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Markus N. Preising, Caroline Pasquay, Birgit Lorenz; Localization of the Human P-Protein to a Subset of the Microtubuli Network Indicates Involvement in Intracellular Transport of L-DOPA Carrying Vesicles. Invest. Ophthalmol. Vis. Sci. 2011;52(14):877.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the human OCA2 gene underlie autosomal recessive ocular and oculocutaneous albinism. OCA2 encodes the P-protein a transmembrane protein of the melanosomal membrane. No function could be associated with the P-protein by now. Predictions based on the domain structure of P-Protein indicate a function in transmembrane transport of small molecules or ions. To approach the impact of mutations in OCA2 we investigated the localization of intrinsic P-protein expression in ARPE19 cells.
ARPE19 cells (ATCC No. CRL-2302) were seeded in 6-well plates at passages three, four and 10. After growth to sub-confluence the cells were probed with a P-protein specific antibody (Biozol, OCA2, H00004948-M02) to show the localization of the intrinsic expression of OCA2 in ARPE19 cells. Antibodies against α-tubulin (Santa Cruz, α-tubulin (YOL1/34), sc-53030) and L-DOPA (Abnova, Anti- Dopamin, 9201036372) were applied to identify possible structures involved. Antibodies were probed with fluorescence labeled secondary antibodies and visualized using an inverse fluorescence microscope. All antisera were applied in a dilution of 1:100. Experiments were performed in triplicate.
Anti-P-ab labeled structures within ARPE19 cells indicating microtubule co-localization and final distribution to the plasma membrane. Probing with α-tubulin showed a distribution over the plasma membrane which was also seen with L-DOPA-ab. α-tubulin and L-DOPA immunofluorescence correlated with P-protein immunofluorescence at the plasma membrane. The mircotubuli-like structures could not be targeted with the referral antibodies applied.
Colocalization of P-protein from intrinsic OCA2 expression with α-tubulin and L-DOPA in ARPE19 cells was shown at the plasma membrane. Further structures labeled by anti-P-ab resembled microtubule structures indicating a function of P-protein in transport processes in ARRP19 cells. Further correlations including actin and actin-interacting proteins are underway.
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