- Email: [email protected]
Immunofluorescent study of a chick lens fiber cell membrane polypeptide
Exp. Eye Res. (1978) 27, 151-157
Immunofluorescent Study of a Chick Lens Fiber Cell Membrane Polypeptide
A vxter-insoluble membrane rich fraction was isolated from the lenses of three-month-old chickens and subjected to electrophoresis on 513O o polyacrylamide gels containing lo,, sodium dodecyl sulfate. The major polypeptide (mol. wt. 26 UOO daltons) was removed from several gels and used to immunize rabbits. The specific antiserum was used for the indirect imm~mofluorescent detection of the first appearance and localization of the major membrane polypeptide in embryonic and post-embryonic chick lenses. The membrane antigen was tirst detected after four days of incubation in the forming lens fibers just beneath the epithclium and it was restricted to the cell membranes. Thereafter, a positive reaction in the fiber membranes was found throughout the lens fiber mass but’ never in the epithelium. The lens cell membranes were seen to first become positive in the equatorial zone as the wlls began to elongate and form fibers. Kie?/ mm7s: lens; fiber; cell; membrane ; polypeptide : immunofluorescence; ontogeny.
1. Introduction In rcccnt years there has been a growing interest in the morphology and IJiOcltcinir;tr,y of the lens fiber cell inend~ranes (Bloci~~e~~da~l, Zweers, Vermorken, Dunia and Ucuedetti, 1972 ; Dunia, Sen Ghosh, Benedetti. Zweers and Bloemendal, 19id; Broekhuyse and Kuhlman, 1974 ; Alcala. Lieska and Illaisel, 1975; Maisel, Alcala an31 Lieski).. 1976; Broekhuyse, Kuhlrnan and Stols, 1976 ; Bloemendal, 1977 ; Bloementlal, Vermorken, Kibbelaar, Dunia and Benedetti, 1977). It has been established that the lens fiber cell plasma membranes of the chick and l)ovine lens contain a polypeptide of ~nolecular weight 26 000-27 500 daltons as a major intrinsic membrane component (Blocrncndal, 1977; Alcala et a,l., 1975; Naisel et al., 1976; Broekhuyse et al., 1976). It has lIeen suggested that this membrane component be designated MP26 (Bloemendal et al.. 1977). In the chick this polypeptide comprises 540$ of the total membrane protein. Our laboratory has succeeded in producing an antibody that is specific for this chick membrane polypeptide (MP26) when compared to lens crystallin (Alcala and JIa,isel, 1978). Since the ontogeny of the chick lens crystallins has been so successfully studied using the immunofluorescent technique (van Doorenmaalen, 1966; Ikeda and Zwaan, 1966, 1967 ; Zwaan and Ikeda, 1965; Brahms and van Doorenmaalen, 1971; Waggoner, Lie&a, Alcala and Maisel, 1976) we have applied the immunofluorescent method to detect the earliest appearance and distribution of MP26 in embryonic and postembryonic chick lenses.
2. Materials and Methods Antigelz preparation Lenses were removed from J-month-old chickens within 2 hr after slaughter of the animal. The fiber mass,free of capsule and epithelium was homogenized at 4°C in a standard salt solution (0.1 M-KCl, 0.01 ivf-%mercaptoethanol, 0.006 M-sodium phosphate buffer, 0014-4835/78/2702-0151
$01.00/O
0 1978 Academic 151
Press Inc. (London)
Limited
152
P. R.
WAGGOSEB
AND
H.
MBISEL
pH 72). The homogenate was centrifuged at 37 000 ;< g for 20 min and the water-insoluble pellet was collected. Plasma membranes were isolated from the pellet as described hi Naisel et al. (1976). The pellet was thoroughly washed in buffer to remove the crystallius and then treated with 8 M-urea to soluhilize the intracellular matrix. The urea insoluble material, enriched in membrane, was washed with buffer to remove urea, and analyzed by SDS-polyacrylamide gel electrophoresis (Maisel et al., 1976). The major polypeptide of the lens fiber membrane was isolated in the following manner. Following electrophoresis of t,he membrane material, the SDS-gels were subjected to repeated changes of a solution of ammonium sulfate, at 40 “/‘A saturation, to reversibly precipitate the polypeptide bands and to remove non-protein bound SDS. The ammonium sulfate precipitated band corresponding in mobility to the membrane polypeptide of molecular weight 26 000 daltons (JIP26; Fig. 1) was sliced from 12 such gels. The gel slices were pooled in 6 ml of a solution of 0.05 M-Tris-HCl, 0.005 nz-MgCl, and 0.01 &I-2mercaptoethanol, pH ‘7.4 and dialyzed against repeated changes of the buffer to elute protein from the gel and remove proteinbound SDS (minimal amounts of SDS remain protein-bound following this procedure). Productiorz. and characterization
of antiserum
The protein solution from the dialysis of the gel segments was mixed with Freund’s complete adjuvant (1:2) and used for immunization of rabbits. The antibody titer and its specificity was monitored by double immunodiffusion (Ouchterlony, 1953) and immunoelectrophoresis (Scheidegger, 1955) in agar. The antiserum was tested against the antigen that was used to produce the antiserum and against a water-soluble lens fraction. The antiserum was collected when an acceptable titer was reached.
