RT Journal Article
SR Electronic
T1 A high-content endogenous GLUT4 trafficking assay reveals new aspects of adipocyte biology
JF Life Science Alliance
JO Life Sci. Alliance
FD Life Science Alliance LLC
SP e202201585
DO 10.26508/lsa.202201585
VO 6
IS 1
A1 Alexis Diaz-Vegas
A1 Dougall M Norris
A1 Sigrid Jall-Rogg
A1 Kristen C Cooke
A1 Olivia J Conway
A1 Amber S Shun-Shion
A1 Xiaowen Duan
A1 Meg Potter
A1 Julian van Gerwen
A1 Harry JM Baird
A1 Sean J Humphrey
A1 David E James
A1 Daniel J Fazakerley
A1 James G Burchfield
YR 2023
UL https://www.life-science-alliance.org/content/6/1/e202201585.abstract
AB Insulin-induced GLUT4 translocation to the plasma membrane in muscle and adipocytes is crucial for whole-body glucose homeostasis. Currently, GLUT4 trafficking assays rely on overexpression of tagged GLUT4. Here we describe a high-content imaging platform for studying endogenous GLUT4 translocation in intact adipocytes. This method enables high fidelity analysis of GLUT4 responses to specific perturbations, multiplexing of other trafficking proteins and other features including lipid droplet morphology. Using this multiplexed approach we showed that Vps45 and Rab14 are selective regulators of GLUT4, but Trarg1, Stx6, Stx16, Tbc1d4 and Rab10 knockdown affected both GLUT4 and TfR translocation. Thus, GLUT4 and TfR translocation machinery likely have some overlap upon insulin-stimulation. In addition, we identified Kif13A, a Rab10 binding molecular motor, as a novel regulator of GLUT4 traffic. Finally, comparison of endogenous to overexpressed GLUT4 highlights that the endogenous GLUT4 methodology has an enhanced sensitivity to genetic perturbations and emphasises the advantage of studying endogenous protein trafficking for drug discovery and genetic analysis of insulin action in relevant cell types.