Proc. Natl. Acad. Sci. USA Vol. 76, No. 9, pp. 4414-4416, September 1979 Biophysics Cytoplasmic phase separation in formation of galactosemic in lenses of young rats () COE ISHIMOTO*, PATRICK W. GOALWINt, SHAO-TANG SUNt, IZUMI NISHIOt, AND TOYOICHI TANAKAtt *Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; and tDepartment of Physics and Center for Materials Science & Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Communicated by John Ross, June 8, 1979 ABSTRACT We have determined the age dependence of represents transparency. The opaque area at 1 day is a solid the characteristics of the cytoplasmic phase separation of lenses spheroid with the boundary at different temperatures at the from normal and galactosemic young rats. In the normal , positions indicated by the curve. As the temperature is lowered, the temperature at which the phase separation occurs decreases monotonically with age. In the lenses of rats fed with a high the area of opacity first appears at the center at approximately diet, the phase separation temperature becomes in- 30'C and then grows toward the periphery. Note that for creasingly higher with the development of galactosemia. When newborn rats, the maximum phase separation temperature is the phase separation temperature becomes higher than the oc- higher than the ocular temperature. In fact, we observed a ular temperature, the nuclear opacity appears in vivo. The pinpoint opacity in the lenses of newborn rats in vivo by cutting opacity is the result of light scattering by spatial fluctuations open the of the rats. In older lenses, the center is clear of the refractive index formed by interspersed regions of two separated phases in the fiber cell cytoplasm. This shows that the and the opaque region is a spheroidal shell with the outside nuclear opacity that develops in the lens of galactosemic rats boundary at the positions indicated in the figure. As the tem- is the manifestation of phase separation of the lens fiber cyto- perature is lowered, the region of opacity initially appears as plasm. a thin shell which then increases in thickness toward the pe- riphery. It is difficult to observe the inside boundary of the shell The galactosemic cataract that develops in the lens of a young of opacity, but observation of the cross section of a lens that had rat when it is fed a high galactose diet has been studied exten- been cut open showed that there was extremely little movement sively because it is an excellent model of human as- of the inside boundary of the shell toward the center as the sociated with defects in sugar metabolism such as and temperature was lowered. The curves in Fig. 1A shift with age galactosemia (1). A "cold cataract" appears in the lens of a away from the center of the lens and toward lower tempera- young rat when it is removed from the animal and cooled (2). tures. The opacity of a cold cataract, which disappears on warming, The galactose-fed animals developed nuclear opacity in vivo is the result of light scattering by spatial fluctuations of the re- at around 35 days or after 14 days of galactose feeding (Fig. 2). fractive index formed by interspersed regions of two separated We emphasize that the present observations concern only the phases of the protein-water mixture of the fiber cell cytoplasm nuclear opacity and not the cortical vacuolar opacity that also (3). We examined this cytoplasmic phase separation during the develops prior to the nuclear opacity in these animals. The development of galactosemic cataract. We present evidence vacuolar opacity is not as intense as the nuclear opacity and did that the nuclear opactiy in galactosemic cataracts is such a phase not interfere with our observations (4). We have examined the separation of the cytoplasm. progressive change with age of the characteristics of cold cat- aract in the galactose-fed animals. There were no differences MATERIALS AND METHODS from the cold cataract in normal animals until around 30 days. Female Sprague-Dawley rats of different ages were obtained After this date, the phase separation temperature started to rise from Charles River Breeding Laboratories. The animals were and became progressively higher. When it became higher than given free access to tap water and Purina Rat Chow. Galac- ocular temperature, the opacity could then be seen in vivo. This tose-fed animals were given, in place of whole chow, a mixture situation is clearly seen in Fig. 3, in which the maximum phase of equal parts of galactose and ground chow starting at 21 days separation temperature of normal and galactosemic lenses is of age. The animals were asphyxiated in a CO2 chamber. The plotted as a function of age. In the normal lenses the maximum lenses were then removed and placed in a temperature-con- phase separation temperature decreases monotonically with trolled cell filled with silicone oil. The cell temperature was age, whereas in the galactosemic lenses, it begins to increase at regulated to an accuracy of ±0.050C. Each lens was viewed 30 days and then increases rapidly at around 35 days, after 14 from the anterior axial direction through a dissecting micro- days of feeding. Fig. 1B shows some typical phase diagrams of scope having a calibrated eyepiece. the cold cataract at various stages of the development of galactosemia. Note that the phase boundary of the opacity not RESULTS only rises in temperature, but also moves toward the center of the lens. The phase separation temperature at which the opacity appears upon cooling was determined as a function of the radial position DISCUSSION in the lenses of rats of various ages (Fig. 1A). The area under each curve of the diagram represents opacity and the remainder We suggest that the changes of the radial position of the phase boundary with age and galactosemia can be understood qual- itatively by considering the dependence of the phase separation The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- temperature on protein concentration. The lens grows by laying vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. $ To whom all correspondence should be addressed. 4414 Downloaded by guest on September 26, 2021 Biophysics: Ishimoto et al. Proc. Natl. Acad. Sci. USA 76 (1979) 4415