Fertile chicken eggs were incubated at 38°C and 85% relative humidity. Embryos were collected at daily intervals from two days of incubation through hatching. Eyes from 5-day-old and adult chickens were also collected. Processing of the tissue and indirect immunofluorescence, using fluorescien isothiocyanate-conjugated goat IgG against rabbit IgG, was carried out according to Ikeda and Zwaan (1966, 1967) and Zwaan and Ikeda (1968). Control experiments consisted of replacing the lens tissue, the antiserum or the fluorescent-labelled goat IgG with a non-specific reactant. The lens tissue was replaced by liver, antiserum by normal rabbit serum and fluorescent labelled goat IgG by a saline solution of the fluorescent dye. In addition, the surrounding ocular tissues (iris, cornea, retina) served as control tissues. A series of dilutions of the antiserum was made to test the specificity of our method and determine the optimal dilution of our antiserum (Petrusz, Sar, Ordronneau and DiMeo, 1976). The antiserum was subsequently used at a dilution of 1 part in 50 of phosphate buffered saline, pH 7.2.
3. Results ImmunoEogicul
reactions
in agar
Immunoelectrophoretic analysis showed that the antiserum produced against ME’26 (Fig. 1) reacted only with this component. No reaction was obtained with soluble lens crystallins. Furthermore, an antiserum prepared against the soluble lens crystallins
did not react
with
the membrane
polypeptide
(Fig.
2). The
membrane
polypeptide antiserum did not cross-react with any lens urea-soluble proteins nor with lens crystallins that had been treated with 1% SDS.
IMMUNOFLUORESCENT
STUDY
OF
LENS
153
MEMBRANE
1mmunoJuorescence A positive immunofluorescent reaction was first detected in the lens at four days of incubation (Fig. 3). The positive reaction was seen in the anterior part of the forming fiber mass just beneath the epithelium and appeared to be conked to the fiber membranes. The epithelium and other ocular tissues were negative. After five days of incubation the fluorescence in the lens fibers had increased dramatically but the f
FIG. 1. An SDS polyacrylamide daltons) was isolated from several
gel of the urea-insolulx gels for the production
membrane fraction. of an antiserum.
MP26
(mol.
wt. 26 000
FIG. 2. An immunoelectrophoresis plate to show that our antiserum reacts with the antigen isolated from the SDS gel but it does not react with any of the soluble lens components. MP26 = MP26 antigen; MP26a = antiserum to MP26; sf = soluble lens fraction; tla = total lens antiserum (antiserum to total water soluble lens fraction); d = delta crystallin precipitin arc; x = alpha crystallin precipitin arc: ,5’ = beta crystallin precipitin arc.
epithelium and surrounding tissues remained negative (Fig. 4). This pattern of a positive reaction in the fiber mass and a negative reaction in the lens epithelium continues into the adult lens and indeed the fluorescence is restricted to the fiber cell membranes as evidenced by Figs 5-10. The epithelial cell membranes at all developmental stages and fiber cell membranes prior to four days of incubation never gave a
FIG. 3. Immunofluorescence of a chick lens after 4 days of ilzubatinn treated with antjiscruln tn MP6. Note that the positive fluorescence is wsttictrtl to the cells ill thv nntrrior rryion 11f’ the lihrr mass. E = epithelium. :: 300. FIG. 4. Immunotluorescence of a chick lens after five dars of incubation and exposctl to antiserum to MP26. Note the brightly fluorescent fiber mass and negativr epithclium (E) and negative nuclei in the forming fibers. :,: 150. FIG. 5. Immunofluorescent staining of a frontal section of a chick lens after nine days of incubation and exposure to antiserum to MP26 antigen. The epithelium (E) is still negative and the restriction of a positive reaction to the membranes is apparent. x 380. FIG. 6. Immunofluorescence of a frontal section of a chick lens after 20 days of incubation and exposure to antiserum to MP26. The restriction of the fluorescence to the membrane is readily apparent in these cortical fibers. The epithelium (E) is negative. x225.
