Golgi localized Arl15 regulates cargo transport, cell adhesion and motility

Abstract

Arf-like GTPases (Arls) regulate membrane trafficking and cytoskeletal organization. Genetic studies predicted a role for Arl15 in type-2 diabetes, insulin resistance, adiposity, and rheumatoid arthritis. Recent studies indicate a possible role for Arl15 in multiple physiological processes, including magnesium homeostasis. However, the molecular function of Arl15 is poorly defined. We evaluated the role of Arl15 in vesicular transport using techniques to quantify cargo trafficking, to mechanobiology. Fluorescence microscopy of stably expressing Arl15-GFP HeLa cells showed its localization to the Golgi and cell surface, including filopodia, and a cohort to recycling endosomes. The dissociation of Golgi, using small molecular inhibitors or the expression of Arf1 dominant-negative mutant, completely mislocalized Arl15 to the cytosol. Interestingly, site-directed mutagenesis analysis identified a novel V80A mutation in the GTP-binding domain that turns Arl15 into a dominant-negative form with a reduced number of filopodia. Depletion of Arl15 in HeLa cells caused mislocalization of cargo, such as caveolin-2 and STX6, from the Golgi. Arl15 knockdown cells displayed reduced filopodial number, altered focal adhesion kinase organization, and enhanced soluble and receptor-mediated cargo uptake without affecting the TfR recycling. Arl15 knockdown decreased cell migration and enhanced cell spreading and adhesion strength. Traction force microscopy experiments revealed that Arl15 depleted cells exert higher tractions and generate multiple focal adhesion points during the initial phase of cell adhesion as compared to control cells. Collectively, these studies demonstrated a functional role for Arl15 in the Golgi, which includes regulating cargo transport to organize membrane domains at the cell surface.

Cleft palate and minor metabolic disturbances in a mouse global Arl15 gene knockout

“…The shorter length ARL15 translated from isoform Arl15 ‐202 (missing exon 3 of the Arl15 ‐201 transcript) does not have a Switch II domain therefore might result in a protein that has a diverged function. Also, the Gln (Q) of DxxGQ motif which normally carries intrinsic GTP hydrolysis activity is not conserved in ARL15 (Glut(Q) to Ala (A)), thus ARL15 may not have any intrinsic GTP hydrolysis activity 14,15 . Comparing human ARL15 and KRAS proteins, the latter has a conserved Q residue and shows weak intrinsic GTP hydrolysis activity (Figure S3B).…”

“…22 2. 15 | Mouse embryonic fibroblast (MEFs) culture Timed matings were set up and females were plug checked. Embryos that at 13.5 days post coitum (dpc)…”

“…Also, the Gln (Q) of DxxGQ motif which normally carries intrinsic GTP hydrolysis activity is not conserved in ARL15 (Glut(Q) to Ala (A)), thus ARL15 may not have any intrinsic GTP hydrolysis activity. 14,15 Comparing human ARL15 and KRAS proteins, the latter has a conserved Q residue and shows weak intrinsic GTP hydrolysis activity (Figure S3B). In order to evaluate ARL15 activity further, we then carried out a GTPases assay comparing the mouse wildtype A86A with A86Q and a mutant A86L that locks ARL15 in a GTP bound active (GTP nonhydrolysable) form.…”

“…14 A recent study revealed its molecular function in cargo transport, adhesion and cell motility which is ARF1 dependent. 15 However, its functional role in a mammalian in vivo system remains unknown.…”

“…In addition, ARL15 function has also been linked to insulin secretion in a human pancreatic β cell line, 9,10 insulin signaling, 11 tumor suppression in the mouse, 12 upregulation of adiponectin in synovial tissue, 13 Mg 2+ uptake in kidney cell lines 4 and upregulation of TGFβ signaling 14 . A recent study revealed its molecular function in cargo transport, adhesion and cell motility which is ARF1 dependent 15 . However, its functional role in a mammalian in vivo system remains unknown.…”