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L. Qian, C.M. Rose, J. Xie, E. Sou, T. Nakamura, S.F. Hamm-Alvarez, A.K. Mircheff; Glycolipid-rich Membrane Microdomains in Lacrimal Acinar Cell Endomembrane Compartments . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2521.
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Purpose: Rabbit lacrimal acinar cells express unusually high levels of ß-galactosidase (Nowroozi et al., 2000, Mircheff et al., 2000), which implies the presence of significant glycosphingolipid (GSL)-rich membrane microdomains. Our goal was to determine the presence of GSL-rich microdomains and whether these microdomains might play important roles in some aspect of endomembrane traffic. Methods: GSL-rich membrane microdomains resist solubilization in Triton X-100, and we used this phenomenon to assay for their presence. On the other hand, methyl-ß-cyclodextran (MBCD) extracts the cholesterol from membrane lipids and disrupts the lateral segregation of membrane microdomains. Primary cultured lacrimal acinar cell were pretreated with 1mM MBCD for 20 m and then incubated with [125I]-EGF for 20 m or [125I]-BSA for 120 m. Cells were washed, lysed, and analyzed by isopycnic centrifugation on sorbitol density gradients. Results: GSL-rich microdomains contributed most substantially to the composition of the basal-lateral membrane (blm), basal-lateral recycling endosome (blre) and blm related trans-Golgi network (tgn-blmr) compartments, but they were also present in the basal-lateral sorting endosome (blse) and Golgi compartments, and they contributed in progressively smaller amounts to the tgn compartments involved in traffic to secretory vesicles and pre-lysomes (tgn-lr,svr), the secretory vesicles (svm), and the pre-lysosomes (preLys). Triton-insoluble microdomains accounted for 50% the acid phosphatase in the blse, 40% in the Golig, 28% in the svm, 23% in the tgn-lr,svr, and 15% in the preLys. MBCD treatment increased accumulation of [125I]-EGF and [125I]-BSA in the blre, tgn and Golgi compartments, apparently by impairing traffic to the lysosomal compartments. Conclusion: Our results suggest that multiple glycosphingolipid-rich microdomains exist in acinar cell endomembrane compartments. Lateral partitioning between GSL-rich and phospholipid-rich microdomains may play a role in sequestration of intracellular autoantigens by determining sorting events related to formation of transport vesicles in the sorting endosome or to retention in the recycling endosome.
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