IMiWUNOFLUORESCENT
STUDY
OF
LENS
MEMBRANE
155
FIG. 7. An enlargement of Fig. 6 to give a better appreciation of the specificity of the antiserum to the fiber membranes. There is no reaction in the epithelium (E) or the cytoplasm of the fibers. x 340. FIG. 8. Immunofluorescence of a frontal section through the nuclear region of a chick lens after 20 days incubat,ion and exposure to antiserum to MP26. The membranes of the nuclear region are very reactive. x 340. FIG. 9. Immunofluorescence of a sagittal section through the chick lens after 18 days of incubation and exposure to antiserum to MP26. Note that the membranes first become reactive as the cells begin to elongate at the equatorial zone (lower left corner) and that the fiber cytoplasm and nuclei are negative. !< 350. FIG. 10. Immunofluorescence of a sagittal section through a chick lens exposed to beta crystallin antiserum. Note that in contrast to Fig. 9 the membranes are negative. ~425.
1.56
P. R,. WAGGONER
AND
H.
MAISEL
positive reaction even at higher concentrations of antiserum. While the fluorescence was brightest in the cortical region of the older lenses where the cell columns were distinct, the nuclear fibers were also very reactive even in the late embryonic and post-embryonic lenses (Fig. 8). The lens cell membra.nes first become positive as the cells begin to elongate in the equatorial zone (Fig. 9). One may get the false impression that the cytoplasm in Fig. 9 is also fluorescent. This can be accounted for by the relatively thick sections (7 pm) that were used. There are undoubtedly some membranes lying deep in the cytoplasm which could account for the apparent reaction. However, one need only refer back to Fig. 7 to be convinced that the reaction is not the result of the cytoplasm. The fiber cell membrane fluorescence is in stark contrast to the results found with an antiserum directed toward one of the lens crystallins which produces a fluorescent cytoplasm and negative membranes and fiber nuclei (Fig. 10). A11 of our control experiments gave negative results.
4. Discussion The first appearance of the lens fiber cell membrane antigen at 4 clays incubation, as detected by immunofluorescence? does not differ greatly from the time of appearance of some of the crystallins. Ikeda and Zwaan (1967) and Brahma and van Doorenmaalen (1971) both reported that alpha crystallin was first detected by immunofluorescence at about 3.5 days of incubation and Waggoner et al., (1976) first detected an anodal beta crystallin and a cathodal beta crystallin at 3 and 5 days of incubation, respectively. While the time of first appearance of crystallins and the membrane antigen are similar, their sites of first appearance are different. The crysfallins are first detected in the parts of forming fiber cells that are nearest the retina (Ikeda and Zwaan, 1966; Brahma and van Doorenmaalen, 1971; Waggoner et al., 1976) but the membrane antigen was first detected in the parts of forming fiber cells that are furthest from the retina. We know from the studies of many authors that the formation of the lens is induced by the presence of the optic cup (Coulombre, 1965) and since the crystallins are first detected in the cell nearest the retina it has been assumed that it reflects an inductive influence from the retina. However, on the basis of the present study. that does not seem to be the case with the membrane antigen. Another striking difference between the membrane antigen and the crystallins is the reactivity of the membrane antigen in the nuclear region as compared to the crystallins. Zwaan and Ikeda (1968), Brahma and van Doorenmaalen (1971) and Waggoner et al., (1976) found that with increasing age the central lens fiber cells become non-reactive in immunofluorescent tests for the presence of the crystallins even though they can be detected immunoelectrophoretically. The nuclear fibers in the present investigation never weakened in their reactivity as the age of the animal increased suggesting that the membrane proteins are more stable than the crystallins and/or their antigenic sites are not masked. The lack of a positive reaction in the lens epithelium at all stages of development is quite different from what has been found for the crystallins. All the classes of chick lens crystallins have been shown to appear in the epithelium after they first appear in the fiber cells of the lens (Zwaan and Ikeda, 1968; Brahma and van Doorenmaalen, 1971; Waggoner, et al., 1976). The lack of an epithelial reaction in the present investigation is in agreement with Bloemendal et al. (1977) and Bloemendal
IMMUKOFLUORESCENT
STUDY
OF
LER’S
157
MEMBRANE
(1977) who reported that MP26 is not found in the epithelium of the bovine. However, Broekhuyse et al. (1976) reported the presence of the main intrinsic polypeptide (MIP), which is presumably identical to MP26, in epithelial membranes. The present data suggest that the antigen detected in the fiber cell membrane is truly characteristic of the terminally differentiated lens cell and is unique to lens fibers. The fact that the membrane antigen is restricted to the fiber cells and appears as the lens cells begin to elongate suggests that it plays a role in fiber cell elongation and/or the development of specialized junctions between the lens fibers. In this regard it is interesting to note that Broekhuyse et al. (1976) found MIP to be the predominant protein in junction enriched preparations. ACKNOWLEDGMENTS
This investigation was supported by Research Grant National Eye Institute, Bethesda, Maryland, U.S.A.