0) 30 41 lo- ~ ~ ~ . o C) E *A 20

10 0

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00 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 Radial position, mm FIG. 1. (A) Progressive change of the characteristics of cold cataract in lenses of normal animals during normal aging on day 1 (-), 10 (0), 18 (A), 25 (A), 42 (0), and 56 (o). (B) Change in lenses of galactose-fed animals at different stage of galactosemia at 34 (A, A, E0) and 35 (0) days and in two lenses of normal animals at 31 (u) and 38 (0) days. Each curve in A and B represents the radial position ofthe boundary between the clear and opaque areas of the lens. The part of the diagram under each curve represents opacity and the remainder, transparency. Thus, another way of reading the diagram is to regard the curve as representing the temperature at which opacity appears at each radial position upon cooling. The symbols at the bottom of the diagrams mark the subcapsular radius of each lens. Experimental details are described in the text. down long, thin fiber cells in concentric layers at the periphery temperature on protein concentration has been observed in of the lens. The protein concentration in the fiber cells at each lysozyme/salt/water mixtures, which undergo phase separa- radial position increases with age due to synthesis and dehy- tion, producing opacity (6). The maximum phase separation dration (5). Thus, the protein concentration increases along the temperature corresponds to the critical point (6). The pro- radius from the periphery to the center of the lens due to the gressive shift away from the center with age of the radial po- increasing age of the fiber cells (5). Fig. 1A shows that except sition of the critical point is due to the general increase in pro- at very young ages, there is an intermediate radial position, or tein concentration throughout the lens (5). Because of the mo- protein concentration, at which the phase separation temper- notonic decrease of protein concentration with radial position, ature is highest. This type of dependence of the phase separation the critical concentration appears at a larger radial position

I

0 80P / (A CD 0 W 60- 0)a) I.II 0/" CD 0 1OR 40F 0 Start of 20F- galactose feeding

