Incorporating phototransduction proteins in zebrafish green cone with pressure-polished patch pipettes.

The neuronal Ca2+-sensor guanylate cyclase-activating protein 3 (zGCAP3) is a major regulator of guanylate cyclase (GC) activity expressed in zebrafish cone cells. Here, the zGCAP3, or a monoclonal antibody directed against zGCAP3, was injected in the cone cytoplasm by employing the pressure-polished pipette technique. This technique allows to perform "real time" zGCAP3 (or of any other phototransduction protein) over-expression or knock-down, respectively, via the patch pipette. Photoresponses were not affected by purified zGCAP3, indicating that GC was already saturated with endogenous zGCAP3. The cytosolic injection of anti-zGCAP3 produced the slowing down kinetics of the flash response recovery, as theoretically expected by a minimal phototransduction model considering the antibody acting exclusively on the maximal GC activation by low Ca2+. However, the antibody produced a progressive current decay toward the zero level, as if the antibody affected also the basal GC activity in the dark.

Zebrafish guanylate cyclase type 3 signaling in cone photoreceptors.

The zebrafish guanylate cyclase type 3 (zGC3) is specifically expressed in cone cells. A specifc antibody directed against zGC3 revealed expression at the protein level at 3.5 dpf in outer and inner retinal layers, which increased in intensity between 3.5 and 7 dpf. This expression pattern differed from sections of the adult retina showing strong immunostaining in outer segments of double cones and short single cones, less intense immunoreactivity in long single cones, but no staining in the inner retina. Although transcription and protein expression levels of zGC3 are similar to that of the cyclase regulator guanylate cyclase-activating protein 3 (zGCAP3), we surprisingly found that zGCAP3 is present in a 28-fold molar excess over zGC3 in zebrafish retinae. Further, zGCAP3 was an efficient regulator of guanylate cyclases activity in native zebrafish retinal membrane preparations. Therefore, we investigated the physiological function of zGCAP3 by two different behavioral assays. Using the morpholino antisense technique, we knocked down expression of zGCAP3 and recorded the optokinetic and optomotor responses of morphants, control morphants, and wild type fish at 5-6 dpf. No significant differences in behavioral responses among wild type, morphants and control morphants were found, indicating that a loss of zGCAP3 has no consequences in primary visual processing in the larval retina despite its prominent expression pattern. Its physiological function is therefore compensated by other zGCAP isoforms.

Operation profile of zebrafish guanylate cyclase-activating protein 3.

The expression pattern and property profile of the neuronal Ca(2+) sensor guanylate cyclase-activating protein 3 (zGCAP3) was studied by immunochemical approaches, biophysical methods and enzymatic assays. Using affinity purified antibodies immunoreactivity towards zGCAP3 was weakly detected in the outer and strongly in the inner segments of cone cells as well as in the outer plexiform layer, to a lesser degree also in the inner plexiform and ganglion cell layer of the zebrafish retina. This cellular distribution was independent of a dark/light cycle. Some neuronal Ca(2+) sensors are acylated (mainly myristoylated) at the amino-terminus. Probing larval and adult stages of the developing zebrafish retina indicated that zGCAP3 was first expressed in a non-myristoylated form, but was finally present in the adult retina as a myristoylated protein. While zGCAP3 did not undergo a classical Ca(2+) -myristoyl switch as investigated by surface plasmon resonance spectroscopy, myristoylation had two main other consequences: it enhanced the Ca(2+) -sensitivity of the Ca(2+) -induced conformational change and it stabilized the protein conformation. Differences between myristoylated and non-myristoylated zGCAP3 were also observed in modulating the kinetic and catalytic parameters of the GCAP-target, a membrane bound guanylate cyclase. Thus, the stabilizing effect of the myristoyl group is apparently less important in the larval than in the adult fish.

Dynamic cellular translocation of caldendrin is facilitated by the Ca2+-myristoyl switch of recoverin.

Caldendrin and recoverin are Ca(2+)-sensor proteins operating in neuronal systems. In a search for novel binding partners of recoverin, we employed an affinity column and identified caldendrin as a possible interaction partner. Caldendrin and recoverin co-localized in the retina in a subset of bipolar cells and in the pineal gland as revealed by immunofluorescence studies. The binding process was controlled by Ca(2+) as revealed by pull-down assays, and surface plasmon resonance studies. Importantly, caldendrin existed as a Ca(2+)-independent homodimer whereas a complex of recoverin and caldendrin formed with low to moderate affinity in the presence of Ca(2+). Co-transfection of COS-7 cells with plasmids harboring the gene for fluorescently labeled recoverin and caldendrin was used to study the cellular distribution by time-lapse fluorescence microscopy. Apparently, the increase of intracellular Ca(2+) facilitates the translocation of caldendrin to intracellular membranes, which is under control of complex formation with recoverin.