No. EY-01755-01
from
the
REFERENCES Alcala.
J., Lieska, N. and Maisel, H. (1975). Protein composition of bovine lens cort,ical fiber cell membranes. Ex~. Eye Res. 21,581-95. Alcala, J. and Maisel, H. (1978). Specific antiserum to the main intrinsic polypept.ide of chick lens fiber cell plasma membranes. Exp. Eye Res. 26, 219-21. Bloemendal, H. (1977). The vertebrate eye lens. Science lw, 127-38. Bloemendal, H., Zweers, A., Vermorken, F., Dunia, I. and Benedetti, E. L. (1972). The plasma membranes of eye lens fibers. Biochemical and structural characterization. Cell Dijf. 1,91-106. Bloemendal, H., Vermorken, A. J. M., Kibbelaar, M., Dunia, I. and Benedetti, E. L. (1977). Nomenclature for the polypeptide chains of lens plasma membranes. Exp. Eye Res. 24,413-15. Brahma, S. K. and van Doorenmaalen, W. J. (1971). Immunofluorescence studies of chick lens FIX and a-crystallin antigens during lens morphogenesis and development. Ophthal. Res. 2, 344-57. Broekhuyse, R. M. and Kuhlman, E. D. (1974). Lens membranes. I. Composition of urea treated plasma membranes of calf lens. Exp. Eye Res. 19, 297-302. Broekhuyse, R. M.. Kuhlman, E. D. and Stols, A. L. H. (1976). Lens membranes II. Isolation and characterization of the main int,rinsic polypeptide (MJP) of bovine lens fiber membranes. Erp. Eye Res. 23, 365-71. Coulombre, A. J. (1965). Experimental embryology of the vertebrate eye. Invest. Ophthalmol. 4, 411-19. Dunia, I., Sen Ghosh, C., Benedetti, E., Zweers, A. and Bloemendal, H. (1974). Isolation and protein patterns of eye lens fiber junctions. FEBS Letters 45, 13944. Ikeda, A. and Zwaan, J. (1966). Immunofluorescence studies on induction and differentiation of the chicken eye lens. Invest. OphthaZmoZ. 5, 40412. Ikeda. A. and Zwaan, J. (1967). The changing cellular localization of a-crystallin in the lens of the chick embryo, studied by immunofluorescence. Develop. BioZ. l&348-67. Maisel, II., Alcala. ,J. and Lieska, N. (1976). The protein structure of chick lens fiber cell membranes and intracellular matrix. Dot. Ophthalmol. 8, 121-33. Onchterlony, 0. (1953). Antigen-antibody reactions in gels. IV. Types of reactions in coordinated systems of diflusion. Acta Path. microbial. &and. 32, 23140. Petrusz, P., Sar, M., Ordronneau, P. and DiMeo, P. (1976). Specificity in immunocyt,ochemical staining. J. Histochem. Cytochem. 24, 1110-15. Scheidegger, J. J. (1955). Une micro-methode de l’immuno-electrophorese. Int. Archs Allergy AppZ. Immun. 7, 103-10. van Doorenmaalen, W. J. (1966). Immunohistological demonstration of adult lens antigens in the embryonic chick lens (II) Exp. Eye Res. 5, 151-55. Waggoner, P. R., Lieska, N., Alcala, J. and Maisel, H. (1976). Ontogeny of chick lens @rystallin polypeptides by immunofluorescence. Ophthal. Res. 8,292-301. Zwaan, J. and Ikeda, A. (1968). Macromolecular events during differentiation of the chicken lens. Exp. Eye Res. 7, 301-11.
