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Michael Robichaux, Theodore G Wensel; Uncovering the layers of the connecting cilium with super-resolution nanoscopy. Invest. Ophthalmol. Vis. Sci. 2017;58(8):360.
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© ARVO (1962-2015); The Authors (2016-present)
The microtubule doublets of the axoneme in the connecting cilium (CC) of retinal rods create subcellular boundaries or layers whose arrangement enables essential but poorly understood trafficking and signaling events. To date, analysis of these layers has been limited mostly to electron microscopy. Our goals were to test the ability of immunofluorescence and super-resolution nanoscopy to determine the locations of specific proteins within these layers and to use this approach to observe changes subcellular localization of CC proteins in mutant ciliopathy models of Bardet-Biedl syndrome (BBS).
STORM (STochastic Optical Reconstruction Microscopy) nanoscopy was performed on ultra-thin sections of WT mouse retinae immunostained with the following antibodies as markers for the subcellular layers within the CC of rods: centrin-2, acetylated-α tubulin, and IFT88. Dye-conjugated WGA was used as a CC surface marker. Antibodies for BBS5 and syntaxin-3 were used to localize these proteins within the cell and the CC layers in WT and BBS2, BBS4 and BBS7 knockout retinae.
In WT mouse rods, we developed 2-color STORM to elucidate 4 distinct layers of the CC by measuring reconstructed super-resolution clusters for each structural marker. The average diameter of the centrin-2 cluster, marking the interior layer of the axoneme, was 152 nm, while the microtubule axoneme cluster average diameter was 268 nm. IFT88 clusters extend ~50 nm from the outside edge of the axoneme, and the WGA CC membrane surface cluster extends the diameter of the final CC layer to >300 nm. We localized syntaxin-3 and BBS5 to the IFT88 subcellular layer on the outside of the axoneme in WT CC. In the BBS mutant retinae, syntaxin-3 is grossly mislocalized to the OS; however, it is still correctly localized within the CC. Surprisingly, BBS5 is not mislocalized grossly or subcellularly in any of the three BBS mutant models.
These results establish the power of super resolution nanoscopy to localize proteins to subcompartments within the CC and elucidate novel subcellular detail. Localizing syntaxin-3 and BBS5 in WT and BBS ciliopathy mutant rods provides new subcellular information for each protein. Specifically, BBS2, BBS4 and BBS7 were found to be essential for preventing improper syntaxin-3 accumulation in the OS, but not for correct subcompartment localization of either syntaxin-3 or BBS5 within the CC.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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