Separation of photoreceptor cell compartments in mouse retina for protein analysis - PubMed (original) (raw)
Separation of photoreceptor cell compartments in mouse retina for protein analysis
Kasey Rose et al. Mol Neurodegener. 2017.
Abstract
Background: Light exposure triggers movement of certain signaling proteins within the cellular compartments of the highly polarized rod photoreceptor cell. This redistribution of proteins between the inner and outer segment compartments affects the performance and physiology of the rod cell. In addition, newly synthesized phototransduction proteins traverse from the site of their synthesis in the inner segment, through the thin connecting cilium, to reach their destination in the outer segment. Processes that impede normal trafficking of these abundant proteins lead to cell death. The study of movement and unique localization of biomolecules within the different compartments of the rod cell would be greatly facilitated by techniques that reliably separate these compartments. Ideally, these methods can be applied to the mouse retina due to the widespread usage of transgenic mouse models in the investigation of basic visual processes and disease mechanisms that affect vision. Although the retina is organized in distinct layers, the small and highly curved mouse retina makes physical separation of retinal layers a challenge. We introduce two peeling methods that efficiently and reliably isolate the rod outer segment and other cell compartments for Western blots to examine protein movement across these compartments.
Methods: The first separation method employs Whatman® filter paper to successively remove the rod outer segments from isolated, live mouse retinas. The second method utilizes ScotchTM tape to peel the rod outer segment layer and the rod inner segment layer from lyophilized mouse retinas. Both procedures can be completed within one hour.
Results: We utilize these two protocols on dark-adapted and light-exposed retinas of C57BL/6 mice and subject the isolated tissue layers to Western blots to demonstrate their effectiveness in detecting light-induced translocation of transducin (GNAT1) and rod arrestin (ARR1). Furthermore, we provide evidence that RGS9 does not undergo light-induced translocation.
Conclusions: These results demonstrate the effectiveness of the two different peeling protocols for the separation of the layered compartments of the mouse retina and their utility for investigations of protein compositions within these compartments.
Keywords: Arrestin; Phototransduction; Protein trafficking; Protein translocation; RGS9; Retina; Transducin.
Figures
Fig. 1
Diagram of retinal cell layers in the mouse retina. a Retinal layers and associated cell types: rod (pink), cone (purple), bipolar (lilac), Müller (gray), ganglion (blue) cells. RPE: retinal pigmented epithelium, OS: outer segment, CC: connecting cilium, IS: inner segment, ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer, GCL: ganglion cell layer. Rhodopsin and Gβ5L are localized to the OS. GNAT1 (rod transducin α-subunit), ARR1 (rod arrestin) and RGS9 are also localized in rod cells. Actin, cytochrome C (cyt C) and Gβ5S are expressed in all retinal layers except the OS. b GNAT1 and ARR1 are localized to different rod cell compartments under different lighting conditions. c The dimension of a central cross section from the posterior pole of the mouse eye containing the neural retina
Fig. 2
Light-induced movement of GNAT1 and ARR1 in rod photoreceptors. a Frozen retinal sections prepared from dark-adapted or light-exposed mice incubated with GNAT1 or ARR1 antibodies (green). The location of the rod outer segment is visualized with an antibody against rhodopsin (red), shown at the right of each panel. The diagram of the rod cell depicts the position of each rod compartment on the retinal section. Scale bar = 20 μm. b Representative immunoblots of ROS collected by filter paper peeling of retinas obtained from dark-adapted (D) or light exposed (L) mice. The -ROS fraction is the ROS-depleted tissue. c Quantified signals from light exposed (n = 6) and dark adapted (n = 5) +ROS and -ROS samples plotted as mean ± SD. There was a statistically significant difference between +ROS (D)/+ROS (L) (p < 0.0003) and -ROS (D)/-ROS (L) respectively for GNAT1 (p < 0.0001) and ARR1 (p < 0.0001) using unpaired _t_-test. There was no statistically significant difference between +ROS (D)/+ROS (L) and -ROS (D)/+ROS (L) for RGS9 (p = 0.8)
Fig. 3
Scanning electron micrographs of lyophilized retina. a Surface of lyophilized retina, photoreceptor side up, before peeling by adhesive tape. b The inner segment layer beneath the ROS. c Photoreceptor cell nuclear layer shows the uniform appearance of rod cell nuclei
Fig. 4
Western blots of retinal layers isolated by Scotch™ tape peels of lyophilized retinas. a Representative immunoblots from +ROS, +RIS and ROS/RIS-depleted tissue (-OIS). b Signals from the Western blots were quantified and plotted as mean ± SD for light (n = 9) and dark (n = 5) conditions. Dark (D) and light (L) samples showed statistically significant differences for +ROS, +RIS and -OIS for GNAT1 (p < 0.0001) and ARR1 (p < 0.0001) using unpaired _t_-test. Light/dark differences were not statistically significant in all samples for RGS9 (p = 0.2) c Signals from the lyophilized isolations of +RIS and -OIS were combined and plotted for comparison with the filter paper peeling method (n = 19 (L) and n = 10 (D)). Light and dark samples were found to be statistically different for +ROS and -ROS for GNAT1 (p < 0.0001) and ARR1 (p < 0.0001) respectively. There was no statistically significant difference between RGS9 light and dark samples (p = 0.7)
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