Immunofluorescent Study of a Chick Lens Fiber Cell Membrane Polypeptide
A vxter-insoluble membrane rich fraction was isolated from the lenses of three-month-old chickens and subjected to electrophoresis on 513O o polyacrylamide gels containing lo,, sodium dodecyl sulfate. The major polypeptide (mol. wt. 26 UOO daltons) was removed from several gels and used to immunize rabbits. The specific antiserum was used for the indirect imm~mofluorescent detection of the first appearance and localization of the major membrane polypeptide in embryonic and post-embryonic chick lenses. The membrane antigen was tirst detected after four days of incubation in the forming lens fibers just beneath the epithclium and it was restricted to the cell membranes. Thereafter, a positive reaction in the fiber membranes was found throughout the lens fiber mass but’ never in the epithelium. The lens cell membranes were seen to first become positive in the equatorial zone as the wlls began to elongate and form fibers. Kie?/ mm7s: lens; fiber; cell; membrane ; polypeptide : immunofluorescence; ontogeny.
1. Introduction In rcccnt years there has been a growing interest in the morphology and IJiOcltcinir;tr,y of the lens fiber cell inend~ranes (Bloci~~e~~da~l, Zweers, Vermorken, Dunia and Ucuedetti, 1972 ; Dunia, Sen Ghosh, Benedetti. Zweers and Bloemendal, 19id; Broekhuyse and Kuhlman, 1974 ; Alcala. Lieska and Illaisel, 1975; Maisel, Alcala an31 Lieski).. 1976; Broekhuyse, Kuhlrnan and Stols, 1976 ; Bloemendal, 1977 ; Bloementlal, Vermorken, Kibbelaar, Dunia and Benedetti, 1977). It has been established that the lens fiber cell plasma membranes of the chick and l)ovine lens contain a polypeptide of ~nolecular weight 26 000-27 500 daltons as a major intrinsic membrane component (Blocrncndal, 1977; Alcala et a,l., 1975; Naisel et al., 1976; Broekhuyse et al., 1976). It has lIeen suggested that this membrane component be designated MP26 (Bloemendal et al.. 1977). In the chick this polypeptide comprises 540$ of the total membrane protein. Our laboratory has succeeded in producing an antibody that is specific for this chick membrane polypeptide (MP26) when compared to lens crystallin (Alcala and JIa,isel, 1978). Since the ontogeny of the chick lens crystallins has been so successfully studied using the immunofluorescent technique (van Doorenmaalen, 1966; Ikeda and Zwaan, 1966, 1967 ; Zwaan and Ikeda, 1965; Brahms and van Doorenmaalen, 1971; Waggoner, Lie&a, Alcala and Maisel, 1976) we have applied the immunofluorescent method to detect the earliest appearance and distribution of MP26 in embryonic and postembryonic chick lenses.
2. Materials and Methods Antigelz preparation Lenses were removed from J-month-old chickens within 2 hr after slaughter of the animal. The fiber mass,free of capsule and epithelium was homogenized at 4°C in a standard salt solution (0.1 M-KCl, 0.01 ivf-%mercaptoethanol, 0.006 M-sodium phosphate buffer, 0014-4835/78/2702-0151
$01.00/O
0 1978 Academic 151
Press Inc. (London)
Limited
152
P. R.
WAGGOSEB
AND
H.
MBISEL
pH 72). The homogenate was centrifuged at 37 000 ;< g for 20 min and the water-insoluble pellet was collected. Plasma membranes were isolated from the pellet as described hi Naisel et al. (1976). The pellet was thoroughly washed in buffer to remove the crystallius and then treated with 8 M-urea to soluhilize the intracellular matrix. The urea insoluble material, enriched in membrane, was washed with buffer to remove urea, and analyzed by SDS-polyacrylamide gel electrophoresis (Maisel et al., 1976). The major polypeptide of the lens fiber membrane was isolated in the following manner. Following electrophoresis of t,he membrane material, the SDS-gels were subjected to repeated changes of a solution of ammonium sulfate, at 40 “/‘A saturation, to reversibly precipitate the polypeptide bands and to remove non-protein bound SDS. The ammonium sulfate precipitated band corresponding in mobility to the membrane polypeptide of molecular weight 26 000 daltons (JIP26; Fig. 1) was sliced from 12 such gels. The gel slices were pooled in 6 ml of a solution of 0.05 M-Tris-HCl, 0.005 nz-MgCl, and 0.01 &I-2mercaptoethanol, pH ‘7.4 and dialyzed against repeated changes of the buffer to elute protein from the gel and remove proteinbound SDS (minimal amounts of SDS remain protein-bound following this procedure). Productiorz. and characterization
of antiserum
The protein solution from the dialysis of the gel segments was mixed with Freund’s complete adjuvant (1:2) and used for immunization of rabbits. The antibody titer and its specificity was monitored by double immunodiffusion (Ouchterlony, 1953) and immunoelectrophoresis (Scheidegger, 1955) in agar. The antiserum was tested against the antigen that was used to produce the antiserum and against a water-soluble lens fraction. The antiserum was collected when an acceptable titer was reached.
