α- and β-crystallins modulate the head group order of human lens membranes during aging

Abstract

Purpose: To examine the physical properties of human lens cell membranes as a function of age.

Methods: The environment of the phospholipid head groups in fiber cell membranes from human lenses, aged 22 to 83 years, was assessed with Laurdan and two-photon confocal microscopy. The effect of mild thermal stress on head group order was studied with lens pairs in which one intact lens was incubated at 50 °C. Dihydrosphingomyelin vesicles were preloaded with Laurdan, α-, β-, or γ-crystallin was added, and surface fluidity was determined.

Results: The membrane head group environment became more fluid with age as indicated by increased water penetration. Furthermore, these changes could be replicated simply by exposing intact human lenses to mild thermal stress; conditions which decreased the concentration of soluble α- and β-crystallins. Vesicle binding experiments showed that α- and β-, but not γ-, crystallins markedly affected head group order.

Conclusions: The physical properties of cell membranes in the lens nucleus change substantially with age, and α- and β-crystallins may modulate this effect. β-Crystallins may therefore play a role in lens cells, and cells of other tissues, apart from being simple structural proteins. Age-dependent loss of these crystallins may affect membrane integrity and contribute to the dysfunction of lenses in older people.

Figure 1.

The effect of age on membrane GP values across the diameter of the lens. (A) Representative pseudocolored GP images of lens sections across the age range. A number of confocal images (775 × 775 μm², 512 × 512 pixels) were acquired, and the edges overlapped to enable the whole length of the lens section to be covered. (B) Membrane properties as a function of lens age. GP values (mean ± SEM) at 0, 0.75, 2, 3, 3.5, 4, and 4.5 mm from the geometric center of the lenses were calculated from the reconstructed GP images. Lenses: 20 to 40 years, n = 5; 40 to 50 years, n = 4; 50 to 60 years, n = 5; 60 to 70 years, n = 5; older than 70 years, n = 4.

Figure 2.

Effect of age on membrane GP values in the lens center and outer region. Age dependence of GP values in the very center of the lens with those 3.5 mm from the lens center. 3.5 mm: y = −0.0001x + 0.4786; R² = 0.0129. Center: y = −0.0028x + 0.5453; R² = 0.8004.

Figure 3.

Cholesterol content in lens regions as a function of age. Cholesterol concentration (mean ± SEM) was determined using electron ionization mass spectrometry.

Figure 4.

The influence of mild thermal stress on (A) membrane GP values in intact human lenses. Three lens pairs (30 years, 41 years, and 63 years) were used with one lens of each pair incubated at 50°C (20 hours) and the other not heated. (B) Soluble proteins. The nuclear regions of three lens pairs were extracted, and the soluble proteins were separated by gel filtration HPLC with absorbance monitored at 280 nm. (A, B) Lenses were sectioned and treated with Laurdan (mean ± SD).

Figure 5.

Effect of exposure time to mild thermal stress on lens membrane GP values and soluble protein content. Concentration of the HMW form of α-crystallin and β-crystallin in portions of a 38-year-old human lens core, as a function of time of incubation at 50°C, compared with GP values of sections from the contralateral lens heated at 50°C. In this lens nucleus, α-crystallin was present only in the HMW form. HMW: y = −0.2264x + 6.3907; R² = 0.9287. β-crystallins: y = −0.8574x + 44.044; R² = 0.7369. GP: y = −0.0055x + 0.4316; R² = 0.8756.