A 70000 Mr membrane protein (MP70) has previously been identified as a specific component of lens intercellular junctions. In this paper we use anti-MP70 immunofluorescence microscopy of dissected fibre bundles to study the formation, distribution and dissociation of junctional plaques in the outer cortex region of the sheep lens. Abundant, small junctional plaques are assembled de novo in the broad sides of the elongating fibres near the equatorial lens periphery. In fully elongated, pole-to-pole fibres, junctional plaques are generally larger, and while dispersed on the broad sides of the fibres in the equatorial lens plane, these junctions line up in the middle of the broad and narrow sides of the fibres in the lens polar regions. This precisely defined positioning is independent of junction size and hence cannot solely be explained by the constraints of fibre width. Junctional plaques fragment to smaller sizes and MP70 is cleaved to MP38 in mature, enucleated fibres located in the deeper portions of the lens outer cortex. These results demonstrate a dynamic aspect of lens intercellular junctions and show that they are positioned in a precise fashion, possibly in association with other membrane or cytoskeletal components.

Lens intercellular junctions, also referred to as lens 16–17 nm junctions, lens gap junctions or lens fibre junctions, are clusters of transmembrane channels (Goodenough, 1979; Kuszak et al. 1982; Paul & Goodenough, 1983; Kistler et al. 1987) and most probably serve in intercellular communication (Goodenough et al. 1980). They contain specifically a 70 000 Mr membrane protein (MP70) and junctional plaques have thus been visualized by anti-MP70 immunofluorescence microscopy (Kistler et al. 1985) and anti-MP70 immunoelectron microscopy (Gruijters et al. 1987).

The vertebrate lens consists of radial stacks of flat hexagonal, pole-to-pole fibres, with young fibres successively forming new concentric layers at the lens equator, somewhat similar to the growth rings in wood. This is because epithelial cells near the lens equator continue to divide during the entire life span of the animal. The daughter cells elongate to form lens fibres, which, when mature, stretch between the two lens poles. This elongation is accompanied by massive membrane synthesis and assembly of intercellular junctions predominantly on the broad sides of the fibres. Mature fibres are enucleated (Kuwabara, 1975) and protein synthesis becomes negligible in deeper regions of the lens (Wannemacher & Spector, 1968).

Hence, the crystalline lens is a good model for the study of the formation, distribution and dissociation of intercellular junctions. In this paper, we use anti-MP70 immunofluorescence microscopy of dissected lens fibres to visualize junctional plaques at various, chronologically ordered stages of formation and dissociation. Our results show a dynamic aspect of fibre junctions: their shape, size, frequency and arrangement changes as a function of fibre maturation and position along the length of an individual fibre. Most importantly, the mapping data from the lens polar regions show precise positioning of junctions, which may indicate an involvement of other membrane or cytoskeletal components.

Irnniunoreagents

Anti-MP70 antibodies were drawn from a collection of monoclonal antibodies against sheep-lens fibre plasma membranes (Kistler et al. 1986). They are secreted in cultures of hybridoma line 6–4-B2-C6 and have been shown to bind specifically to junctional plaques (Kistler et al. 1985; Gruijters al. 1987). Fluorescein isothiocyanate-conjugated sheep antibodies against mouse Ig, used as secondary label, were from Serotec (Bicester, Oxon, England).

Mapping of fibre junctions

Lenses were extracted from the eyes of sheep less than one year old and placed directly into 2% formaldehyde (made fresh from paraformaldehyde) in phosphate-buffered saline (PBS). Fixed lenses were stored at 4°C for less than a week and washed with PBS prior to use.