Fertile chicken eggs were incubated at 38°C and 85% relative humidity. Embryos were collected at daily intervals from two days of incubation through hatching. Eyes from 5-day-old and adult chickens were also collected. Processing of the tissue and indirect immunofluorescence, using fluorescien isothiocyanate-conjugated goat IgG against rabbit IgG, was carried out according to Ikeda and Zwaan (1966, 1967) and Zwaan and Ikeda (1968). Control experiments consisted of replacing the lens tissue, the antiserum or the fluorescent-labelled goat IgG with a non-specific reactant. The lens tissue was replaced by liver, antiserum by normal rabbit serum and fluorescent labelled goat IgG by a saline solution of the fluorescent dye. In addition, the surrounding ocular tissues (iris, cornea, retina) served as control tissues. A series of dilutions of the antiserum was made to test the specificity of our method and determine the optimal dilution of our antiserum (Petrusz, Sar, Ordronneau and DiMeo, 1976). The antiserum was subsequently used at a dilution of 1 part in 50 of phosphate buffered saline, pH 7.2.
3. Results ImmunoEogicul
reactions
in agar
Immunoelectrophoretic analysis showed that the antiserum produced against ME’26 (Fig. 1) reacted only with this component. No reaction was obtained with soluble lens crystallins. Furthermore, an antiserum prepared against the soluble lens crystallins
did not react
with
the membrane
polypeptide
(Fig.
2). The
membrane
polypeptide antiserum did not cross-react with any lens urea-soluble proteins nor with lens crystallins that had been treated with 1% SDS.
IMMUNOFLUORESCENT
STUDY
OF
LENS
153
MEMBRANE
1mmunoJuorescence A positive immunofluorescent reaction was first detected in the lens at four days of incubation (Fig. 3). The positive reaction was seen in the anterior part of the forming fiber mass just beneath the epithelium and appeared to be conked to the fiber membranes. The epithelium and other ocular tissues were negative. After five days of incubation the fluorescence in the lens fibers had increased dramatically but the f
FIG. 1. An SDS polyacrylamide daltons) was isolated from several
gel of the urea-insolulx gels for the production
membrane fraction. of an antiserum.
MP26
(mol.
wt. 26 000
FIG. 2. An immunoelectrophoresis plate to show that our antiserum reacts with the antigen isolated from the SDS gel but it does not react with any of the soluble lens components. MP26 = MP26 antigen; MP26a = antiserum to MP26; sf = soluble lens fraction; tla = total lens antiserum (antiserum to total water soluble lens fraction); d = delta crystallin precipitin arc; x = alpha crystallin precipitin arc: ,5’ = beta crystallin precipitin arc.
epithelium and surrounding tissues remained negative (Fig. 4). This pattern of a positive reaction in the fiber mass and a negative reaction in the lens epithelium continues into the adult lens and indeed the fluorescence is restricted to the fiber cell membranes as evidenced by Figs 5-10. The epithelial cell membranes at all developmental stages and fiber cell membranes prior to four days of incubation never gave a
FIG. 3. Immunofluorescence of a chick lens after 4 days of ilzubatinn treated with antjiscruln tn MP6. Note that the positive fluorescence is wsttictrtl to the cells ill thv nntrrior rryion 11f’ the lihrr mass. E = epithelium. :: 300. FIG. 4. Immunotluorescence of a chick lens after five dars of incubation and exposctl to antiserum to MP26. Note the brightly fluorescent fiber mass and negativr epithclium (E) and negative nuclei in the forming fibers. :,: 150. FIG. 5. Immunofluorescent staining of a frontal section of a chick lens after nine days of incubation and exposure to antiserum to MP26 antigen. The epithelium (E) is still negative and the restriction of a positive reaction to the membranes is apparent. x 380. FIG. 6. Immunofluorescence of a frontal section of a chick lens after 20 days of incubation and exposure to antiserum to MP26. The restriction of the fluorescence to the membrane is readily apparent in these cortical fibers. The epithelium (E) is negative. x225.
