PARTICULARIDADES DEL TORO DE LIDIA

My results indicate that Psidin is required at two steps of nervous system development: ORN survival and axonal pathfinding. Therefore, I next investigated the molecular mechanisms Psidin uses to regulate neuron number and axon targeting, respectively. Psidin is the homologue of yeast MDM20, the non-catalytic subunit of the NatB complex. In budding yeast, natB subunit mutants show defects including mitochondrial inheritance, budding, and cell division (Hermann et al., 1997). For the human homologue a role in cell cycle control or cell growth was found using analysis in tissue culture (Starheim et al., 2008). Essentially, the authors suggested that the anti-apoptotic protein p21 is involved in regulating downstream targets of NatB. Therefore I studied the Drosophila gene dNAA20,

which encodes the predicted homologue of the catalytic subunit of NatB of yeast and human. I used RNAi in order to study the effect of dNAA20 knockdown on psidin phenotypes

in vivo. RNAi against dNAA20 was driven by act-GAL4 in eyFlp clones. The effect of knocking down dNAA20 on targeting and cell number was analyzed. Expression of dNAA20 RNAi in

wild type, psidin1 _{and psidin}IG978 _{background had no impact on the targeting pattern of Or59c}

and Or42a neurons (Figure 7.10A, Figure 7.11A). However, the expression of dNAA20 RNAi resulted in a strong functional interaction in the cell number of ORNs. Knockdown of dNAA20 showed a significant reduction of the respective ORN class in psidinIG978 background. Levels were reduced to levels comparable to Or59c and Or42a psidin1 mutants, respectively. Importantly, Or42a and Or59c neuron numbers in psidin1 null mutants were not further decreased by dNAA20 RN Ai expression (Figure 7.10B, Figure 7.11B). Expression of RNAi against dNAA20 in wild type clones did not result in targeting or cell number defects of

Figure 7.10 | Psidin and dNAA20 interact in Or59c neurons

RNAi against dNAA20 was driven by act-GAL4 in eyFlp clones. Axons were visualized using a Or59c::mCD8-GFP direct fusion construct. (A) Targeting pattern of Or59c neurons with psidin1 and

psidinIG978 background (circles and arrowhead indicate mistargeting) in the presence and absence of RNAi against dNAA20. Knock down had no influence on Or59c axons. (B) Total cell number of Or59c and positive neurons in wild type, psidin1 and psidinIG978 background, in the presence and

Or42a or Or59c neurons, possibly because the amount of dNAA20 still present after knock- down was sufficient to maintain wild type cell numbers. In contrast to the reduction in cell number in psidinIG978 mutants, Or59c and Or42a ORNs did not show any enhancement in mistargeting or ectopic synapse formation when dNAA20 RNAi was expressed in psidinIG978

and psidin1 mutants (Figure 7.10, Figure 7.11). I could verify the knock-down of dNAA20

using S2 cell culture and western blot analysis. Protein levels of dNAA20 were reduced by 80% upon co-expression of RNAi against dNAA20-myc (Figure 7.12). These results indicate that dNAA20 is dispensable for ORN targeting, but it is required for the formation of the correct number of ORNs in a Psidin-dependent manner. These data are consistent with a role in cell cycle progression similar to the function of the yeast and human NatB complexes.

In vitro experiments in both yeast and human cell lines demonstrated that the catalytic and non-catalytic subunits of NatB form a physical complex (Polevoda et al., 2003; Starheim et al., 2008). The data suggest that Psidin and dNAA20 might act in the same molecular pathway in vivo.

Figure 7.11 | Psidin and dNAA20 interact in Or42 neurons

RNAi against dNAA20 was driven by act-GAL4 in eyFlp clones. Axons were visualized using a Or59c::mCD8-GFP direct fusion construct. (A) Targeting pattern of Or42a neurons with psidin1 and

psidinIG978 background in the presence and absence of RNAi against dNAA20. Knock down in

psidinIG978 neurons reduced the cell number, which in parallel caused a reduced innervation in the AL (circles). (B) Total cell number of Or42a positive neurons in wild type, psidin1 _{and psidin}IG978 background, in the presence and absence of dNAA20 RNAi. Knock down in psidinIG978 neurons caused a reduction of neuron number. Bar graphs: One-way ANOVA, Bonferroni post-test for normally distributed values (* p<0.05, ** p<0.01, *** p<0.001). Error bars ± SEM.

Next, I addressed whether Psidin and dNAA20 also interact physically as their human and yeast homologues using co-immunoprecipitation assays (Figure 7.13). An N-terminal fusion of Psidin to an HA-protein tag and N-terminal myc fusion of dNAA20 were expressed in S2 cells. The pull-down of Psidin using anti-HA antibody resulted in co-immunoprecipitation of dNAA20 (Figure 7.13). These data show that also in Drosophila, Psidin and dNAA20 form a complex. Given that psidinIG978 contains a point mutation that selectively influences axon targeting, but not ORN survival, it was interesting to investigate whether psidinIG978 still interacts with dNAA20 as I would have predicted. I found that PsidinIG978 still pulls down dNAA20 at levels comparable to Psidin wild type (Figure 7.13), consistent with the fact that the psidinIG978 mutant allele in the presence of dNAA20 (or absence of dNAA20 RNAi) does not affect the cell number of ORNs.

Figure 7.12 | Knock down of dNAA20 in S2 cells

Expression of UAS-Psidin-HA and UAS-dNAA20-myc was driven by ub-GAL4 in S2 cells. Protein levels of dNAA20-myc are significantly (80%) reduced upon co-expression of RNAi against dNAA20. Bar graphs: One-way ANOVA, Bonferroni post-test for normally distributed values (* p<0.05, ** p<0.01, *** p<0.001). Error bars ± SEM.

Figure 7.13 | Psidin and dNAA20 interact in vitro

UAS-Psidin-HA and UAS-dNAA20-myc were expressed in S2 cells under the control of a ubiquitous GAL4 (ub-GAL4). Using Co-IP, dNAA20 pulled down together with Psidin (arrowhead). Also PsidinIG978 _{is able to bind to dNAA20 at levels comparable to wild type}