Unravelling the functional role of Arf-like GTPases 14 and 15 in mammalian cells

Abstract

Small G-proteins of Arf-like (Arl) GTPase subfamily are shown to regulate several cellular processes including intracellular trafficking, cytoskeletal organization, organelle biogenesis, cell adhesion and migration. Around 21 genes belong to this family have been identified in human. However, the critical function of Arl14 and Arl15 in cargo transport was unclear. In this study, we have attempted to characterize the role of Arl14 and Arl15 in multiple cellular processes, including intracellular trafficking using HeLa cells.

Objective I: Elucidating the role of Arl15 in modulating cell adhesion, motility and filopodia biogenesis.

Our study characterized the intracellular localization of Arl15 using epitope tagged Arl15-GFP in multiple mammalian types. We have observed that Arl15-GFP localizes to Golgi, plasma membrane (PM) including filopodia, and a cohort to recycling endosomes in HeLa, A549, neuro 2a, and primary keratinocytes. Additionally, we noticed the localization of Arl15 to long extracellular tube structures (resembling tunneling nanotubes, TNTs) connecting the Neuro 2a cells. The dual localization of Arl15 to Golgi and PM is independent of the actin cytoskeleton, but it is dependent on Golgi integrity. The dissociation of Golgi using small molecular inhibitors or the expression of Arf1 dominant-negative mutant completely mislocalizes Arl15 to the cytosol. We identified a novel V80A mutation in the GTP-binding domain that turns the Arl15 into a dominant-negative form and results in a reduced number of filopodia. Depletion of Arl15 in HeLa cells causes mislocalization of cargo such as caveolin-2, STX6, and ectopically expressed GFP-GPI from Golgi and accumulation of lipid droplets. Further, Arl15 knockdown cells display reduced filopodial number, dispersion of vinculin localization (focal adhesion kinase), and enhanced soluble and receptor-mediated cargo uptake without affecting the recycling kinetics. In addition, Arl15 knockdown decreases cell migration and increases cell adhesion, and displays enhanced cell spreading. Traction force microscopy studies revealed that Arl15 depleted cells exert higher traction force and generate multiple focal adhesion points. These studies demonstrated a function to Arl15 in Golgi, which regulates cargo transport to organize membrane domains at the cell surface to control cell migration, spreading and adhesion, including filopodial biogenesis.

Objective II: Studying the role of Arl14 in vesicular trafficking

Studies have suggested that Arl14 regulates the movement of MHC-II vesicles along the actin cytoskeleton in dendritic cells. We have studied the localization of Arl14 using epitope tagged Arl14-GFP in HeLa cells. Arl14 localizes to REs, late endosomes, and lysosomes. Expression of Arl14S27N-GFP showed no change in its localization, indicating that Arl14S27N-GFP is not acting as a dominant negative mutant while constitutive active mutant of Arl14 (Arl14Q68L-GFP) majorly localized as punctate structures that are clustered and positive for RFP-STX13 (represents endosomal structures). Depletion of Arl14 showed an enhanced number of LAMP-1-positive lysosomes without changing the localization of lysosome biogenesis transcription factors TFEB and TFE3. Immunoblotting analysis showed no change in Rab5, STX13, Rab11, annexinA2, LAMP-1, and LAMP-2 expression in siArl14 compared to siControl. Overall, these studies showed that Arl14 localizes to endo-lysosomal organelles, and its depletion altered the number of LAMP-1 positive compartments.