IMiWUNOFLUORESCENT
STUDY
OF
LENS
MEMBRANE
155
FIG. 7. An enlargement of Fig. 6 to give a better appreciation of the specificity of the antiserum to the fiber membranes. There is no reaction in the epithelium (E) or the cytoplasm of the fibers. x 340. FIG. 8. Immunofluorescence of a frontal section through the nuclear region of a chick lens after 20 days incubat,ion and exposure to antiserum to MP26. The membranes of the nuclear region are very reactive. x 340. FIG. 9. Immunofluorescence of a sagittal section through the chick lens after 18 days of incubation and exposure to antiserum to MP26. Note that the membranes first become reactive as the cells begin to elongate at the equatorial zone (lower left corner) and that the fiber cytoplasm and nuclei are negative. !< 350. FIG. 10. Immunofluorescence of a sagittal section through a chick lens exposed to beta crystallin antiserum. Note that in contrast to Fig. 9 the membranes are negative. ~425.
1.56
P. R,. WAGGONER
AND
H.
MAISEL
positive reaction even at higher concentrations of antiserum. While the fluorescence was brightest in the cortical region of the older lenses where the cell columns were distinct, the nuclear fibers were also very reactive even in the late embryonic and post-embryonic lenses (Fig. 8). The lens cell membra.nes first become positive as the cells begin to elongate in the equatorial zone (Fig. 9). One may get the false impression that the cytoplasm in Fig. 9 is also fluorescent. This can be accounted for by the relatively thick sections (7 pm) that were used. There are undoubtedly some membranes lying deep in the cytoplasm which could account for the apparent reaction. However, one need only refer back to Fig. 7 to be convinced that the reaction is not the result of the cytoplasm. The fiber cell membrane fluorescence is in stark contrast to the results found with an antiserum directed toward one of the lens crystallins which produces a fluorescent cytoplasm and negative membranes and fiber nuclei (Fig. 10). A11 of our control experiments gave negative results.
4. Discussion The first appearance of the lens fiber cell membrane antigen at 4 clays incubation, as detected by immunofluorescence? does not differ greatly from the time of appearance of some of the crystallins. Ikeda and Zwaan (1967) and Brahma and van Doorenmaalen (1971) both reported that alpha crystallin was first detected by immunofluorescence at about 3.5 days of incubation and Waggoner et al., (1976) first detected an anodal beta crystallin and a cathodal beta crystallin at 3 and 5 days of incubation, respectively. While the time of first appearance of crystallins and the membrane antigen are similar, their sites of first appearance are different. The crysfallins are first detected in the parts of forming fiber cells that are nearest the retina (Ikeda and Zwaan, 1966; Brahma and van Doorenmaalen, 1971; Waggoner et al., 1976) but the membrane antigen was first detected in the parts of forming fiber cells that are furthest from the retina. We know from the studies of many authors that the formation of the lens is induced by the presence of the optic cup (Coulombre, 1965) and since the crystallins are first detected in the cell nearest the retina it has been assumed that it reflects an inductive influence from the retina. However, on the basis of the present study. that does not seem to be the case with the membrane antigen. Another striking difference between the membrane antigen and the crystallins is the reactivity of the membrane antigen in the nuclear region as compared to the crystallins. Zwaan and Ikeda (1968), Brahma and van Doorenmaalen (1971) and Waggoner et al., (1976) found that with increasing age the central lens fiber cells become non-reactive in immunofluorescent tests for the presence of the crystallins even though they can be detected immunoelectrophoretically. The nuclear fibers in the present investigation never weakened in their reactivity as the age of the animal increased suggesting that the membrane proteins are more stable than the crystallins and/or their antigenic sites are not masked. The lack of a positive reaction in the lens epithelium at all stages of development is quite different from what has been found for the crystallins. All the classes of chick lens crystallins have been shown to appear in the epithelium after they first appear in the fiber cells of the lens (Zwaan and Ikeda, 1968; Brahma and van Doorenmaalen, 1971; Waggoner, et al., 1976). The lack of an epithelial reaction in the present investigation is in agreement with Bloemendal et al. (1977) and Bloemendal
IMMUKOFLUORESCENT
STUDY
OF
LER’S
157
MEMBRANE
(1977) who reported that MP26 is not found in the epithelium of the bovine. However, Broekhuyse et al. (1976) reported the presence of the main intrinsic polypeptide (MIP), which is presumably identical to MP26, in epithelial membranes. The present data suggest that the antigen detected in the fiber cell membrane is truly characteristic of the terminally differentiated lens cell and is unique to lens fibers. The fact that the membrane antigen is restricted to the fiber cells and appears as the lens cells begin to elongate suggests that it plays a role in fiber cell elongation and/or the development of specialized junctions between the lens fibers. In this regard it is interesting to note that Broekhuyse et al. (1976) found MIP to be the predominant protein in junction enriched preparations. ACKNOWLEDGMENTS
This investigation was supported by Research Grant National Eye Institute, Bethesda, Maryland, U.S.A.
