Characterizing molecular diffusion in the lens capsule

Abstract

The lens capsule compartmentalizes the cells of the avascular lens from other ocular tissues. Small molecules required for lens cell metabolism, such as glucose, salts, and waste products, freely pass through the capsule. However, the lens capsule is selectively permeable to proteins such as growth hormones and substrate carriers which are required for proper lens growth and development. We used fluorescence recovery after photobleaching (FRAP) to characterize the diffusional behavior of various sized dextrans (3, 10, 40, 150, and 250 kDa) and proteins endogenous to the lens environment (EGF, gammaD-crystallin, BSA, transferrin, ceruloplasmin, and IgG) within the capsules of whole living lenses. We found that proteins had dramatically different diffusion and partition coefficients as well as capsule matrix binding affinities than similar sized dextrans, but they had comparable permeabilities. We also found ionic interactions between proteins and the capsule matrix significantly influence permeability and binding affinity, while hydrophobic interactions had less of an effect. The removal of a single anionic residue from the surface of a protein, gammaD-crystallin [E107A], significantly altered its permeability and matrix binding affinity in the capsule. Our data indicated that permeabilities and binding affinities in the lens capsule varied between individual proteins and cannot be predicted by isoelectric points or molecular size alone.

Keywords: FRAP; basement membrane; binding affinity; diffusion coefficient; lens capsule; partition coefficient; permeability.

Figure 1

Helium ion microscopy of the anterior lens capsule of an adult mouse lens A) Top down view of the anterior capsule, bar=200nm. B) Surface image acquired at a 40 degree tilt. Representative diameters are provided above selected pores, bar=20nm.

Figure 2

Behavior of fluorescein labeled dextrans and protein in the anterior mouse lens capsule as a function of Stoke’s radius. A) The equilibrium partition coefficient of these molecules calculated by dividing the fluorescence intensity within the capsule (I_capsule) by the fluorescent intensity in solution (I_solution). B) The diffusion coefficients for the studied molecules within the capsule evaluated from FRAP recovery curves. C) The calculated permeability of dextrans and proteins in the lens capsule. These values are compared to permeability values determined for Ficoll in the glomerular basement membrane (adjusted for thickness) determined by Edwards et al, 1997.

Figure 3

Lens capsule matrix interactions of fluorescein labeled dextrans and proteins. A) Comparison of the immobile fractions of each molecule and B) their binding affinity ratios evaluated from FRAP recovery curves.

Figure 4

Change in protein behavior in mouse anterior capsules modified by neutralizing carboxyl or amine groups, removal of heparan sulfate side chains, or blocking hydrophobic interactions. Proteins are listed from smallest to largest. A) Percent change of partition coefficients from values determined in unmodified capsules. B) Percent change of diffusion coefficients from values determined in unmodified capsules. C) Percent change of permeability values from values determined in unmodified capsules. (E=EGF, G=γD-crystallin, G^m=γD-crystallin [E107A], B=BSA, T=transferrin, C=ceruloplasmin, I=IgG) (*p<=0.05)

Figure 5

Change in matrix interactions in modified lens capsules. A) Percent change of immobile fractions of proteins from values determined in unmodified capsules. B) Change in binding affinity ratios. (E=EGF, G=γD-crystallin, G^m=γD-crystallin [E107A], B=BSA, T=transferrin, C=ceruloplasmin, I=IgG) (*p<=0.05)