LIMK1 activity regulates Rab5 endosome trafficking

Abstract

The endosomal recycling pathway involves the regulated balance of two processes; recycling endocytosed proteins to the cell membrane and targeting such proteins for lysosomal degradation. The recycling pathway alters the composition of plasma membrane proteins such as cell-adhesion molecules (CAMs). While cadherin endocytosis is essential for developmental processes, its deregulation has been known to contribute to cancer metastasis. Motor proteins are required for endocytic trafficking by tethering to membranes and moving them along cytoskeletal filaments, thus providing the force required for membrane deformation and scission which is responsible for promoting endosomal sorting and the formation of transport intermediates. LIM domain kinase 1 (LIMK1) has been extensively described to regulate actin dynamics by phosphorylating cofilin. However, its role on microtubule cytoskeleton remains to be elucidated. Hence, investigation is done on the role of LIMK1 activity on dynein and endocytic recycling in this study. As an initial step to elucidate the role of LIMK1 on dynein, GST pull-down and co-immunoprecipitation is performed to demonstrate LIMK1 association with dynein. In addition, live cell imaging of HeLa cells over-expressing constitutively active LIMK1-T508EE displayed a marked retardation in the trafficking speed of Rab5 particles, which are present on early endosomes. Internalisation of N-cadherin was also perturbed in LIMK1-T508EE overexpressing cells.