Phase Separation and Lens Cell Age

113

To determine the molecular mechanisms for cold cataract formation in the nucleus of the young mammalian lens, we have investigated the thermally reversible opacification of -crystallin solutions isolated from calf lens. Coexistence curves (plots of opacification temperature Tc versus protein concentration) were determined for the individual y-crystallin fractions II, III, and IV as well as for the unfractionated -crystallin mixtures isolated from the nucleus and cortex. The coexistence curve of yIV-crystallin is remarkably elevated above those of y4I-and yIII-crystallin and the -crystallin mixtures. The yIV-crystallin fraction is the major determinant of the opacification temperature within the whole lens or isolated cytoplasm. Quasielastic light-scattering spectroscopy of yIV-crystallin solutions indicates that above Tc there are two populations of protein aggregates of distinctly different mean size. As the temperature is lowered towards Tc, both populations increase in size. Opacification occurs when the population of large scatterers, which is composed of <0.1% protein by weight, reaches an average radius of about 20,000 A.The cytoplasm in eye lens cells contains a highly concentrated solution of lens-specific proteins, the crystallins. Their concentration increases from =250 to 400 g/liter going from cortex to nucleus, thus establishing the refractive index gradient required for proper focusing of incident light (1). The cytoplasm is normally transparent because of short-range ordering of the crystallins (2, 3). Mammalian lenses contain primarily a-crystallins (Mr 700,000-1,200,000), l3-crystallins (Mr 50,000-300,000) and -crystallins (Mr 20,000) (1,4,5). Their relative proportions vary with age and location within the lens as a result of both differential synthesis during development and selective degradation and insolubilization with aging (4-15). The lens nucleus is highly enriched in -crystallins, which are monomeric, basic proteins rich in sulfhydryl groups (8,(16)(17)(18)(19). The majority of human cataracts represent an irreversible opacification of the lens nucleus that progresses with age, a condition that is accompanied by oxidation of sulfhydryl groups, aggregation, and insolubilization of the various crystallins (4,8,13,(20)(21)(22)(23).The cold cataract phenomenon observed in most young mammalian lenses (e.g., calf, rat, and rabbit) is a convenient model system to study the relationship between nuclear opacification and -crystallin aggregation. A temperaturedependent reversible opacification can be induced in the nucleus of these young lenses (24)(25)(26)(27)(28). This behavior appears to be an intrinsic property of the concentrated mixture of crystallins, since it also can be induced in purified, membranefree cytoplasmic extracts of lens nucleus (27,(29)(30)(31)(32). Cold cataract has been modeled as a reversible phase-separation phenomenon (26)(27)(28)(29)(30)(31)(32). Upon lowering the temperature below a critical value Tc, the cytoplasm separates into protein-rich and protein-poor d...

Section: Introductionmentioning

confidence: 94%

mentioning

confidence: 99%

Opacification of gamma-crystallin solutions from calf lens in relation to cold cataract formation.

Siezen¹,

Fisch²,

Slingsby³

et al. 1985

113

“…The laser spectrometer consisted of a 10-mW Melles-Griot He-Ne laser, a temperature-controlled chamber where lenses are placed, and a United Detector Technology (UDT, Santa Monica, CA) PIN 10-D photodiode that detected light transmitted through lenses in the chamber (10,22). The chamber held several lenses, as seen in Fig.…”

mentioning

confidence: 99%

Phase separation in lens cytoplasm is genetically linked to cataract formation in the Philly mouse.

Clark¹,

Carper²

1987

The variation of the phase-separation temperature, T,, in In the mammalian lens, a reorganization of the cellular structure is necessary for the transition from the normal transparent state to the opaque, cataractous state (1-3). The transition in early stages of cataract development is associated with a phase separation in the lens cytoplasm (4-6). A phase separation is a low-energy mechanism of cytoplasmic restructuring that is reversible and is characterized by a temperature, Tc, at which the cytoplasm undergoes a transition from a transparent state to an opaque state (2, 4, 5, 7). In the transparent state, short-range order in the organization of cytoplasmic protein allows the cytoplasm to exist as a single homogeneous phase (8). In the opaque state, the short-range order is disrupted, and the cytoplasm exists as

“…Below Tc two separate cytoplasmic phases were responsible for light scattering and opacification (3,4). Normally, Tc in lens cells is well below body temperature, and an abnormal increase in the Tc toward body temperature is a fundamental feature of the earliest stages of opacification in several animal models for cataract formation (5)(6)(7)(8)(9).…”

mentioning

confidence: 99%

Phase-separation inhibitors and prevention of selenite cataract.

Clark

Steele

1992

The variation of the phase-separation temperature (Td) was studied in lenses during formation of cataracts induced by a subcutaneous ihfection of sodium selenite. In normal control animals, the Tc decreased monotonically with increasing age. Approximately 2 days after administration of the selenite the T, decreased sharply to a minimum, and then at day 4 the Tc increased dramatically toward body temperature. Mature irreversible cataracts formed -6 days after injection of the selenite. (18)(19)(20)(21). The effect of WR-77913 on x-ray cataract was not entirely unexpected because the amino phosphorothioates are believed to behave as free-radical scavengers (17). The inhibition of the x-ray cataract could be explained as protection against oxidative damage to lens cells caused by free radicals as well as an effect on T, (17,22).It was not anticipated that WR-77913 would prevent cataract produced by selenite. In contrast to the x-ray cataract, T, was reported to decrease rather than increase in the selenite cataract (23). Selenite-induced opacity was associated with lenticular accumulation of calcium (24, 25) rather than free-radical formation. Oxidation of protein sulfhydryls was observed in the late stages of cataract formation (24,26,27), although oxidation of epithelial cells may be important at earlier stages of cataract formation. The differences between the x-ray and selenite mechanisms make the selenite model an interesting test for the effectiveness of WR-77913 as a PSI in vivo. In this report we show a remarkable observation: the chemical inhibition of abnormal changes in Tc, in vivo, is sufficient for the effective prevention of selenite cataract in rats. While it is possible that the properties of WR-77913 as a PSI may have nothing to do with its mode of action in preventing selenite cataract, we present direct evidence that reagents acting in vivo on a cytoplasmic phase separation have the potential to control and prevent formation of cataract. was administered by intraperitoneal injection at a dose of 820 mg/kg 15 min prior to the injection ofPBS only (control group B) or sodium selenite (group D). At various times, upl to 9 days after treatments, animals from each group were euthanized. Lenses were removed and placed in silicone oil in the temperature-controlled sample chamber (see Fig. 2). The T" was measured immediately. MATERIALS AND METHODS