Abstract
Purpose :
Light is detected by the outer segment compartment of retinal photoreceptors. The outer segment is an adapted primary cilium, which are thin microtubule-based organelles that relay extracellular signals to the cell through receptors enriched in the ciliary membrane. The distinct protein composition of the cilium is maintained by a membrane diffusional barrier, formed by transition zone proteins. While previous work has shown that membrane proteins utilize ciliary transport carriers (such as TULP, IFT, and BBSome) to enter and exit the cilium, the molecular components of the diffusion barrier remain unknown. Tectonic1 (Tctn1), an extracellular protein, forms a large complex with other ciliary proteins at the transition zone and has been shown to regulate ciliary membrane composition in primary cilia. We hypothesize that Tctn1 forms a diffusion barrier that maintains the unique membrane-associated protein composition of the photoreceptor outer segment.
Methods :
To study Tctn1 in rod photoreceptors, a conditional Tctn1floxed mouse was generated through CRISPR gene targeting. This Tctn1flox/flox mouse was then crossed to an iCre75 mouse, which specifically expresses Cre recombinase in rod photoreceptors, to then generate our rod-specific Tctn1 knock-out mouse (iCre;Tctn1f/f).
Results :
We found that ciliary outer segment formation is normal in the absence of Tctn1; however, proteins typically excluded from the outer segment are now accumulating within the outer segment of iCre;Tctn1f/f rods. Mislocalization of these proteins in the outer segment is detrimental to rod health as photoreceptor degeneration occurs by 6 months. Interestingly, we show that the abundance and localization of ciliary transport carriers are not affected by the loss of Tctn1 which would suggest that Tctn1 acts as a physical gate at the transition zone.
Conclusions :
Together, our results show that Tctn1 is part of the membrane ciliary gate. We are now testing whether the loss of Tctn1 impacts the diffusional rate of membrane proteins through the transition zone, allowing them to sneak by the exit machinery. Ultimately, my work has helped to understand how the transition zone functions as a membrane gate within photoreceptors and primary cilia in general.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.