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Michael A Robichaux, Theodore G Wensel; Localization of the Bardet-Biedl syndrome protein complex in photoreceptor cilia.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5524.
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© ARVO (1962-2015); The Authors (2016-present)
Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy characterized by the dysfunction and loss of primary cilia, including the rod outer segment (ROS) sensory cilia of photoreceptor cells. BBS causes progressive retinal degeneration among other devastating symptoms. 8 essential BBS genes form the ciliary BBSome protein complex (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9 & BBIP10), which is hypothesized to regulate ciliary trafficking events within ROS cilia and other ciliated cells. One key limitation to determining the precise function of the retinal BBSome is the lack of sub-cellular localization data and co-localization with specific ciliary structures of photoreceptor cells, including the basal body centrioles and the connecting cilium (CC) (which measures 1000 nm in length and ~300 nm in diameter). Localization within submicron structures requires advanced imaging technologies such as cryo-electron tomography (cryo-ET), or super resolution imaging fluorescence microscopy, such as STORM. These tools will test our hypothesis that the retinal BBSome is specifically localized to the photoreceptor basal body and CC.
We have developed a protocol to use STORM super resolution imaging to image isolated photoreceptor cells, which enables the precise localization of the BBSome and other sub-cellular targets to a resolution of 20-30nm. Isolated photoreceptor cells were carefully dissociated from fresh mouse retinas and processed to preserve their morphology for STORM immunolabeling and imaging with validated antibodies.
Using this novel application of STORM, we have found that the retinal BBSome complex is specifically localized near the ROS basal body and CC. To validate the length and location of ROS structural markers, we imaged the centrin-2 positive CC, the acetylated microtubules of the ROS axoneme, and PCM-1, a marker for the basal body. Using these structural guides, we localized BBS4, BBS8 and BBS9 to discrete puncta at both ends of the CC, including near the PCM-1 basal body marker.
These results implicate a potential dual role for the BBSome within the basal body and at the base of the outer segment. Future localization analyses of the BBSome with other key ciliary targets will continue to inform functional studies of the retinal BBSome in photoreceptor cilia.
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