Technical Reference #2069

2069.

The N-Myc Down Regulated Gene1 (NDRG1) Is a Rab4a Effector Involved in Vesicular Recycling of E-Cadherin Sushant K. Kachhap; Dennis Faith; David Z. Qian; Shabana Shabbeer; Nathan L. Galloway; Roberto Pili; Samuel R. Denmeade; Angelo M. DeMarzo; Michael A. Carducci, Johns Hopkins University School of Medicine, PLoS ONE, (2007)
Link To Paper

Abstract:
Cell to cell adhesion is mediated by adhesion molecules present on the cell surface. Downregulation of molecules that form the adhesion complex is a characteristic of metastatic cancer cells. Downregulation of the N-myc down regulated gene1 (NDRG1) increases prostate and breast metastasis. The exact function of NDRG1 is not known. Here by using live cell confocal microscopy and in vitro reconstitution we report that NDRG1 is involved in recycling the adhesion molecule E-cadherin thereby stabilizing it. Evidence is provided that NDRG1 recruits on recycling endosomes in the Trans Golgi network by binding to phosphotidylinositol 4-phosphate and interacts with membrane bound Rab4aGTPase. NDRG1 specifically interacts with constitutively active Rab4aQ67L mutant protein and not with GDP-bound Rab4aS22N mutant proving NDRG1 as a novel Rab4a effector. Transferrin recycling experiments reveals NDRG1 colocalizes with transferrin during the recycling phase. NDRG1 alters the kinetics of transferrin recycling in cells. NDRG1 knockdown cells show a delay in recycling transferrin conversely NDRG1 overexpressing cells reveal an increase in rate of transferrin recycling. This novel finding of NDRG1 as a recycling protein involved with recycling of E-cadherin will aid in understanding NDRG1 role as a metastasis suppressor protein.

Materials & Methods:
HEK293 cells were transfected with NDRG1DsRed2 constructs using Lipofectamine 2000 reagent and stable clones were selected using G418. For recycling experiments HEK293-DsRed2 cells were plated on glass bottom petridishes (MatTek Corporation) allowed to adhere for 12 h. Cells were serum starved for 1h before being pulsed with Alexa flour-488 conjugated transferrin (45 mg/ ml) for 5 min to load the early endosomes and 60 min to load the recyling endosomes. Recycling was initiated after washing cells in PBS and adding excess of unconjugated iron-saturated holo transferrin (1mg/ml). Live Cell confocal microscopy was carried out using Utraview LCI (Perkin Elmer) equipped with Spinning Nipkow disk with microlenses. Cells were viewed using a 1006 objective. Images were grabbed every second in the temporal module for 1–5 min using a LSI-cooled 12-bit CCD camera and processed using the NIH ImageJ software (http://rsb.info.nih. gov/ij/download.html) before being made into a movie. For experiments involving E-cadherin trafficking E-cadherinEGFP constructs (a kind gift by Dr. James Nelson Stanford University) were transfected in NDRG1DsRed2-HEK293 cells. Twenty four hours post transfection cells were imaged as above or chelated using EDTA (2.5 mM) for 30 min and plated on glass bottom petri dishes and then imaged as above.

Microscopic Technique
Live Cell Confocal Microscopy

Cell Type(s)
HEK293 cells