For dissection the lens was cut pole-to-pole into four similar portions. Using fine forceps, fibres were dissected from just below the cut surface under a dissecting microscope fitted with an eyepiece graticule to measure the distance (±0·1 mm) from the equatorial outer surface to where the fibres were to be removed. In this way fibres from 3 to 0 mm in depth, in 0·5 mm steps, were removed from several lenses (see scale diagram, Fig. 1). Depending on the depth from the equator of the mature fibres and the degree of suture involvement, fibre bundles from 7 mm to over 12 mm in length were obtained. Anti-MP70 labelling and immunofluorescence microscopy of fibre bundles was done according to immunocytochemical protocols described previously (Kistler et al. 1985). Subsequent to the fluorescence microscopy we confirmed, in several cases, the structural integrity of individual fibres in the bundles by viewing them in the scanning electron microscope. For this, specimens were dismounted, fixed in 6% glutaraldehyde, 1 % OsO4 for 1 h, dehydrated in an ethanol series, critical point dried and gold coated. In general, bundles consisted of predominantly intact fibre cells along the entire length.

Fig. 1.

Schematic drawing (to scale) of a lens from a sheep less than 1 year old, indicating the positions in the outer cortex from which fibre bundles were microdissected for junction mapping. Broad and narrow sides are indicated on the schematic drawing of the fibre on the right.

Fig. 1.

Schematic drawing (to scale) of a lens from a sheep less than 1 year old, indicating the positions in the outer cortex from which fibre bundles were microdissected for junction mapping. Broad and narrow sides are indicated on the schematic drawing of the fibre on the right.

Freeze-fracture

Fibres dissected from defined lens regions were sandwiched between two precleaned glass slides in a drop of 30 % glycerol in PBS and subsequently frozen in liquid nitrogen. The slides were fractured apart under liquid nitrogen and shadowed w’ith platinum/carbon according to Bullivant (1973). Replicas were reinforced with collodion prior to floating them off the glass slide onto hydrofluoric acid. Replicas were cleaned on chromic acid for 1 h. To remove the collodion, replicas suspended on a water droplet in a platinum loop were submersed in amyl acetate. Replicas at the water dropletamyl acetate interface remained intact after solubilization of the collodion and after transferring to distilled water, replicas were picked up onto coated grids in the normal way.

Freeze-fracture electron microscopy shows that fibre junctions are abundant in the outer cortex and strongly reduced in number deeper in the lens. Similarly, anti-MP70 immunofluorescence microscopy shows a strong macular staining pattern in the outer cortex, which fades abruptly at the outer–inner cortex transition (Kistler et al. 1985). De novo formation and dissociation of fibre junctions all occur within the outer cortex. To study these processes we have used anti-MP70 immunofluorescence microscopy to visualize junctions in fibre bundles derived from four sheep-lens cortical regions from single lenses (Fig. 1): (1) outer bow region, immediately below dividing epithelial cells, depth approximately 0mm below the equator; (2) middle bow region, fibres partially elongated, depth approximately 0·5 mm, (3) inner bow region, fibres completely elongated and becoming involved in suture formation at the lens poles, depth approximately 1 mm; (4) inner zone of outer cortex, elongated fibres form sutures at poles, depth 1·5 mm. Cleavage of MP70 to MP38 and loss of the epitope for monoclonal antiM P70 occur at the interface of the inner-outer cortex at approximately 2 mm depth (Kistler & Bullivant, 1987).

The junctional appearance will now be described individually for those four regions.

(1) Elongating fibres, 0mm (Fig. 2). Junctions on the broad sides of the fibres have generally round or oval shapes with diameters up to 2μtm. They are abundant and dispersed in an apparently random fashion.

Fig. 2.

Outer bow region of lens (depth Omm). Anti-MP70 fluorescence microscopy of dissected fibres reveals a macular staining pattern, which represents the dispersed arrangement of 16–17 nm intercellular junctions in this region. Rows of small junctions are on the narrow sides of the fibres. ×1400.

Fig. 2.

Outer bow region of lens (depth Omm). Anti-MP70 fluorescence microscopy of dissected fibres reveals a macular staining pattern, which represents the dispersed arrangement of 16–17 nm intercellular junctions in this region. Rows of small junctions are on the narrow sides of the fibres. ×1400.

