Defining the layers of a sensory cilium with STORM and cryoelectron nanoscopy

Abstract

Primary cilia carry out numerous signaling and sensory functions, and defects in them, “ciliopathies,” cause a range of symptoms, including blindness. Understanding of their nanometer-scale ciliary substructures and their disruptions in ciliopathies has been hindered by limitations of conventional microscopic techniques. We have combined cryoelectron tomography, enhanced by subtomogram averaging, with superresolution stochastic optical reconstruction microscopy (STORM) to define subdomains within the light-sensing rod sensory cilium of mouse retinas and reveal previously unknown substructures formed by resident proteins. Domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of subcompartments, including the lumen and walls of the axoneme, the membrane glycocalyx, and the intervening cytoplasm. Within this framework, we report spatial distributions of key proteins in wild-type (WT) mice and the effects on them of genetic deficiencies in 3 models of Bardet–Biedl syndrome.

Compartmentalization of Photoreceptor Sensory Cilia

“…Interestingly, the tomograms also revealed a structure resembling the terminal plate found in motile cilia ( Figure 4A ). Super-resolution microscopy of immunolabeled proteins revealed concentric layering of the CC ( Figure 4B ) (Robichaux et al, 2019 ). Overlapping localization of IFT88, IFT81, and BBS5 distribution across CC was also observed ( Figure 4C ), suggestive of IFT trains.…”

“…Recent advances in super-resolution fluorescence microscopy and high-resolution cryo-TEM tomography have revealed detailed structure of the TZ and localized protein composition at nanometer resolution ( Figure 4 ) (Gilliam et al, 2012 ; Yang et al, 2015 , 2018 ; Shi et al, 2017 ; Robichaux et al, 2019 ; Sun et al, 2019 ). An elegant STORM-based study showed detailed localization of 12 distal appendage-associated molecules and 4 additional proteins surrounding the distal appendages (Yang et al, 2018 ) and super-resolution STED imaging mapped the distribution of TZ complex proteins in primary cilia (Yang et al, 2015 ).…”

“…An elegant STORM-based study showed detailed localization of 12 distal appendage-associated molecules and 4 additional proteins surrounding the distal appendages (Yang et al, 2018 ) and super-resolution STED imaging mapped the distribution of TZ complex proteins in primary cilia (Yang et al, 2015 ). Studies of the CC demonstrated structural details of basal bodies and the axoneme, showing microtubule triplets and doublets, respectively, along with distal appendages (Gilliam et al, 2012 ; Robichaux et al, 2019 ). Nine-fold averaging of cryo-EM tomograms revealed Y link-like structures in the lumen of the CC ( Figure 4A ), a hallmark of the TZ, although the structures appeared to be formed from two independent structures rather than the single structure observed in standard TEM studies in primary cilia (Robichaux et al, 2019 ).…”

“…Studies of the CC demonstrated structural details of basal bodies and the axoneme, showing microtubule triplets and doublets, respectively, along with distal appendages (Gilliam et al, 2012 ; Robichaux et al, 2019 ). Nine-fold averaging of cryo-EM tomograms revealed Y link-like structures in the lumen of the CC ( Figure 4A ), a hallmark of the TZ, although the structures appeared to be formed from two independent structures rather than the single structure observed in standard TEM studies in primary cilia (Robichaux et al, 2019 ). Interestingly, the tomograms also revealed a structure resembling the terminal plate found in motile cilia ( Figure 4A ).…”

“…In photoreceptors, IFT is involved in the development and maintenance of photoreceptor OSs (Pazour et al, 2002 ) and is proposed to be involved in the trafficking of opsins and membranes destined for the OS discs or lamellae (Pearring et al, 2013 ; Wheway et al, 2014 ; Imanishi, 2019 ). IFT52, 57, 88, and 20 have been identified along the OS axoneme and within the IS of Xenopus photoreceptors (Luby-Phelps et al, 2008 ) and IFT88 and 81 in the CC of mouse photoreceptors (Robichaux et al, 2019 ) ( Figure 4 ). Mutations in IFT proteins have been shown to cause mislocalization of rhodopsin and accumulation of membrane vesicles in the IS.…”