Intracellular transport of ganglioside GD3 in CHO-K1 cells

RAMIRO IGLESIAS- BARTOLOMÉ; PILAR M. CRESPO; JOSÉ L. DANIOTTI

Carlos Paz, Córdoba, Argentina

Workshop; International Workshop on Membrane Trafficking; 2005

INTRACELLULAR TRANSPORT OF GANGLIOSIDE GD3 IN CHO-K1 CELLS Ramiro Iglesias-Bartolomé, Pilar M. Crespo y José Luis Daniotti CIQUIBIC (UNC-CONICET), Fac. de Cs. Químicas, UNC, Cba., Argentina. E-mail: ramiro@dqb.fcq.unc.edu.ar Gangliosides, complex glycosphingolipids containing sialic acids, are synthesized in the lumen of the Golgi complex. We previously found that ganglioside GD3 traffics from this organelle to plasma membrane by a BFA-insensitive exocytic pathway in CHO-K1 cells. Moreover, dominant negative form of Rab11, which prevent exit of VSVG from the Golgi complex, did not influence the capacity of GD3 to reach the cell surface. After their arrival to the plasma membrane, gangliosides can undergo endocytosis. Internalization involves sorting processes and has an important role in cells, since accumulation of glycosphingolypids in numerous glycosphingolypid storage diseases disrupts normal endocytic transport. The movement of plasma membrane glycolipids along the endocytic pathway has been examined by using fluorescent-labeled lipid analogues, labeleded toxins or radioctive-labeled lipids. In addition, antibody-binding techniques were extensible used to follow endocytic transport of many proteins. In this work we characterize the endocytic transport of ganglioside GD3 by using the well-known anti-GD3 mouse monoclonal antibody R-24 in CHO-K1 clones expressing GD3. CHO-K1 cells were incubated with R24 antibody at 4ºC to inhibit endocytosis. Then, cells were washed and transferred at 37°C to allow endocytosis for different times. By biochemical techniques, immunofluorescence and confocal microscopy we observe that 55% of GD3-R24 complex was endocyted in 15 min and began to accumulate in an intracellular perinuclear compartment. This compartment colocalized  with two markers for the recycling endosome: Rab11 and transferrine, but not colocalized significantly with GalNacT, a trans Golgi network (TGN) marker. After 60 min of endocytosis it was not possible to detect R24 antibody, unless the antibody was constantly present in the culture medium. In order to test if the bulk of endocyted R-24 antibody is being rapidly degraded at lysosomes, we used the inhibitors of lysosome degradation NH4Cl and chloroquine. None of the drogues could prevent R24 depletion, indicating that the antibody is not degraded in lysosomas. Besides, we demonstrated that GD3-R24 complex exit the recycling compartment by a Brefeldin A and Monensine sensitive pathway. These results indicate that GD3-R24 complex is rapidly endocyted in CHO-K1 cells, accumulate in recycling endosome and could return directly to plasma membrane or indirectly throw through Golgi complex. Financiado por: Fundación Antorchas, CONICET, SECyT-UNC, FONCYT.