(2) Elongating fibres, 0·5 mm (Fig. 3). While dispersed on the broad sides of the fibres, junctions vary dramatically in size, with diameters up to 7fim. Some particularly large junctional plaques have diameters approaching the width of the broad side. Though these very large junctions have generally oval outlines, they often contain MP70 exclusion zones. Similar particle-free islands have been demonstrated in lens fibre junctions by freeze-fracture electron microscopy (Lo & Harding, 1986). Most recently, using anti-MP70 freeze-fracture replica immunogold labelling (FR1L, Gruijters et al. 1987), we have found that in general these particle-free islands indeed do not bind anti-MP70 antibodies (unpublished data) and are thus likely to be identical to the MP70 exclusion zones observed by immunofluorescence microscopy. Their meaning is at present unclear.

Fig. 3.

Middle bow region of lens (depth 0·5 mm). Junctional plaques are highly variable in size and often contain MP70 exclusion zones, which appear as dark islands within brightly and homogeneously anti-MP70-staincd junctions. × 1400.

Fig. 3.

Middle bow region of lens (depth 0·5 mm). Junctional plaques are highly variable in size and often contain MP70 exclusion zones, which appear as dark islands within brightly and homogeneously anti-MP70-staincd junctions. × 1400.

(3) Pole-to-pole fibres, 1mm (Figs 49). In the equatorial region (Figs 4, 7), fibre junctions are dispersed on the broad sides of the fibres. Somewhat irregularly shaped, they vary in size with diameters up to 4–5 gm. Proceeding polewards along a fibre bundle, a tendency for a more ordered junction arrangement on the broad sides becomes apparent (Figs 5, 8). In the polar regions, still on the same fibre bundle, junctions of various sizes (up to 5·5μm) line up in the middle of the broad sides. In the particular fibre bundle, shown in Figs 6, 9, lining up of both small and large junctions is most prominent at the anterior lens pole (Fig. 6) and somewhat less precise at the posterior pole (Fig. 9). This difference is however not consistent when comparing different fibre bundles and may therefore not be significant. Fig. 10 is from another fibre bundle and demonstrates that even the smallest junctions generally line up in the middle of the broad sides. Similarly, small junctions line up in the middle of the narrow sides of the fibres, in agreement with the results of a recent freeze-fracture study (Lo & Harding, 1986).

Fig. 4.

Equatorial region: junctional plaques are dispersed on broad sides of fibres.

Fig. 4.

Equatorial region: junctional plaques are dispersed on broad sides of fibres.

Fig. 5.

Halfway to anterior pole; junctions tend to line up in the middle of broad sides.

Fig. 5.

Halfway to anterior pole; junctions tend to line up in the middle of broad sides.

Fig. 6.

Anterior polar region; junctions generally line up in the middle of broad and narrow sides.

Fig. 6.

Anterior polar region; junctions generally line up in the middle of broad and narrow sides.

Fig. 7.

Equatorial region, as for Fig. 4.

Fig. 7.

Equatorial region, as for Fig. 4.

Fig. 8.

Halfway to posterior pole; junctions are dispersed in this case, but often show a tendency for lining up similar to Fig. 5. Small junctions line up in the middle of narrow sides.

Fig. 8.

Halfway to posterior pole; junctions are dispersed in this case, but often show a tendency for lining up similar to Fig. 5. Small junctions line up in the middle of narrow sides.

Fig. 9.

Posterior polar region, junctions tend to line up in the middle of broad sides although less precisely than in Fig. 6.

Fig. 9.

Posterior polar region, junctions tend to line up in the middle of broad sides although less precisely than in Fig. 6.

Fig. 10.

Selected example of junction mapping in lens polar region: small and large junctions are equally precisely lined up in the middle of the broad sides, and small junctions line up in the middle of narrow sides of the fibres. × 1400.

Fig. 10.

