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Michael Robichaux, Theodore G Wensel; STORM super resolution imaging of the connecting cilium in thin retinal sections. Invest. Ophthalmol. Vis. Sci. 2016;57(12):571.
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© ARVO (1962-2015); The Authors (2016-present)
The connecting cilium (CC) is a 1µm portion of the primary cilium in rod photoreceptor cells that is the site of protein trafficking to and from the outer segment membrane disks. Imaging localization of ciliopathy proteins, which are essential for visual protein trafficking and rod photoreceptor cell viability, to the CC is particularly difficult because conventional microscopy with limited resolution power is unable to localize targets within the 300 nm diameter of the CC or the adjacent basal body complex. The goal of this study is to adapt immunostaining methods for STORM imaging of the CC in the context of mouse retinal sections to overcome this resolution limit for super resolution localization.
Isolated unfixed retinas are specially processed for immunostaining and penetration into the restrictive CC with specific antibodies and secondary antibodies that are conjugated to photoswitching fluorophores necessary for STORM (e.g. Alexa647). Stained retinas are post-fixed and embedded into epoxy resin blocks for ultramicrotome sectioning and subsequent STORM acquisition.
As a marker of the CC, centrin-2 is accurately reconstructed by STORM analysis along the 1µm length of the structure and within the 100nm width of the luminal space between the axonemal microtubules of the CC. To test the possibility of localizing ciliopathy proteins to the CC with this method, retinal sections were co-immunostained with a validated BBS5 antibody and centrin-2. BBS5 positive puncta localized along the edge of the CC and to within a distance of 20nm from the centrin-2 positive CC lumen.
By establishing the feasibility of this STORM localization approach, sub-localization of ciliopathy proteins, such as those associated with the Bardet-Biedl syndrome (BBS), can be more accurately studied in the physiological context the mouse retina. The function of these and many other photoreceptor factors associated with the CC are characterized incompletely, in part, because accurate localization, as presented here, was not available. Specific to these findings, the measured distance between the BBS5 reconstructed puncta, which likely represent the BBSome complex of multiple BBS proteins, and centrin-2 matches the width of the microtubule doublets within the CC, suggesting that BBS5 traffics along the CC on the outer face of the axonemal microtubules.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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