Drosophila Rhodopsin 7 can partially replace the structural role of Rhodopsin 1, but not its physiological function
Drosophila Rhodopsin 7 can partially replace the structural role of Rhodopsin 1, but not its physiological function
Rudi Grebler 0 1 2 3
Christa Kistenpfennig 0 1 2 3
Dirk Rieger 0 1 2 3
Joachim Bentrop 0 1 2 3
Stephan Schneuwly 0 1 2 3
Pingkalai R. Senthilan 0 1 2 3
Charlotte Helfrich‑Förster 0 1 2 3
0 Developmental Biology, Institute of Zoology, University of Regensburg , Regensburg , Germany
1 Cell- and Neurobiology, Zoological Institute, Karlsruhe Institute of Technology (KIT) , Karlsruhe , Germany
2 Neurobiology and Genetics, Biocenter, Theodor Boveri Institute, University of Würzburg , 97074 Würzburg , Germany
3 Present Address: Oxitec Ltd , 71 Innovation Drive, Milton Park, Oxford OX14 4RQ , UK
Rhodopsin 7 (Rh7), a new invertebrate Rhodopsin gene, was discovered in the genome of Drosophila melanogaster in 2000 and thought to encode for a functional Rhodopsin protein. Indeed, Rh7 exhibits most hallmarks of the known Rhodopsins, except for the G-protein-activating QAKK motif in the third cytoplasmic loop that is absent in Rh7. Here, we show that Rh7 can partially substitute Rh1 in the outer receptor cells (R1-6) for rhabdomere maintenance, but that it cannot activate the phototransduction cascade in these cells. This speaks against a role of Rh7 as photopigment in R1-6, but does not exclude that it works in the inner photoreceptor cells.
Phototransduction; Electroretinograms; Rhodopsins; Compound eyes; Drosophila melanogaster
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Vision begins with the absorption of photons by visual
pigment molecules. Rhodopsins are membrane-bound
G-protein-coupled receptors that absorb photons, undergo
conformational changes, and activate a G-protein to initiate
visual signal transduction. Animal genomes typically
contain multiple Rhodopsin genes coding for Rhodopsins with
different spectral properties providing the basis for color
vision. Each photoreceptor cell usually expresses a single
rhodopsin, but exceptions are known in both vertebrates
and invertebrates (Applebury et al. 2000; Hu et al. 2011,
2014; Mazzoni et al. 2004; Stavenga and Arikawa 2008).
The fruit fly Drosophila melanogaster possesses six
different well-characterized Rhodopsin molecules, Rh1 to Rh6.
With the exception of Rh2, all Rhodopsins are found in the
receptor cells of the compound eyes: Rh1 is expressed in
the six outer receptor cells (R1–6) of each eye unit and Rh3
to Rh6 are expressed in the two inner receptor cells (R7,
R8) (reviewed in Rister et al. 2013; Behnia and Desplan
2015). A seventh Rhodopsin, Rh7, of still unknown
location and function was predicted from the genome in 2000
(Adams et al. 2000; Terakita 2005). qPCR studies showed
that Rh7 is expressed at low levels in the compound eyes,
suggesting that it may be co-expressed with one or several
of the other Rhodopsins (Posnien et al. 2012; Senthilan and
Helfrich-Förster 2016).
The aim of the present study was to investigate a
potential function of Rh7 in the compound eyes. A suited
method to reveal the properties of an unknown Rhodopsin
is to express it in R1–6 instead of Rh1 (Feiler et al. 1988;
1992; Townson et al. 1998; Salcedo et al. 1999; Knox et al.
2003; Hu et al. 2014). Rh1 is required for proper
rhabdomere morphogenesis and maintenance, in addition to
its role as photopigment (O’Tousa et al. 1985; Kumar and
Ready 1995; Kumar et al. 1997; Zuker et al. 1985). Thus,
loss of Rh1 (in ninaE17 mutants) leads to the collapse of
rhabdomeric microvilli inside the photoreceptor cytoplasm
(Ahmad et al. 2007; Bentrop 1998; Kurada and O’Tousa
1995; Leonard et al. 1992). This can be prevented by
expressing other functional Rhodopsins in R1–6 of ninaE17
mutants (Kumar et al. 1997). Here, we expressed Rh7
instead of Rh1 under the Rh1 promotor (Rh1–Rh7;ninaE17
flies) and investigated whether Rh7 can (1) rescue the
retinal degradation provoked by the ninaE 17 mutation and (2)
lead to normal electroretinogram (ERG) responses. We also
expressed Rh7 in addition to Rh1 (Rh1–Rh7 flies) to see
whether this increases the ERG responses.
Materials and methods
Wild-type CantonS (WTCS) as well as flies with yellow
body color and white eyes (yellow− white1118 = y− w1118)
served as control for semi-thin sections and
immunocytochemistry. For qPCR, deep pseudopupil, and ERG
measurements, only flies in the y− w1118 background were used.
In addition, ninaE17 and sevLY3 mutants were in the y−
w1118 background. ninaE (=neither inactivation nor
afterpotential E) codes for Rh1 and y− w1118; ninaE17 mutants
are Rh1 null mutants (O’Tousa et al. 1985). sev (sevenless)
codes for a tyrosin kinase that is critical for the
development of the photoreceptor cell R7 (Basler and Hafen 1988)
and y− w1118 sevLY3 mutants lack the inner photoreceptor
cell R7 (Harris et al. 1976). In the following, we will omit
“y− w1118” and simply use ninaE17 and sevLY3.
Generation of Rh1–Rh7 transgenic flies
To generate flies expressing the Rh7 coding region under
control of the Rh1 promotor, the full-length Rh7 CDS
was amplified by (...truncated)