Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Summary

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Abstract

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 µm) and thin (~2 µm) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

Introduction

Rod photoreceptor cells, tightly packed in the outermost layer of the neural retina, are an integral part of dim light vision. To function as faithful photon counters, rods utilize a G-protein-based signaling pathway, termed phototransduction, to generate rapid, amplified, and reproducible responses to single photon capture. This response to light ultimately triggers a change in the current at the plasma membrane and is subsequently signaled to the rest of the visual system¹. As their name implies, each rod cell has a distinct rod-like shape and exhibits a highly polarized cellular morphology, consisting of an outer segment (OS), inner segment ....

Protocol

All experiments were performed according to the local institutional guidelines of the committee on research animal care from the University of Southern California (USC).

1. Live cell retinal peeling method

1. Preparation of Ames' buffers, peeling papers, and dissection dish
   1. Using blunt tip iris scissors (or equivalent scissor type), cut the cellulose filter paper (Grade 413) into rectangles measuring approximately 5 mm x 2.5 mm. Store the cuttings for future use.
   2. Prepare 1 L of Ames' HEPES buffer by combining one bottle of Ames' Medium, 2.38 g HEPES, and 0.877 g of NaCl. Dissolve ....

Results

The present strategies were developed to provide relatively rapid and simple methods to isolate and analyze proteins among specific rod subcellular compartments for western blot analysis. We applied two sequential peeling techniques (Figure 1 and Figure 2) followed by immunoblotting to demonstrate that these methods could reliably be used to detect the known distribution of rod transducin (GNAT1) and arrestin (ARR1) in both dark- and light-adapted animals. To va.......

Discussion

Many retinal diseases affect rod photoreceptor cells, leading to rod death and, ultimately, complete vision loss³⁷. A significant portion of the genetic and mechanistic origins of human retinal degeneration have been successfully recapitulated in numerous mouse models over the years. In that context, the ability to easily and selectively separate individual rod subcellular compartments from the small mouse retina would greatly enhance our understanding of the localized biochemical and molecular un.......

Materials

Name	Company	Catalog Number	Comments
100 mL laboratory or media bottle equipped with a tubing cap adapter	N/A	N/A
100% O2 tank	N/A	N/A
1000mL Bottle Top Filter, PES Filter Material, 0.22 μm	Genesee Scientific	25-235
4X SDS Sample Buffer	Millipore Sigma	70607-3
50 mL Falcon tube	Fisher Scientific	14-432-22
95% O2 and 5% CO2 tank	N/A	N/A
Ames’ Medium with L-glutamine, without bicarbonate	Sigma-Aldrich	A1420
CaCl2 (99%, dihydrate)	Sigma	C-3881
Drierite (Anhydrous calcium sulfate, >98% CaSO4, >2% CoCl2)	WA Hammond Drierite Co LTD	21005
Falcon Easy-Grip Petri Dish (polystyrene, 35 x 10 mm)	Falcon-Corning	08-757-100A
Falcon Easy-Grip Tissue Culture Dish (60 x 15 mm)	Falcon-Corning	08-772F
Feather Scalpel (No. 10, 40 mm)	VWR	100499-578
Feather Scalpel (No. 11, 40 mm)	VWT	100499-580
KCl (99%)	Sigma	P-4504
Kimble Kontes pellet pestle	Sigma	z359971
Labconco Fast-Freeze Flasks	Labconco	N/A
LN2 (liquid nitrogen) + Dewar flask or similar vacuum flask	N/A	N/A
MgCl2	Sigma	M-9272
Milli-Q/de-ionized water	EMD Millipore	N/A
Na2HPO4 (powder)	J.T. Baker	4062-01
NaCl (crystal)	EMD Millipore	Sx0420-3
NaHCO3	Amresco	0865
OmniPur EDTA	EMD	4005
OmniPur HEPES, Free Acid	EMD	5320
Parafilm M	Sigma-Aldrich	P7793
Reynolds Wrap Aluminum Foil	Reynolds Brands	N/A
Scotch Magic Tape (12.7 mm x 32.9 m)	Scotch-3M	N/A
Sodium deoxycholate	Sigma-Aldrich	D67501
Spectrafuge mini centrifuge	Labnet International, Inc	C1301
Tissue incubation chamber (purchased or custom made)	N/A	N/A
Tris-HCl	J.T.Baker	4103-02
Triton X-100	Signma-Aldrich	T8787
VirTis Benchtop 2K Lyophilizer or equivalent machine	SP Scientific	N/A
VWR Grade 413 Filer Paper (diameter 5.5 cm, pore size 5 μm)	VWR	28310-015
Whatman Grade 1 Qualitative Filter Paper (diameter 9 cm, pore size 11 μm)	Whatman/GE Healthcare	1001-090
Wide bore transfer pipet, Global Scientific	VWR	76285-362