Steric volume exclusion sets soluble protein concentrations in photoreceptor sensory cilia

Abstract

Proteins segregate into discrete subcellular compartments via a variety of mechanisms, including motor protein transport, local binding, and diffusion barriers. This physical separation of cell functions serves, in part, as a mechanism for controlling compartment activity by allowing regulation of local protein concentrations. In this study we explored how soluble protein size impacts access to the confined space within the retinal photoreceptor outer segment signaling compartment and discovered a strikingly steep relationship. We find that GFP monomers, dimers, and trimers expressed transgenically in frog rods are present in the outer segment at 1.8-, 2.9-, and 6.8-fold lower abundances, relative to the cell body, than the small soluble fluorescent marker, calcein. Theoretical analysis, based on statistical-mechanical models of molecular access to polymer meshes, shows that these observations can be explained by the steric hindrance of molecules occupying the highly constrained spaces between outer segment disc membranes. This mechanism may answer a long-standing question of how the soluble regulatory protein, arrestin, is effectively excluded from the outer segments of dark-adapted rods and cones. Generally, our results suggest an alternate mode for the control of protein access to cell domains based on dynamic, size-dependent compartmental partitioning that does not require diffusion barriers, active transport, or large numbers of immobile binding sites.

Fig. 1.

The density of structures in rod photoreceptor compartments is heterogeneous. (A) Transmission EM of an amphibian rod. E, ellipsoid; IS, inner segment (cell body); M, myoid; N, nucleus; OS, outer segment. The OS is densely packed with lamellar disc membranes (Inset) whereas the IS myoid is relatively sparsely populated with membranous vesicles and organelles (© Townes-Anderson et al., 1985. Originally published in Journal of Cell Biology, 100:175–188). (Scale bar, 5 μm.) (B) The geometry of the outer segment with disc membranes (DM), the major transduction proteins (pymol representations), and the plasma membrane (PM), drawn to scale. Rhodopsins, present at an average spatial density of ∼6 nm in the disc membrane, possess cytoplasmic loops that extend ∼2 nm into the interdiscal space. Transducins, tethered to the disc membranes by acylation of the α-subunits and farnesylation of the γ-subunits, are approximately spherical with radius 2.9 nm (34) and are present at ∼1:10 with respect to rhodopsin. PDE6, the largest protein in the interdiscal space spanning almost the entire gap, is ∼1:200 per rhodopsin. (C) The blue shaded region represents the volume of the outer segment aqueous cytoplasm accessible to the center of mass of a spherical solute with r_h ∼ 3 nm.

Fig. 2.

Soluble molecule levels in the rod outer segment depend on molecular size. (A) Images of live retinal slices. (B) Axial distributions of fluorescent molecules in rods. (Left) Central confocal z-sections; lines indicate positions from which fluorescence was read. S, presynaptic spherule. (Scale bars, 10 μm.) (Right) Fluorescence intensities plotted as a function of distance from the IS-OS interface (Left arrows). (C) Intensity distributions normalized to maximum fluorescence and averaged across multiple cells. (D) Fraction of fluorescence in the OS as a function of estimated hydrodynamic radii. Vertical error bars, F/F_max SEM. n = 5, 14, 7, and 7 for calcein and 1×, 2×, and 3× GFP. Horizontal error bars represent SEM of r_h approximation. Line was found from linear regression of the data.

Fig. 3.

Soluble protein access to outer segment interdiscal spaces is molecular size dependent. (A) End-on imaging of the outer segment in live rods expressing GFP variants. (Left) Red dots in confocal images indicate position of FRAPa experiments shown at Right. F₀, fluorescence before photobleach. (B) EM of a Rana catesbeiana rod showing incisures.[Reprinted from Experimental Eye Research, 45/1, Yoshihiko Tsukamoto; The number, depth and elongation of disc incisures in the retinal rod of Rana catesbeiana, 105–116, Copyright 1987, with permission from Elsevier (ref. 17)]. (C) Median voxel intensities and their SDs, averaged from 10, 4, 5, and 6 1×, 2×, 3×, and 3× hypotonic (ht) rods, respectively. (D) Diffusion coefficients in isotonic and hypotonic rods plotted as a function of hydrodynamic radii. n = 7, 7, and 6 for 1×, 2×, and 3× isotonic and 9, 7, and 6 for hypotonic. (A–D) Error bars, SEM. Horizontal error bars are as in Fig. 2. (Scale bars, 5 μm.) Hypotonic medium was 0.25 isotonic.

Fig. 4.

The steric volume exclusion model for regulation of soluble protein concentrations in spatially constrained cellular compartments. (A) F_OS/F_max vs. r_h from Fig. 2D (blue circles). Lines are referenced to the right ordinate: green, calculated f_ac,OS(r_h); red, estimated f_ac,IS(r_h); dashed line, f_ac,OS(r_h)/f_ac,IS(r_h). (B) Predicted OS/IS concentration ratios for monomer (M), dimer (D), and tetramer (T) arrestin in dark-adapted rods. Gray diamonds represent maximum and minimum values calculated for dimers of various shapes (text). Dashed line indicates arrestin–GFP ratio (from C). (C) Arrestin–GFP distribution in dark adapted rods is similar to that of 3× GFP (cf. ref. 32).