No. EY-01755-01
from
the
REFERENCES Alcala.
J., Lieska, N. and Maisel, H. (1975). Protein composition of bovine lens cort,ical fiber cell membranes. Ex~. Eye Res. 21,581-95. Alcala, J. and Maisel, H. (1978). Specific antiserum to the main intrinsic polypept.ide of chick lens fiber cell plasma membranes. Exp. Eye Res. 26, 219-21. Bloemendal, H. (1977). The vertebrate eye lens. Science lw, 127-38. Bloemendal, H., Zweers, A., Vermorken, F., Dunia, I. and Benedetti, E. L. (1972). The plasma membranes of eye lens fibers. Biochemical and structural characterization. Cell Dijf. 1,91-106. Bloemendal, H., Vermorken, A. J. M., Kibbelaar, M., Dunia, I. and Benedetti, E. L. (1977). Nomenclature for the polypeptide chains of lens plasma membranes. Exp. Eye Res. 24,413-15. Brahma, S. K. and van Doorenmaalen, W. J. (1971). Immunofluorescence studies of chick lens FIX and a-crystallin antigens during lens morphogenesis and development. Ophthal. Res. 2, 344-57. Broekhuyse, R. M. and Kuhlman, E. D. (1974). Lens membranes. I. Composition of urea treated plasma membranes of calf lens. Exp. Eye Res. 19, 297-302. Broekhuyse, R. M.. Kuhlman, E. D. and Stols, A. L. H. (1976). Lens membranes II. Isolation and characterization of the main int,rinsic polypeptide (MJP) of bovine lens fiber membranes. Erp. Eye Res. 23, 365-71. Coulombre, A. J. (1965). Experimental embryology of the vertebrate eye. Invest. Ophthalmol. 4, 411-19. Dunia, I., Sen Ghosh, C., Benedetti, E., Zweers, A. and Bloemendal, H. (1974). Isolation and protein patterns of eye lens fiber junctions. FEBS Letters 45, 13944. Ikeda, A. and Zwaan, J. (1966). Immunofluorescence studies on induction and differentiation of the chicken eye lens. Invest. OphthaZmoZ. 5, 40412. Ikeda. A. and Zwaan, J. (1967). The changing cellular localization of a-crystallin in the lens of the chick embryo, studied by immunofluorescence. Develop. BioZ. l&348-67. Maisel, II., Alcala. ,J. and Lieska, N. (1976). The protein structure of chick lens fiber cell membranes and intracellular matrix. Dot. Ophthalmol. 8, 121-33. Onchterlony, 0. (1953). Antigen-antibody reactions in gels. IV. Types of reactions in coordinated systems of diflusion. Acta Path. microbial. &and. 32, 23140. Petrusz, P., Sar, M., Ordronneau, P. and DiMeo, P. (1976). Specificity in immunocyt,ochemical staining. J. Histochem. Cytochem. 24, 1110-15. Scheidegger, J. J. (1955). Une micro-methode de l’immuno-electrophorese. Int. Archs Allergy AppZ. Immun. 7, 103-10. van Doorenmaalen, W. J. (1966). Immunohistological demonstration of adult lens antigens in the embryonic chick lens (II) Exp. Eye Res. 5, 151-55. Waggoner, P. R., Lieska, N., Alcala, J. and Maisel, H. (1976). Ontogeny of chick lens @rystallin polypeptides by immunofluorescence. Ophthal. Res. 8,292-301. Zwaan, J. and Ikeda, A. (1968). Macromolecular events during differentiation of the chicken lens. Exp. Eye Res. 7, 301-11.