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Evan Boitet; NUDC is involved in protein trafficking to the primary cilium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5516.
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© ARVO (1962-2015); The Authors (2016-present)
Nuclear distribution protein C (NUDC) is known to localize to motile cilia however, it has long been accepted that this protein is excluded from primary cilia. Prior studies from our lab have shown NUDC is involved in a direct interaction with rhodopsin, which is dependent on the accessibility of rhodopsin’s C-terminus. To determine if NUDC is critical for the proper trafficking of rhodopsin from Golgi to the primary cilium we preformed in vitro assays altering expression levels of NUDC and analyzing the resulting localization of rhodopsin.
hTERT-RPE cells were cultured in 10cm dishes in DMEM/F-12 (1:1) medium with 10% fetal bovine serum (FBS) and 1% antibiotics. Cells were grown to 70% confluency before transfection via electroporation with shRNAs directed against NUDC, mCherry-NUDC, rhodopsin and rhodopsin ADRP mutants including: Q344X, Ter349E, and P23H. Directly after transfection, cells were placed in 6-well plates in media described above. Serum starvation of the transfected cells was carried out 24-hours post-transfection to induce cell polarization. Cells were kept in DMEM/F-12 (1:1) medium with 1% antibiotics and no FBS for 48 hours, at which time cells were then processed for immunohistochemical analysis. Cells were fixed in 4% paraformaldehyde at room temperature and labeled for rhodopsin (B6-30N), NUDC (rabbit α-NUDC monoclonal), and cilia (arl13b).
Based on results from immunohistochemical cell analysis, expression of NUDC is required for the formation of the primary cilium in hTERT-RPE cells. Surprisingly, we also found that NUDC localizes to the primary cilium when full-length, properly folded rhodopsin is expressed, however NUDC was not detected in the primary cilium when mislocalizing rhodopsin mutants were expressed in these cells.
Formation of the primary cilium in hTERT-RPE cells is dependent on the expression of NUDC. We provide evidence for the first time that NUDC localizes to the primary cilium, however this interaction was dependent upon rhodopsin being co-expressed. These data support our previous findings that NUDC and rhodopsin directly interact and strengthens the hypothesis that NUDC is critical to proper rhodopsin trafficking.
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