Abstract
Purpose :
Retinal degeneration is a common feature of ciliopathies, syndromes caused by impairment of primary cilia. ARL13B is one of the small G-proteins that function in ciliary protein transport and signaling. Mutations of ARL13B were found in patients with Joubert syndrome associated in some cases with retinal anomalies (Cantagrel et al, 2008; Thomas et al, 2015).
To understand how ARL13B is transported into the cilium and distributed within the organelle we sought to identify its interaction partners.
Methods :
For tandem affinity purification (TAP), mouse GFP-S-tagged ARL13B was expressed in retinal pigment epithelium RPE-1 cells. GST-tagged human ARL13B and its mutated variants were expressed in E. coli. Binding to tubulin or to purified axonemes was tested by co-sedimentation analysis. For functional analyses GFP-tagged ARL13B and its mutated variants were expressed in mouse embryonic fibroblasts (MEFs). Confocal microscopy images were analyzed using ImageJ and GraphPad Prism.
Results :
Using TAP and mass spectrometry, we found that ARL13B interacted with tubulin though a region located within the G-domain. To address the function of the G-domain within the ciliary compartment, we obtained an ARL13B mutant lacking the G-domain (ARL13B-ΔGD). GFP-ARL13B-ΔGD localized to cilium in wt MEFs and Arl13b null (hnn) MEFs. Both the GTPase mutant ARL13B T35N and ARL13B-ΔGD failed to rescue shortened cilia in hnn MEFs.
In hnn MEFS, full-length ARL13B variants including ARL13B T35N distributed evenly (mean % of cilia with even distribution was 75.0 -93.0). In contrast, ARL13B-ΔGD accumulated in bulges located mostly at the distal tip and dramatically diminished along the cilium (13.5+2.1% of cilia had even distribution, one-way ANOVA test, p< 0.01).
By in vitro co-sedimentation we showed that ARL13B interacted with purified axonemes through its G-domain indicating that in the cilium the G- domain is involved in anchoring to the axoneme. Treatment of ciliated MEFs with a detergent demonstrated lower retention of GFP-ARL13B-ΔGD compared to GFP-ARL13B (two-way ANOVA test, p= 0.028) suggesting that the association of ARL13B-ΔGD with cilia was weakened.
Conclusions :
The G-domain of ARL13B is not required for trafficking to the cilium but it is indispensable for interaction with the axoneme and proper distribution of ARL13B and other signalin proteins of the ciliary membrane
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.