Characterization of Cystinosin Intraellular Trafficking

Characterization of Cystinosin Intracellular Trafficking
Progress report – March 2007

Research project conducted at Inserm U574 (Necker Hospital, Paris)
Principal investigator: Corinne Antignac
Persons working on the project:
            Dr Anne Bailleux (PhD – funded by the Cystinosis Research Foundation)
            Nathalie Nevo (technician, Inserm funded)

Background and objectives
The global aim of the research project is to characterize intracellular trafficking of cystinosin. The specific aims of the projects are:
1. To characterize how cystinosin is sorted to the lysosome:

2. To identify proteins interacting with cystinosin

Update on the progress of research plan

Aim 1 : Sorting of cystinosin to the lysosome
- MDCK and CaCo-2 cell lines stably expressing cystinosin-GFP or cystinosine-HA have been generated in the laboratory. In all cases, the tag is located at the C-terminus of the protein, since we have been unable to express constructs with HA inserted in the second inter-TM loop.
- To assess which pathway is used by cystinosine to reach the lysosomes and to identify which adaptor protein complexes are used, we want to inactivate each component of the adaptor protein complexes by RNA interference. Transfection of plasmids which contain sequences encoding short-hairpin RNA (shRNA) molecules specific of AP1 ?-adaptin and of AP2 µ2-adaptin has no measurable effect in MDCK and Caco-2 cells probably because of the poor efficiency of transfection. We thus decided to use a lentiviral vector system derived from HIV-1 to express shRNAs directed against AP1 and AP2 in tagged-cystinosin containing cells. In addition, enhanced GFP (EGFP) is incorporated in the system as a reporter gene, allowing easy check of transduction efficacy. In preliminary experiments, efficient transduction was observed, with more than 90% of cells transduced. We are now producing high-titre lentivirus batches in order to proceed to the next steps of the project.

Aim 2: Identification of proteins interacting with cystinosin<
A lot of effort has been put on the setting up of the experimental procedures, which needed many adjustments, given the structure of cystinosin as a highly glycosylated, seven-transmembrain domain containing protein.
During the last six months, we have made the following progresses:
- We had initially planned to work on lysosomes purified by Percoll gradients, but we were unable to obtain a dry pellet of lysosomes, which was needed for BN-PAGE. We thus turned to the use of intracellular membrane pellets, which, as an additional advantage, contain not only lysosomes, but the various cell fractions involved in the intracellular traffic of membranes proteins. To obtain intact lysosomes in the pellet, we have tested several experimental conditions, especially by testing numerous detergents, various conditions of centrifugation… and have shown that using DDM 0.05% allows the isolation of a membrane pellet that contains, on average, 46% of intact lysosomes (mean obtained from 27 experiments), with a three time enrichment in cystinosin-GFP compared to the total cell extract.
- Blue-native polyacrylamide gel electrophoresis (BN-PAGE) permits to separate intact and functional membrane protein complexes. This technique offers the advantage of separating native protein complexes present in membrane protein samples without dissociating them.
The pellet of intracellular membranes obtained with DDM 0.05% was solubilized with NP40 1%, sonicated and submitted to BN-PAGE. Preliminary experiments revealed the presence of cystinosine-GFP on the BN-PAGE. Now, we have to analyze the size of the cystinosine-GFP containing complexes and to define the quantity of materiel needed to identify the partners of cystinosine-GFP.
- Co-immunoprecipitation is the alternative method we choose to identify the partners of cystinosine. HA- and GFP- tagged-cystinosin was immunoprecipited using antibodies directed against the tag (HA or GFP), but the experiments were hampered by the heavy background. We think we have overcome this difficulty by using cleaner protein preparation (see above) and by using magnetic beads directly coupled to monoclonal antibodies directed against GFP or HA, avoiding the secondary antibody.