, I I I I ,I,;/ L 1 10 20 30 40 50 60 Age, days FIG. 2. Percentage of galactosemic rat lenses having in vivo nuclear opacity as a function of age. The rats were fed a 50% galactose diet starting at the age of 21 days. Downloaded by guest on September 26, 2021 4416 Biophysics: Ishimoto et al. Proc. Nati. Acad. Sci. USA 76 (1979) Some preliminary results on normal rat lenses incubated in salt .,, ::: IIt., solutions indicate that there is a strong dependence of the phase ::::, In In separation temperature on salt concentrations. The decrease X:in of the maximum phase separation temperature with age in normal rat lenses may be related to the change of ionic strength 0~60 during the dehydration process. For galactose-fed animals, the Galactosernic lenses lens membrane becomes weak and eventually begins to mal- Sc " function after approximately 2 weeks of feeding (10). This I a)0. breakdown of the membrane leads to changes in protein con- Csa centrations and salt composition inside the lens fiber cells (10). 0c 40 Indeed, we have observed a strong similarity between the ev- olutions of phase boundaries of the galactosemic lens and nor- Cs mal lenses incubated in saline solution. Further study is needed Q*3C to identify the factors that are important to the change of the 0)CA phase separation temperature. It is now clearly desirable to make in vivo determinations of l 20 the phase separation temperature of the cytoplasm. The dif- E ference of the phase separation temperature from the ocular E temperature is a direct indicator of the susceptibility of the lens mx lo to cytoplasmic phase separation and, therefore, to nuclear opacity. These determinations may be achieved by measuring 0 Start of the protein diffusivity in the cytoplasm by laser light scattering galactose feeding spectroscopy. The protein diffusivity is zero at the critical point I and becomes larger when the ocular temperature is higher than 0 10 20 30 40 50 60 the critical temperature (3, 11). Age, days In summary, we have presented evidence that the nuclear FIG. 3. Maximum phase separation tempoerature of normal (open opacity in galactosemic cataract is the result of light scattering circles) and galactosemic (dots) rat lenses as3 a function of age. The by spatial fluctuations of the refractive index formed by in- lenses were heated only to 600C. Observations3 at higher temperatures terspersed regions of two separated phases of the lens fiber cell were impossible because of the denaturation of the lens protein. The cytoplasm. numbersIOAOtA{+rat lenses1n examined:A ,..nwas -OAe4, DOCC62, to,, :o3b,ma anaEA .31iJ {A_tor +I,Mne ageseo of 31, 34, 35, 36, and 37 days, respectively. Each lens at the age of 34 We thank Prof. G. B. Benedek for his suggestions and discussions. or 35 days is represented by a single dot. For lenses at the ages of 31, This research was supported by National Institutes of Health Grant 36, and 37 days, each is represented by two connected dots so that the EY01696 and National Science Foundation Grant CHE77-26924. total number of dots, isolated or connected, is approximately the same for each day's measurement. This gives each dot approximately equal 1. Van Heyningen, R. (1971) Exp. Eye Res. 11, 415-428. statistical weight. 2. Zigman, S. & Lerman, S. (1964) Nature (London) 203, 662- 663. 3. Tanaka, T., Ishimoto, C. & Chylack, L. T., Jr. (1977) Science 197, 1010-1012. when the protein concentration increases at every point in the 4. Sippel, T. 0. (1966) Invest. Ophthalmol. 5,568-575. lens. It is well established that there is an osmotic swelling of the 5. Philipson, B. (1969) Invest. Ophthalmol. 8,258-270. lens caused by the accumulation of dulcitol formed from ga- 6. Ishimoto, C. & Tanaka, T. (1977) Phys. Rev. Lett. 39, 474- lactose by lens aldose reductase (7). The general decrease in 477. protein concentration to 7. Kinoshita, J. H. (1974) Invest. Ophthalmol. 13, 713-724. throughout the lens due this swelling 8. Philipson, B. (1969) Invest. Ophthalmol. 8, 281-289. during the development of galactosemic cataract (8) may ex- 9. Benedek, G. B., Clark, J. I., Serrallach, E., Young, C. Y., Mengel, plain why the changes in the characteristics of cold cataract L., Sauke, T., Bagg, A. & Benedek, K. (1979) Philos. Trans. R. during the process correspond to a reversal of the changes that Soc. London 292, 121-132. occur with aging. 10. Kinsey, V. E. & Hightower, K. R. (1978) Exp. Eye Res. 26, The phase separation temperature depends not only on the 521-528. total protein concentration, but also on other factors such as the 11. Tanaka, T. & Ishimoto, C. (1977) Invest. Ophthalmol. 16, composition of proteins and the concentration of ions (6, 9). 135-140. Downloaded by guest on September 26, 2021