Grabe, V.

Hansson, B.

Jain, K.

Lavista-Llanos, S.

Wicher, D.

English

MPG.PuRe

Calmodulin regulates the olfactory performance in Drosophila melanogaster. Kalpana Jain1†, Sofia Lavista-Llanos1†, Veit Grabe1, Bill S Hansson1*, Dieter Wicher1*  Affiliation: 1 Max Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology, Hans-Knöll-Strasse 8, D-07745 Jena, Germany.  † Shared authorship * Shared senior authorship  Correspondence to: Dieter Wicher (dwicher@ice.mpg.de)              Supplementary Information Supplementary information includes four figures with legends, one table, supplementary material and methods, and references.                   Supplementary Figure S1.  Point mutation in Orco CBS does not affect trafficking of Or22a protein in ciliated OSN dendrites. (a-l) Fluorescent images of Drosophila antennae stained with Alexa Fluor 488 goat anti- rabbit. Antennae from a-h were exposed to Or22a primary antibody, those from i-l were not. Antennae marked with white dotted lines are overview projections of OrcoWT-R (a); OrcoK339N (c); OrcoWT (e); UAS-OrcoK339N (g); OrcoWT (i); OrcoK339N (k). (b) Expanded image for OrcoWT-R representing the dotted square in (a), respectively for OrcoK339N (d) from (c), OrcoWT (f) from (e), UAS-OrcoK339N (h) from (g), OrcoWT (j) from (i), OrcoK339N (l) from (k). Scale bar: overview sections (20 µm), detailed sections (10 µm).      Supplementary Figure S2. Effect of CaM inhibition on the odor response in OrX/Orco expressing OSNs. (a) Time course of Ca2+ fluorescence responses of OrX/OrcoWT OSNs under control conditions (OrcoWT) in the presence of 10 µM W7 (OrcoWT+W7) upon stimulation with 100 µM VUAA1 (n = 10 for OrcoWT, n = 8 for OrcoWT+W7). (b) Concentration response relationship for conditions as in (a) (7 ≤ n ≤ 11). Curves represent sigmoidal fits described by Hill coefficients 1.44 (OrcoWT), 0.94 (OrcoWT+W7), and EC50 of 88 µM (OrcoWT), 83 µM (OrcoWT+W7). (c) Maximum fluorescence response (Max ∆F/F0 (%)) obtained with 1 mM VUAA1. n = number of antennae. Two-tailed unpaired t- test, **P < 0.01. Data given as mean ± SEM.      Supplementary Figure S3. Short-range attraction to vinegar and grape juice in the trap assay experiment. Behavioral responses of (OrcoWT, n = 40 for each stimulus) and Orco null-mutants (Orco-/-, n = 40 for each stimulus) flies to grape juice (10–2 diluted in mineral oil, purple) or vinegar (10–2 diluted in mineral oil, green) in a trap-assay. n = number of flies.Two-tailed unpaired t- test to solvent control (** P < 0.01; *** P < 0.001); ns = not significant. Data given as mean ± SEM.           Supplementary Figure S4. Long-range attraction to vinegar and grape juice in the wind tunnel experiment. (a) Landing percentage of OrcoWT flies reaching the odor source (vinegar (green) and grape juice (purple)) as counted within 10 min in wind tunnel bioassay (n = 15). (b) Total percentage of OrcoWT flies landing on to the grape juice and vinegar source (n = 15). n = number of flies. Two-tailed unpaired t-test, *P < 0.05. Data given as mean ± SEM.               Supplementary Table S1: Genotype of flies used in this study 1 Name Figure(s) Control Mutant Ca2+  imaging OrX/Orco Figure 1: c, d, e, f Figure 3: a-f Figure 4: a-d OrcoWT (+; UAS-GCaMP6f/CyO; Orco-GAL4/TM6B) OrcoK339N (w/w; UAS-GCaMP6f/UAS-OrcoK339N; Orco-GAL4,Orco1/Orco-GAL4,Orco1) Or22a/Orco Figure 2: c, d, e, f Figure 5: a-d OrcoWT (+; UAS-GCaMP6f/CyO; Or22a-GAL4/TM6B) OrcoK339N (w/w; UAS-GCaMP6f/UAS-OrcoK339N; Or22a-GAL4,Orco1/Or22a-GAL4,Orco1) OrX/Orco Supplementary Figure S2 OrcoWT (+; UAS-GCaMP6f/CyO; Orco-GAL4/TM6B) - 2 Name Figure(s) Control Mutant                  Wind tunnel bioassay OrX/Orco Figure 6: a, b OrcoWT (Canton-S) OrcoWT-R (OrcoWT rescue) (w/w; UAS-Orco/UAS-Orco; Orco-GAL4,Orco1/Orco-GAL4,Orco1)    OrcoK339N (w/w; UAS-OrcoK339N/UAS-OrcoK339N; Orco-GAL4,Orco1/Orco-GAL4,Orco1)  Orco-/- (Orco1 null mutant) Or22a/Orco Figure 6: c, d OrcoWT-R (OrcoWT rescue) (w/w; UAS-Orco/UAS-Orco; Or22a-GAL4,Orco1/Or22a-GAL4,Orco1)  OrcoK339N (w/w; UAS-OrcoK339N/UAS-OrcoK339N; Or22a-GAL4,Orco1/Or22a-GAL4,Orco1)  Orco-/- (Orco1 null mutant) OrX/Orco Supplementary Figure S4 OrcoWT (Canton-S) - 3 Name Figure