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First published online March 21, 2005
Journal of Experimental Biology 208, 1247-1256 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01527

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Rhodopsin patterning in central photoreceptor cells of the blowfly Calliphora vicina: cloning and characterization of Calliphora rhodopsins Rh3, Rh5 and Rh6

Angelika Schmitt, Andreas Vogt, Katrin Friedmann, Reinhard Paulsen and Armin Huber^*

Institut für Zoologie, Universität Karlsruhe, Haid-und-Neu-Strasse 9, 76131 Karlsruhe, Germany

View larger version (107K): [in a new window]   Fig. 1. Amino acid sequence alignment of Calliphora (Cv) and Drosophila (Dm) rhodopsins. Sequences are shown in single letter code. The seven transmembrane domains (TM1–TM7) are indicated in grey. Black boxes highlight amino acids conserved in all sequences. Accession numbers of the sequences are: DmRh1, P06002; DmRh3, P04950; DmRh4, P08225; DmRh5, P91657; DmRh6, O01668; CvRh1, J05596; CvRh3, Af878411; CvRh5, Af878412; CvRh6, Af878413.

View larger version (24K): [in a new window]   Fig. 2. Autofluorescence of Calliphora rhabdomeres. Confocal images of a frontal region of the intact Calliphora compound eye after simultaneous excitation with 476 nm and 488 nm wavelength laser light show (A) green fluorescence of R7y rhabdomeres and (B) red fluorescence of R1–6 rhabdomeres. (C) Overlay of A and B. A 20x/0.50 water immersion objective was used on a Leica TCS-SP confocal microscope. Scale bar, 20 µm. (D) Ratio of R7p to R7y rhabdomeres. Mean values were determined by evaluating the fluorescence pattern of randomly chosen regions comprising between 55 and 136 ommatidia from 20 Calliphora compound eyes. Bars are ± S.D.

View larger version (90K): [in a new window]   Fig. 3. Specific immunolabelling of Calliphora rhodopsins Rh3, Rh5 and Rh6. Longitudinal cryosections through the compound eye of Calliphora were probed with antibodies directed against Calliphora Rh3 (A,B), Calliphora Rh5 (C,D) and Drosophila Rh6 (E,F) in the absence or presence of peptides as indicated. The peptides used to block the reaction with Calliphora Rh3 (25 µg ml–1) and Rh5 (15 µg ml–1) were the same as used to generate the polyclonal antibodies. Reaction of the monoclonal anti-Drosophila Rh6 antibody was blocked with a peptide of the C-terminal region of Calliphora Rh6 rhodopsin (CGKDDTASDSRTQATA, 25 µg ml–1). Indirect immunofluorescence of antibodies is shown in green. Red fluorescence: labelling of actin filaments with rhodamine-coupled phalloidin. Scale bar, 80 µm.

View larger version (49K): [in a new window]   Fig. 4. Expression pattern of Calliphora rhodopsins Rh3, Rh5 and Rh6. Longitudinal (A,C) and cross (B,D) sections through the compound eye of Calliphora were labelled with antibodies specific for Calliphora Rh3, Rh5, Rh6 and with rhodamine-coupled phalloidin. (A,B) Labelling of Rh5 (green), Rh6 (blue) and the actin cytoskeleton of the rhabdomeres (red). The overlay of Rh6 (blue) and actin (red) labeling appears purple. Rh5 and Rh6 are detected in the basal part of the retina. The cross section in B is at the level of R8 cells. (C,D) Labelling of Rh3 (green), Rh6 (blue) and the actin cytoskeleton of the rhabdomeres (red). Rh3 and Rh6 are detected in the apical and basal portion of the retina, respectively. The cross section in D is at the R7 cell level in which Rh3 but not Rh6 is detected. Scale bars, (A,C) 80 µm; (B,D) 8 µm.

View larger version (72K): [in a new window]   Fig. 5. Quantitative evaluation of isolated Calliphora rhabdoms labelled for Rh3, Rh5 and Rh6. Isolated Calliphora rhabdoms were double labelled with antibodies specific for Rh5 and Rh6 (A,B) or with antibodies against Rh3 and Oregon Green-coupled wheat germ agglutinin (C,D). (A) An overlay of differential interference contrast and indirect immunofluorescence images of two rhabdoms, one labelled with the anti-Rh5 antibody (red) and the other one labelled with the anti-Rh6 antibody (green). (B) The percentage of rhabdoms containing Rh5 or Rh6 was determined by counting the number of rhabdoms stained with either antibody. (C) A few rhabdoms viewed with differential interference contrast optics, one of which is labelled with anti-Rh3 (red). (D) The percentage of rhabdoms stained with anti-Rh3 was determined relative to the total number of rhabdoms stained with wheat germ agglutinin. For the quantitative evaluation, four independent preparations from both male and female flies were analysed. Values are means ± S.D. Scale bar, 40 µm.