Abstract
Purpose: :
Retinal photoreceptor outer segments (OS) are specialized sensory cilia. The appreciation of OS as cilia is important because it connects inherited retinal degenerations (IRDs) at a mechanistic level to a larger class of systemic cilia disorders such as Bardet Biedl syndrome. To study PSCs we performed a detailed proteomic analysis of mouse PSC complexes and identified many novel PSC proteins. We are now investigating the roles of novel proteins from the PSC complex proteome in photoreceptor biology and disease. As part of these studies, we have identified Ttc21b as a novel cilia protein that is required for normal photoreceptor sensory cilia and renal primary cilia formation.
Methods: :
The location of Ttc21b in renal and photoreceptor cilia was determined by expressing V5- epitope tagged Ttc21b in cultured mouse renal (IMCD3) cells, and immunofluorescence analyses using anti-peptide antibodies. Ttc21b expression in IMCD3 cells and photoreceptor cells in vivo was knocked down using shRNAs. The structure of renal cilia following Ttc21b suppression was evaluated by immunostaining. The structure of photoreceptor cilia following Ttc21b knockdown was evaluated in 3D volume reconstructions of confocal images obtained from immunostained sections of transfected retina.
Results: :
The Ttc21b protein is located in the basal bodies and transition zone of renal and photoreceptor cilia. shRNA-mediated suppression of Ttc21b in IMCD3 cells results in notably shortened cilia compared to control shRNA transfected cells. Axonemes in shRNA-Ttc21b treated cells were 1.6±1.1 µm long, compared to 6.4±1.3 µm in control shRNA (non-targeted) treated cells (P<0.0001). Co-transfection of human Ttc21b cDNA with the shRNA-Ttc21b restores cilia to almost full length (4.8±1.1 µm; P<0.0001). Similarly, Ttc21b knockdown in the retina in vivo results in abnormal PSC structure, with "bulbs" in place of or at the distal end of the outer segments of most transfected photoreceptor cells. In contrast, the outer segments of control shRNA transfected photoreceptors are slender rods.
Conclusions: :
These data demonstrate that Ttc21b is required for formation of normal primary and photoreceptor cilia. A recent publication suggests that TTC21B is a retrograde IFT protein, which suggests that the defects we observe in cilia structure follow knockdown of Ttc21b may be due to loss of retrograde IFT activity. Additional studies of the role of TTC21B in photoreceptor sensory cilia biology and disease are warranted.
Keywords: photoreceptors • gene/expression • retinal degenerations: cell biology