Abstract
Primary cilia play critical roles in many aspects of biology. Specialized versions of primary cilia are involved in many aspects of sensation. The single photoreceptor sensory cilium (PSC) or outer segment elaborated by each rod and cone photoreceptor cell of the retina is a classic example. Mutations in genes that encode cilia components are common causes of disease, including retinal degenerations. The protein components of mammalian primary and sensory cilia have not been defined previously. Here we report a detailed proteomics analysis of the mouse PSC complex. The PSC complex comprises the outer segment and its cytoskeleton, including the axoneme, basal body, and ciliary rootlet, which extends into the inner segment of photoreceptor cells. The PSC complex proteome contains 1968 proteins represented by three or more unique peptides, including approximately 1500 proteins not detected in cilia from lower organisms. This includes 105 hypothetical proteins and 60 proteins encoded by genes that map within the critical intervals for 23 inherited cilia-related disorders, increasing their priority as candidate genes. The PSC complex proteome also contains many cilia proteins not identified previously in photoreceptors, including 13 proteins produced by genes that harbor mutations that cause cilia disease and seven intraflagellar transport proteins. Analyses of PSC complexes from rootletin knock-out mice, which lack ciliary rootlets, confirmed that 1185 of the identified PSC complex proteins are derived from the outer segment. The mass spectrometry data, benchmarked by 15 well characterized outer segment proteins, were used to quantify the copy number of each protein in a mouse rod outer segment. These results reveal mammalian cilia to be several times more complex than the cilia of unicellular organisms and open novel avenues for studies of how cilia are built and maintained and how these processes are disrupted in human disease.
Highlights
Primary cilia play critical roles in many aspects of biology
The inner and outer segment compartments are distinguishable by morphology, by function, and no doubt by their protein complements, they cannot be completely separated using standard biochemical techniques because the outer segment is an elaborated cilium, and its ciliary rootlet extends far into the inner segment [2, 33]
photoreceptor sensory cilium (PSC) complexes isolated from wild-type mice contain outer segments plus the portions of their cytoskeletons that extend into the inner segments of photoreceptor cells, including the basal body and ciliary rootlet (Fig. 1)
Summary
Animals—This research was approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Frozen sections of retinas were prepared from mice that were 1) completely dark-adapted, 2) exposed to the same light regimen as mice from which retinas were harvested for PSC complex isolation, or 3) bleached with 15,000 lux of light for 30 min with dilated pupils prior to sacrifice. These sections were embedded adjacent to each other and probed with antibodies to arrestin and ␣-transducin. We used the Expression Analysis Systematic Explorer (EASE) program to determine which functional categories are statistically overrepresented in PSC complex proteome with respect to total protein set annotated in mouse [32]. Potential candidate genes were evaluated manually to determine whether they fell within published intervals for disease loci
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