INTRODUCTION

Macropinocytosis is a regulated form of endocytosis that provides cells with a way to non-selectively uptake large amounts of extracellular fluid and membrane. It is initiated by growth-factor stimulation, leading to an increase in actin polymerization at the cell surface, resulting in actin-mediated ruffling and the formation of macropinosomes.

In response to growth-factor stimulation, Sorting Nexin-5 (SNX-5) is transiently recruited to the plasma membrane, initiating the formation of macropinosomes.

Concentration of PI(4,5)P2 increases on membrane ruffles before decreasing with the conversion to PI(3,4,5)P3 on the cell surface. Sorting Nexin-18 (SNX-18) elevates macropinocytosis by preferentially binding and indirectly facilitating the conversion of PI(4,5)P2 into PI(3,4,5)P3.

The interaction between cytosolic Sorting Nexin-33 (SNX-33) with Wiskott-Aldrich Syndrome protein (WASp) drives actin polymerisation in the pernuclear space, initiating membrane ruffling and macropinocytosis.

The large size of macropinosomes enables easy identification through the use of fluid phase markers such as 10,000 Da dextran.

10,000 Da dextran, upon internalisation by macropinosomes, elicits a bright, red-fluorescence, which can be quantified by epi-fluorescent microscopy.

HYPOTHESIS

Transfection of A431 cells with SNX-5 dominant negative mutant pDNA will inhibit the initiation of macropinosome formation, leading to the inhibition of macropinocytosis and a decrease in 10,000 Da dextran uptake, as quantified by epi-fluorescent microscopy.

Transfection of A431 cells with SNX-18 dominant negative mutant pDNA will inhibit the conversion of PI(4,5)P2 into PI(3,4,5)P3, leading to the inhibition of macropinocytosis and a decrease in 10,000 Da dextran uptake, as quantified by epi-fluorescent microscopy.

Transfection of A431 cells with SNX-33 dominant negative mutant pDNA will inhibit membrane ruffling, leading to the inhibition of macropinocytosis and a decrease in 10,000 Da dextran uptake, as quantified by epi-fluorescent microscopy.

PROPOSED METHODS

A431 cells will be seeded onto 8 glass coverslips in a 24-well dish and maintained in Dulbecco’s Modified Eagle Medium (DMEM)+10% Fetal Bovine Serum (FBS).

Prior to transfection, DMEM+10% FBS in each well will be replaced with 400μL DMEM+10% FBS. Transfection reagents for each well will be prepared separately by incubating 50μL of DMEM+DNA (0.8μg/well) and 50μL of DMEM+Lipofectamine2000

(1.5μL/well) independently for 5 minutes at room temperature. The reagents will be mixed and incubated for another 20 minutes before adding into the wells. The cells will be left to incubate for 24 hours at 37°C.

Uptake of 10,000 Da dextran will be performed by removing transfection reagents from all wells before adding 500μL of DMEM+10% FBS and dextran (0.2mg/mL) into each well. The cells will be left to incubate for 10 minutes at 37°C and washed 3 times with Phosphate-Buffered Saline (PBS) before fixing with 4% Paraformaldehyde (PFA)/PBS. The cells will be left at room temperature and stored for 24 hours at 4°C.

The coverslips will be mounted onto glass slides before epi-fluorescence microscopy. Four images will be collected in green and red channels (2 images/channel) and merged using ImageJ. Analysis will be carried out by quantifying 10,000 Da dextran in successfully transfected cells.