Abstract
Purpose :
Tulp1 is a photoreceptor-specific protein localized to the inner segment and outer plexiform layer (OPL) compartments. In the absence of Tulp1, several outer segment (OS)-specific proteins are mislocalized and synaptic vesicle recycling is impaired. Intraflagellar transport (IFT) and Kinesin-2 are known to regulate anterograde ciliary protein trafficking. Recently, we identified IFT88, IFT57, IFT20, Kif3a and Kif3b as candidate Tulp1 binding partners. We hypothesize that Tulp1 might directly cooperate with IFT and Kinesin-2 in regulating photoreceptor protein transport. Here we analyze the expression of IFT-B and Kinesin-2 proteins in tulp1-/- versus WT retinas to better understand the role of Tulp1 in photoreceptor protein trafficking.
Methods :
IHC and Western blot analysis was used to investigate the expression level and pattern of IFT-B members (IFT88, IFT57, and IFT20) and Kinesin-2 proteins (Kif3a, Kif3b and KAP3) in tulp1-/- versus WT retinas.
Results :
Immunoblot analysis demonstrated a significant reduction of IFT88 and IFT57 in tulp1-/- retinas as compared to WT. Surprisingly, a more severe reduction of IFT88 was seen in the OPL as compared to other photoreceptor compartments. The two motor subunits of Kinesin-2, Kif3a and Kif3b, are also significantly reduced in tulp1-/- retinas, whereas IHC staining does not reveal any difference in their distribution throughout the retinal layers.
Conclusions :
Our results indicate a reduction in the expression levels of two IFT-B proteins (IFT88 and IFT57) as well as the two Kinesin-2 motor proteins (Kif3a and Kif3b) in the absence of Tulp1. Our data suggests that anterograde protein trafficking is altered in Tulp1 mutant retinas leading to mislocalized OS proteins. Of note, the reduction of IFT88 expression was most severe in the OPL, suggesting that protein transport to the synapse is also affected without Tulp1 and may be the cause of synapse malformation seen in tulp1-/- retinas. Overall, our findings suggest that Tulp1 may be a part of the IFT-B and Kinesin-2 complex involved in the transportation of photoreceptor proteins in multiple compartments. Further testing involving Co-IP is ongoing to confirm which proteins are direct Tulp1 binding partners.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.