Abstract
BackgroundChanges in genes coding for ciliary proteins contribute to complex human syndromes called ciliopathies, such as Bardet-Biedl Syndrome (BBS). We used the model organism Paramecium to focus on ciliary ion channels that affect the beat form and sensory function of motile cilia and evaluate the effects of perturbing BBS proteins on these channels.MethodsWe used immunoprecipitations and mass spectrometry to explore whether Paramecium proteins interact as in mammalian cells. We used RNA interference (RNAi) and swimming behavior assays to examine the effects of BBS depletion on ciliary ion channels that control ciliary beating. Combining RNA interference and epitope tagging, we examined the effects of BBS depletion of BBS 7, 8 and 9 on the location of three channels and a chemoreceptor in cilia.ResultsWe found 10 orthologs of 8 BBS genes in P. tetraurelia. BBS1, 2, 4, 5, 7, 8 and 9 co-immunoprecipitate. While RNAi reduction of BBS 7 and 9 gene products caused loss and shortening of cilia, RNAi for all BBS genes except BBS2 affected patterns of ciliary motility that are governed by ciliary ion channels. Swimming behavior assays pointed to loss of ciliary K+ channel function. Combining RNAi and epitope tagged ciliary proteins we demonstrated that a calcium activated K+ channel was no longer located in the cilia upon depletion of BBS 7, 8 or 9, consistent with the cells’ swimming behavior. The TRPP channel PKD2 was also lost from the cilia. In contrast, the ciliary voltage gated calcium channel was unaffected by BBS depletion, consistent with behavioral assays. The ciliary location of a chemoreceptor for folate was similarly unperturbed by the depletion of BBS 7, 8 or 9.ConclusionsThe co-immunoprecipitation of BBS 1,2,4,5,7,8, and 9 suggests a complex of BBS proteins. RNAi for BBS 7, 8 or 9 gene products causes the selective loss of K+ and PKD2 channels from the cilia while the critical voltage gated calcium channel and a peripheral receptor protein remain undisturbed. These channels govern ciliary beating and sensory function. Importantly, in P. tetraurelia we can combine studies of ciliopathy protein function with behavior and location and control of ciliary channels.
Highlights
Changes in genes coding for ciliary proteins contribute to complex human syndromes called ciliopathies, such as Bardet-Biedl Syndrome (BBS)
BBS protein interactions in Paramecium In order to determine the BBS protein interactions in P. tetraurelia, we expressed FLAG-tagged BBS8 and BBS9 genes separately and analyzed the proteins immunoprecipitated from Whole cell extract (WCE) after separation by SDS gel electrophoresis
Taking into consideration the mammalian BBSome and ciliary membrane trafficking, and our results, we propose that the BBS proteins of the putative Paramecium BBS complex and BBS3 interact with protein cargo of the Golgi vesicles and/or plasma membrane that have proteins destined for the ciliary membrane (Figure 8)
Summary
Changes in genes coding for ciliary proteins contribute to complex human syndromes called ciliopathies, such as Bardet-Biedl Syndrome (BBS). We used the model organism Paramecium to focus on ciliary ion channels that affect the beat form and sensory function of motile cilia and evaluate the effects of perturbing BBS proteins on these channels. Seven of the fourteen BBS proteins (BBS1, 2, 4, 5, 7, 8, 9) with BBIP10 form the BBSome, which, with the small GTPase Rab and its exchange factor Rabin, is vital for trafficking of Golgi vesicles to the ciliary transport apparatus (intraflagellar transport, IFT) for ciliogenesis [13,14,15,16]. The BBSome is recruited to the membrane by BBS3 (Arl6) where it forms a coat complex that, with the IFT apparatus, facilitates the incorporation of proteins like the somatostatin receptor into the ciliary membrane [16]. BBS6, 10 and 12 appear to form a complex with the chaperonins that are responsible for BBSome assembly [21] (see [11,12,15,22] for reviews)
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