Control Mutant Trap assay OrX/Orco Supplementary Figure S3 OrcoWT (Canton-S)  Orco-/- (Orco1 null mutant) 4 Name Figure Control Mutant                Immunohistochemistry   anti-Or22a  Supplementary Figure S1: a, b, c, d OrcoWT-R (OrcoWT rescue)   (w/w,; UAS-Orco/UAS-Orco; Orco-GAL4,Orco1/Orco-GAL4,Orco1)  OrcoK339N (w/w,; UAS-OrcoK339N/CyO; Orco-GAL4,Orco1/Orco-GAL4,Orco1) Supplementary Figure S1: e, f, g, h   OrcoWT (Canton-S) - UAS-OrcoK339N (Parental line)  (w118; UAS-DmOrcoK339N/CyO; +)  no primary antibody Supplementary Figure S1: i, j, k, l OrcoWT (Canton-S)  OrcoK339N (w/w,; UAS-OrcoK339N/CyO; Orco-GAL4,Orco1/Orco-GAL4,Orco1) Supplementary Material and Methods  Immunohistochemistry Seven days old mated females from OrX/OrcoWT-R, OrX/OrcoK339N, OrX/OrcoWT (Canton-S), and UAS-OrcoK339N were used. Flies were anesthetised on ice and only one antenna (3rd segment) was excised from the head for each replicate. Antennae were fixed with 4% PFA (paraformaldehyde) + 0.1% Triton X-100 (Carl Roth, Germany) for 2 hours on ice, on a rotator. In the next step, antennae were washed with phosphate-buffered saline solution (PBS) containing 0.1 % Triton X-100 (PBST) 3 times for every 20 min. Next, antennae were blocked in blocking solution (normal goat serum (NGS) in PBST) for one hour at room temperature (RT). Afterwards, antennae were incubated with the primary antibody anti-Or22a (1:1000) in blocking solution for 48 hrs at 4˚C. Later, antennae were washed with PBST for four times for 15 min at RT, and then treated with the secondary antibody (Alexa Fluor488 goat anti rabbit IgG (H+L), 1:250 Invitrogen by Thermo Fisher Scientific) in blocking solution for 36 hrs at 4 ˚C. Lastly, antennae were washed again with PBST three times for 20 minutes, and mounted in vectashield (Vector Burlingame, CA, USA). Anti-Or22a was kindly donated by Dr L. Vosshall (Rockefeller University).   Two-choice trap bioassay We tested the innate attraction of Drosophila melanogaster OrX/OrcoWT (Canton-S) flies and orco null mutant (orco-/-) flies to vinegar and grape juice by two-choice trap assays as described in 1. Shortly, 5 day-old OrX/OrcoWT or Orco-/- flies were starved during 12 hours, with free access to water. Forty samples of twenty five to forty flies each were introduced into a box (10 cm x 8 cm x 10 cm) equipped with two containers (4.5 cm height, 3 cm diameter with a pipette-tip opening of 2 mm), each containing respectively an odor source (grape juice (10-2)  or vinegar (10-2) in mineral oil) or solvent (mineral oil). Flies were allowed to choose during 24 hours at 25°C and 75% humidity. An attraction index (AI) was calculated as AI = (O−C) / 30, where O is the number of flies entering the odorant containing trap and C is the number of flies entered the solvent containing trap. The index ranges from -1 (complete avoidance of the odor source) to 1 (complete attraction to the odor source), whereas a value of 0 characterizes no response, i.e. the odor is not detected, or is neutral. For each stimulus, statistical difference was calculated against 0 (no response) and between genotypes.    Confocal microscopy Antennae images were captured using cLSM 880 (Carl Zeiss, Oberkochen, Germany) using a 40X water immersion objective (C-Apochromat, NA:1.2, Carl Zeiss). Antennae overview stacks were captured at 1024 x 1024 pixel resolution at 0.49 µm intervals, and for the detailed section at 856 x 856 pixel resolution at 0.49 µm intervals. Fiji Image J software2 was used to create the stack projections. NA: Numerical aperture. All images used for the figure representation were of 8 bits.  Supplementary References 1 Dweck, H. K. M. et al. Olfactory channels associated with the Drosophila maxillary palp mediate short- and long-range attraction. eLife 5, e14925, doi:10.7554/eLife.14925 (2016). 2 Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9, 671-675, doi:10.1038/nmeth.2089 (2012).        

Calmodulin regulates the olfactory performance in Drosophila melanogaster

http://hdl.handle.net/21.11116/0000-0007-F709-9

Calmodulin regulates the olfactory performance in Drosophila melanogaster

Abstract

Similar works

Full text

Available Versions

MPG.PuRe