Previous studies of the 9 + 2 motile flagella from Chlamydomonas reinhardtii
, have shown that cryo-ET is a powerful tool for investigating the characteristic geometry of the eukaryotic axoneme (Nicastro et al., 2006
). Our 3D reconstructions of primary cilia in ice reveal a number of surprising features regarding the roles of individual disease genes, as well as insights into previously-known features. The quantitative parameters of the recurring features of the CC, which have not previously been measured accurately due to distortions from sample processing, are presented in . Some of the characteristics of rod cilia are general for primary cilia, and some, like the stacks of disks, are specific for photoreceptors.
Rootlet structures have been identified with a number of different eukaryotic cilia including reproductive tissue (Anderson, 1972
), airway epithelia (Yang et al., 2005
), and hair cell stereocilia (Kitajiri et al., 2010
). While rootlets are found in many ciliated cells, it is not clear whether rootletin is present in every case, nor whether the rootlet is always striated. Transport along the rootlet has not been previously reported, but we identify light-regulated changes in the dynamics of non-vesicle cargoes along the rootlet. Fibers connect the rootlet to the basal bodies, but absence of the rootlet does not alter the position of basal bodies or gross structure of the axoneme. The fragility observed at axonemal sites remote from the rootlet in rootletin-deficient rods suggests an indirect role in supplying components needed for stability. In this regard, the light-dependent dynamism of the rootlet-associated particles suggests a possible role in transport of such components. The fact that rootletin is widely expressed in mammalian tissues (Figure S1
) suggests rootletin may be important for the function of many non-photoreceptor cilia as well.
Our analysis has provided a glimpse into the organization of the periciliary region surrounding the CC and reveals the complexity of these structures. In the distal IS adjacent to the CC there is a dense network of transition fibers, rootlet fibers, and distal appendages associated with the basal bodies, transport vesicles, and periciliary plasma membrane. Transition fibers nucleate directly from basal body C-microtubules, are oriented perpendicular to the axoneme, and anchor the axoneme to the plasma membrane. We observed additional filaments, originating from either the rootlet or the IS cytoplasm, that form a filamentous/tubular structure parallel to the axoneme ( and ). These structures associate with vesicles and may participate in cargo sorting and transport through the CC. Many IFT-associated proteins are localized to this region (Sedmak and Wolfrum, 2010
), and it is likely that the filaments play some role in assembly of cargo-bearing IFT particles, and in vesicle fusion with the plasma membrane. Defects in proteins located at the ciliary membrane, including IFT proteins, are associated with a number of retinal ciliopathies. Mutations found in members of a complex network of structural and signaling proteins result in the combined deaf-blindness phenotype of Usher’s syndrome (Maerker et al., 2008
). This signaling network is located at the periciliary membrane complex (Yang et al., 2010
), a structure in the apical IS adjacent to the CC, which corresponds to the periciliary region in our tomograms. This complex is likely important for docking of rhodopsin transport vesicles for rhodopsin transport through the CC.
The observation of vesicles within the CC and filament-associated particles within the lumen of the axoneme suggests that transport modalities other than IFT are important for OS replenishment. Previous cryo-ET structures of flagella from Chlamydomonas
revealed axoneme microtubules spaced ~20 nm from the flagellar membrane (Nicastro et al., 2005
; Nicastro et al., 2006
). While this limited space may hinder vesicle movement within the motile flagella, the analogous region is expanded to ~50 nm in the CC, which provides the potential for higher vesicle trafficking though the primary cilium. At the base of the OS, a network of actin filaments extending into the distal CC is likely involved in the sorting of molecules between plasma membrane and disks. Defects in these structures and their associated functions may be mechanisms by which ciliary defects lead to human disease. Some of these defects may be specific for photoreceptors, but it is likely that many apply to primary cilia in general and to extra-retinal symptoms of ciliopathies such as Bardet-Biedl syndrome.
The BBSome complex, which forms a membrane coat, was hypothesized to play some role in vesicle budding at the ciliary membrane (Jin et al., 2010
). Our data reveal that a genetic defect in the BBSome, the BBS4 knockout, leads to a massive accumulation of vesicles rather than a dearth of them. Rather than a role in vesicle budding, the accumulation of vesicles and defective disk morphogenesis in Bbs4−/−
rods suggest that a BBSsome component is required either for vesicle fusion with the plasma membrane or for vesicular transport into the CC.
The standard model for disk morphogenesis relies on an assumed continuity of the OS and CC plasma membrane with the first few basal disks, which are proposed to be formed by evagination of the plasma membrane. This standard model has previously been challenged (Chuang et al., 2007
), but the recent model has proven extremely controversial. Our work sheds considerable light on this question by clearly revealing nascent basal disks, distinct from, and enclosed by the plasma membrane. Our work argues against the evagination model, but questions the alternative model (Chuang et al., 2007
) in which vesicle trafficking through the CC and fusion with basal disks plays a major role in transport of rhodopsin. Hence, if rhodopsin and other disk membrane proteins are primarily transported through the plasma membrane of the CC, there must be mechanisms for moving them from the plasma membrane to nascent disks, possibly involving the actin network we describe in the distal CC.
Our work clearly rules out the proposed role of the GARP domain or free GARP as an important mediator of disk spacing or disk-microtubule interactions, as we show this interaction is normal in the absence of GARP, despite grossly misformed disk membranes in Cngb1−/−
rods. Quantitative analysis of wildtype OS, by both Fourier analysis and direct measurements (, , , and S5
), reveal diskal spacings of 32 nm, evenly divisible by the tubulin dimer repeat of 8 nm. Thus, microtubules, rather than interdisk spacers, are likely key to regulating this spacing. Our tomograms provide clear evidence for CNGB1 and GARPs acting as key regulators of disk size by mediating a physical connection between disk rims and the OS plasma membrane as suggested previously (Batra-Safferling et al., 2006
). Loss of this critical inter-membrane connectivity is likely an important factor in the progression of retinal degeneration in Cngb1−/−
Our results demonstrate the utility for using cryo-ET to characterize and compare structures of rod CC from wildtype and mutant retinas, but much more remains to be done. In the future, it seems likely that application of averaging techniques to repeating structures (Koyfman et al., 2011
), with finer sampling, could improve the resolution and level of detail for structures such as the axoneme. Moreover, there are many more mouse ciliopathy models from which rods can be obtained and studied using cryo-ET. Because primary cilia are found in nearly every cell in the body, the structural insights gained from cryo-ET of photoreceptor CC have widespread implications for normal ciliary function and for the etiology of pleiotropic ciliopathies. The identification by cryo-ET of structural perturbations resulting from additional specific gene defects, can help to elucidate the structural and functional roles of the corresponding gene products, and contribute to development of a complete molecular description of primary cilium structure and function.