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Theodore G Wensel, Zhixian Zhang, Feng He, Steven J. Pittler; Cryo-Electron Tomography Reveals Structural Roles for Domains of the Cyclic Nucleotide-Gated Channel Beta Subunit. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1736.
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© ARVO (1962-2015); The Authors (2016-present)
To assess details of morphological phenotypes in rods of mice lacking the WT beta subunit of the cyclic nucleotide-gated cation channel (CNGB1-/-), also expressing a truncated form of the beta subunit lacking its N-terminal GARP2 (aa 1-370) region (T-beta transgenic). The goal was to compare the structural functions of the full-length protein to those of the truncated form.
Mice were constructed using standard transgene introduction of a construct containing the murine opsin promoter driving expression of T-beta. Rod cells were crudely purified from mice of different genotypes, at ages P21-P28. Cells were collected, applied to electron microscope grid and vitrified by plunge-freezing in liquid ethane. They were imaged in a 200 kV and 300 kV cryo-electron microscope over a range of tilt angles, with data collection via a direct electron detector. Images were motion corrected, and processed using Motioncorr ( Li et al., 2013) and IMOD (Mastronarde, 1997) to generate 3-dimensional tomographic maps. Recurring structures such as disk stacks and disk-rim connection complexes, were isolated computationally and combined using sub-tomogram averaging by EMAN2 (Bell et al., 2016) to improve signal to noise.
As observed by other techniques, the severe phenotype of the CNGB1-/- genotype was partially rescued by the T-beta transgene. Some rods with relatively intact outer segments, albeit with altered membrane morphology, could be isolated and good tomograms could be generated. The rod outer segments contained some stacked disks, but these were not properly aligned perpendicular to the long axis of the outer segment. Inter-disk spacing, and spacing between plasma membrane and disk rims were not significantly altered from those in WT. The greatly elongated disks filled with vesicles that are observed in the KO (Gilliam et al., 2012) were not observed in the presence of the transgene.
The truncated form of the beta subunit fulfills at least some of the structure-stabilizing functions of the full-length protein, especially those required for constraining disk size. However, it is not sufficient to prevent mis-orientation of disc membranes in the outer segments, development of aberrant membrane structures, and eventual cell death, indicating that the GARP2 region has a major role in supporting outer segment structural integrity.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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