Abstract
Purpose: :
Primary or sensory cilia are involved in many aspects of cellular biology. All sensory cilia are composed of a central microtubule–based axoneme, surrounded by a membrane sheath. Additional specializations are present in distinct cell types, such as the stack of membrane discs present in the outer segments of photoreceptor cells. As in other cilia, the axoneme of photoreceptor cells is a critical organizing component of the outer segment. To facilitate characterization of these structures in molecular detail, we have conducted proteomic analyses of mouse photoreceptor outer segments and their axonemes.
Methods: :
Outer segments (OS) and basal body–axoneme complexes were isolated from wild–type mouse retinas, and purified by sucrose density gradient centrifugation using established methods. Proteins were separated on 4–12% SDS–PAGE gels, stained with Coomassie blue, and the gel pixelated into 30 pieces. The proteins within each gel pixel were identified by trypsin digestion followed by tandem mass spectrometry using a nano–capillary HPLC interfaced directly with a linear ion trap mass spectometer. Peptide spectra were compared to a composite mouse protein databases using Sequest and DTASelect software to generate peptide and then protein identifications. Only peptides that passed stringent filters for mass accuracy and enzyme specificity were considered to be reliably identified.
Results: :
Immunofluoresence and EM evaluations demonstrated that the OS and basal body–axoneme preparations were highly purified. A total of 232578 spectra were obtained from the OS preparation. Analysis showed these were derived from 13228 peptides, representing a total of 1807 proteins. Similarly, 181752 spectra from 15687 peptides and 2007 proteins were obtained from the basal–body axoneme analyses. The false positive rate for peptide identification was estimated to be< 0.5% using a reverse database. Quantitative analyses of peptide abundance confirmed the purity of the protein preparations. The OS and basal–body axoneme proteomes share1213 proteins; 604 proteins are unique to the OS, and 802 unique to the basal body–axoneme.
Conclusions: :
We have characterized the proteome of a mammalian sensory cilium. The data show that photoreceptor OS and their axonemes are significantly more complex than the other eukaryotic cilium. This proteome data provide the basis for investigations of the molecular mechanisms of photoreceptors. We are hopeful that such studies will improve our understanding of photoreceptor biology and facilitate development of therapies for retinal diseases.
Keywords: photoreceptors • proteomics • cytoskeleton