout formation.

The calcium pump PMCA4b promotes epithelial cell polarization and lumen formation

“…We show that PMCA4b colocalizes both with the wild type and a constitutively active form of Arf6, and that Arf6 inhibition interferes with PMCA4b recycling. Previous studies indicate that Arf6 plays a role in actin cytoskeleton remodeling and lumen formation in epithelial and endothelial cells (Francis et al, 2023;Milanini et al, 2018;Monteleon et al, 2012;Tushir et al, 2010;Zangari et al, 2014) that is in good agreement with our current observation demonstrating that Arf6 inhibition prevent PMCA4b-induced pre-lumen formation in 2D cell cultures (Fig. 6E, F).…”

GLT-1a glutamate transporter nanocluster localization is associated with astrocytic actin and neuronal Kv2 clusters at sites of neuron-astrocyte contact

“…Furthermore, GLT-1 transporters are rapidly internalized under conditions of high extracellular glutamate, such as in ischemia and traumatic brain injury ( Ibáñez et al, 2016 ). Given the potential importance of actin in endocytosis ( Francis et al, 2023 ; Wu and Wu, 2023 ; Yu and Yoshimura, 2023 ), localizing GLT-1 nanoclusters near actin filaments could be a mechanism to swiftly internalize glutamate-bound transporters unable to function due to ionic gradient perturbations in pathophysiological conditions. Additionally, nanocluster perturbation via cholesterol depletion decreased transport efficiency by ∼30%, suggesting that nanoclustering of GLT-1 may be functionally relevant for transporter function ( Raunser et al, 2006 ).…”