This study has resolved the long-standing question concerning the structure and function of the striated ciliary rootlet. To study the in vivo functions of the ciliary rootlet, we created and analyzed lines of rootletin mutant mice. Elimination of rootletin expression ablated ciliary rootlets in all ciliated cells. This finding formally demonstrates that rootletin is the major, and likely only, structural component of the ciliary rootlet. Our analyses of the mutant mice devoid of rootlets help define the in vivo functions of this structure. Thus, the rootlets were found to be dispensable for ciliary development and basal functions but required for the long-term stability of some types of cilia. Among them, the photoreceptors stand out as the type of cells particularly sensitive to a loss of the rootlets. This is almost certainly due to the fact that photoreceptors elaborate the light-sensing outer segments, the largest mammalian cilia by far. Any shear force encountered by the outer segments would be magnified multiple times by the leverage effect when sensed at the point of anchor. Within the connecting cilia and basal outer segments, the tightly packed axonemal microtubule bundles provide rigidity and support. At points proximal to the basal bodies, it appears that the ciliary rootlet assumes a major structural role. Indeed, gentle dissection of the retina in vitro causes massive breakage of the “rootletless” photoreceptors, primarily at the junction between inner and outer segments and sometimes within the inner segments. The age-dependent photoreceptor degeneration in the mutant mice may therefore be attributed, at least in part, to an increased vulnerability to mechanical stress.
The in vitro experiments in the present study lend support to the notion that the rootlets function primarily as a support structure. First, we demonstrate by time-lapse fluorescence recording and FRAP experiments that the rootletin fibrous network in transfected cells is a very stable structure. These findings are consistent with our previous report that recombinant rootletin fibers do not fully disassemble even during mitosis, suggesting that rootletin fibers are among the least dynamic of all cytoskeletons. There are three major fibrous cytoskeletal networks in cells: microtubules, actin filaments, and intermediate filaments. The first two are usually more dynamic, undergoing active changes in their lengths and rapid recovery in FRAP experiments (1
). Intermediate filaments are relatively more stable (8
). Rootletin shares certain structural characteristics with intermediate filaments. Both rely on rod domains for polymerization and lateral interactions. Indeed, the recovery time for rootletin in FRAP experiments is similar to that of keratin, the most stable form of intermediate filaments. Mutations or deletions of keratins cause blistering skin diseases (6
), indicating that keratins confer critical structural stability to epidermis against mechanical stress. Thus, the similarity in structure and dynamics that rootletin shares with intermediate filaments supports the in vivo function of the ciliary rootlet in mechanical support.
Second, we present evidence that the ciliary rootlet integrates with actin filaments, which are known to support cells against mechanical stress. In photoreceptor cells, actin filaments exist throughout the cells. They are especially enriched in the distal end of the inner segments, where the rootlets originate. Thus, the integration between these two cytoskeletal networks in cells probably enhances the structural support of the ciliary rootlet. Because we did not find actin as a rootletin-interacting protein in our previous yeast two-hybrid screen using the rootletin fragments as baits, the integration between rootletin and actin may be indirect, or the interaction occurs only between filamentous forms of the two cytoskeletal proteins. The association between rootletin and actin filaments and a reported interaction between the rootlet and intermediate filaments (11
) may contribute substantially to the role of the rootlet as an important support structure.
The present study confirms that rootlets serve to anchor membranous organelles within the photoreceptor inner segments. Large membranous saccules that normally surround the rootlets in photoreceptor inner segments become randomized in the absence of rootlets. It is possible, though unconfirmed, that an interaction between rootletin and the cortical actin filaments on the cytoplasmic faces of the membranes is responsible for the congregation of membranes around the rootlets. Also unclear at the present is the identity of the membranous saccules and how much, if any, the loss of anchor to these membranous organelles contributes to the photoreceptor degeneration.
The present study also identifies rootletin as a candidate gene for human retinal degeneration. Based on the murine data, the corresponding human retinal degeneration may be one of late onset, possibly with systemic manifestation of a ciliary defect, such as respiratory tract symptoms. The disease course of rootletin-related retinal degeneration may be influenced by cumulative life experiences. For example, sport or job-related activities that are prone to high levels of physical impact might well exacerbate the course of disease. From this perspective, identification of rootletin gene mutations that cause retinal degeneration may provide both prognostic and treatment benefits to patients.