Characterization of ttc-1

ttc-1 encodes a poorly characterised protein, which contains the 30 amino acid length tetratricopeptide TPR1 motif. TPR motifs form helical structure domains, which allow the proteins to be involved in the scaffolding of mutliprotein complexes, thus these complexes can be implicated in many cellular processes such as transcription, protein translocation and protein degradation (Allan and Ratajczak, 2011). This suggests a potential regulatory role for TTC-1 in modulating the interaction and the regulation of proteins in C. elegans. BLASTP revealed that it is the ortholog of the human tetratricopeptide repeat protein 17 (TTC17), which has been recently shown to be involved in primary ciliogenesis in human cells and in modulating actin polymerisation (Bontems et al., 2014). In addition, the study of Bontems et al. also showed a possible implication of TTC17 in zebrafish ciliogenesis and that TTC17 was ubiquitously expressed in rat tissues. This last observation suggests that this protein could be involved in many different cell processes in addition to its implication

time, by using a ceramide synthase inhibitor in Madin Darby Canine Kidney (MDCK) cells, that ceramides were involved in the regulation of ciliogenesis (Wang et al., 2009). More recently, the same group showed the involvement of ceramide in stem cell ciliogenesis and differentiation (He et al., 2014). In fact, their results suggested that ciliogenesis was promoted by long chain fatty acyl ceramides that have an inhibiting effect on an atypical PKC.

These results point to a connexion between TTC proteins and ceramides. It would be interesting to know whether, as in zebrafish or in human, ceramides and TTC-1 play a role in C. elegans ciliogenesis.

3.1 Expression of ttc-1

We were able to show that the expression of ttc-1 in C. elegans was mainly present in the head, the gut, the tail, and in the cell of the vulva during formation. Interestingly this expression pattern partially overlapped with the one of hyl-2 described by Menuz et al., which emphasized the possible interaction of ttc-1 with the ceramide pathway.

It was hard to identify whether ttc-1 expression was within specific cells such as neurons. For instance, we compared this expression with the pattern of URX and BAG neurons but we were not able to find comparable pattern.

The expression of TTC-1 in the vulva during development at L4 stage suggests a potential role of the protein in the development of C. elegans. In addition, its wide expression also implies that this protein may perform multiple tasks.

3.2 Implication of ttc-1(gnv3) in the sphingolipids homeostasis

We have shown that in addition to restoring a normal resistance to anoxia in the ttc-1(gnv3);hyl-2(gnv1) mutant, ttc-1(gnv3) by itself had a resistance to anoxia comparable to the resistance of wild type animals.

An explanation for the accumulation of SMs in hyl-2(gnv1) could be due to an increase in the expression and/or activity of another ceramide synthase, HYL-1. We hypothesized that hyl-1 could be overexpressed in hyl-2(gnv1) mutant and normally expressed or downregulated in the ttc-1(gnv3);hyl-2(gnv1) by the mutation affecting TTC1. However a preliminary analysis of hyl-1 expression did not reveal any striking difference between hyl-2(gnv1), N2, ttc-1(gnv3);hyl-2(gnv1) and ttc-1(gnv3) (Figure 11). This preliminary result invalidates this hypothesis and should be confirmed. It would be also interesting to look at the third ceramide synthase LAGR-1 and at other genes encoding proteins involved in the production of GlcCers and SMs. For instance, pmt-1 and pmt-2 are known to encode phosphoethanolamine methyltransferases required for the biosynthesis of phosphatidylcholine (PCho) (Brendza et al., 2007; Palavalli et al., 2006). From PCho, complex sphingolipids can be produced. Thus we wanted to check whether an increase and a decrease in the expression of pmt-1 and pmt-2 could be found in hyl-2(gnv1) and ttc-1(gnv3);hyl-2(gnv1) respectively. This could have in part elucidated the variations in certain sphingolipids levels observed between these two mutants. Preliminary results were confusing and did not allow us to conclude anything.

Figure 11: Expression of hyl-1 in hyl-2(gnv1), ttc-1(gnv3);hyl-2(gnv1), gnv3#1, gnv3#2. N2 and RB1036 (deletion mutant of hyl-1) were used as controls. No big changes were observed, n = 1.

These preliminary investigation led us hypothesize that TTC-1 could be involved in the control of the breakdown of the sphingolipids and that impairment of this protein could favour the breakdown of certain species of sphingolipids.

3.2.1 Interaction with SR protein kinase homologue spk-1

ttc-1 was predicted to interact with the serine/threonine kinase spk-1 (Zhong and Sternberg, 2006), which has been demonstrated by RNAi experiments to be required for germline development in both hermaphrodites and males, and for embryogenesis (Kuroyanagi et al., 2000). Interestingly spk-1 was also predicted to interact with the nucleosome remodelling factor nurf-1, which has been shown to regulate vulval development (Andersen et al., 2006; Zhong and Sternberg, 2006). These studies potentially relating ttc-1 to the development of the vulva are coherent with our results showing ttc-1 expression within the cells of the vulva during the L4 stage.

We hypothesized that ttc-1 could regulate the activity of spk-1 and thus affect proteins that could be involved in or influence the sphingolipid metabolism. This could

be an explanation of the sphingolipid changes, especially for GlcCer and SM, that we observed in ttc-1(gnv3);hyl-2(gnv1) compared to hyl-2(gnv1). We currently would like to identify, by western blotting, whether we could observe any change in proteins containing SR domains in C. elegans. Unfortunately, preliminary observations were not conclusive (data not shown). In addition, we did not test spk-1 for its resistance to anoxia. It would be interesting to obtain a loss of function mutant or the corresponding RNAi in order to test that.

Negative results related to the hypothetical involvement of spk-1 in the response to anoxia and in the sphingolipid homeostasis would suggest the involvement of other interactors of ttc-1.