Selected example of junction mapping in lens polar region: small and large junctions are equally precisely lined up in the middle of the broad sides, and small junctions line up in the middle of narrow sides of the fibres. × 1400.

(4) Pole-to-pole fibres, 1·5mm (Figs 11, 12). Junctional plaques undergo a process of dissociation. This is indicated in two ways: anti-MP70 immunofluorescence staining within the predominantly oval junctional plaques is punctate rather than homogeneous (Fig. 11); and freeze-fracture electron microscopy reveals small clusters of intramembrane particles instead of the characteristic plaque-like aspect (Fig. 12). This dissociation or fragmentation of fibre junctions follows the enucleation of the lens fibres. It occurs outside of the outer-inner cortex transition and thus apparently precedes the cleavage of MP70 to MP38 (Kistler & Bullivant, 1987).

Fig. 11.

Inner zone of outer cortex (depth 1·5 mm). Spotty rather than homogeneous staining of junctions indicates dissociation or fragmentation of junctional plaques. × 1400.

Fig. 11.

Inner zone of outer cortex (depth 1·5 mm). Spotty rather than homogeneous staining of junctions indicates dissociation or fragmentation of junctional plaques. × 1400.

Fig. 12.

Freeze-fracture electron microscopy of the same lens region as in Fig. 11. Junctional plaques on broad sides fragment to numerous small clusters of intramembrane particles on broad sides. Inset shows intact junctional plaques in lens region immediately before dissociation. ×20000.

Fig. 12.

Freeze-fracture electron microscopy of the same lens region as in Fig. 11. Junctional plaques on broad sides fragment to numerous small clusters of intramembrane particles on broad sides. Inset shows intact junctional plaques in lens region immediately before dissociation. ×20000.

Junctional appearance as a function of fibre distance from the equator of the lens and as a function of position along the length of a fibre is summarized in the schematic drawing (Fig. 13). Formation, enlargement, redistribution and dissociation are thus assigned a chronological order and reveal the dynamic nature of lens fibre junctions.

Fig. 13.

Temporal and spatial chronology of formation, enlargement, distribution and dissociation of intercellular junctions on the broad sides of fibres. Fibre age increases towards the right of the scheme.

Fig. 13.

Temporal and spatial chronology of formation, enlargement, distribution and dissociation of intercellular junctions on the broad sides of fibres. Fibre age increases towards the right of the scheme.

The ocular lens has previously been used as a model to study the formation of junctional plaques. Small clusters of intramembrane particles have been visualized by freeze-fracture electron microscopy of outer cortex tissue and have been identified as newly forming junctional plaques (Benedetti et al. 1974), in agreement with our results from anti-MP70 immunofluorescence microscopy. While the freeze-fracture technique is suitable for identifying and mapping fibre junctions over a small volume of the lens, adequate sampling proves difficult when junctional appearance over wider regions of the lens is to be studied. Anti-MP70 immunofluorescence microscopy permits complete junction mapping in the lens (Kistler et al. 1985), as well as relative junctional positioning along the full length of dissected fibre bundles. It has in addition the great advantage that the staining is specific for the 16–17 nm intercellular junctions and hence is also superior over immunocytochemistry with anti-MIP26 antibodies. MIP26 is another candidate for a fibre junction protein (Sas et al. 1985). However, MIP26 has also been localized in non-junctional lens membranes (Paul & Goodenough, 1983; Fitzgerald et al. 1985) and anti-MIP26 antibodies are thus not suitable for junction mapping. Using anti-MP70 immunofluorescence microscopy, changes in junctional appearance and arrangement related to fibre location in the lens can be shown, and the chronology of junction formation and dissociation can easily be determined.

Our results show that size, shape, frequency and arrangement of fibre junctions change considerably as fibre maturation progresses. In addition to these changes from fibre to fibre in a radial direction, anti-MP70 immunofluorescence microscopy has also revealed dramatic changes in junction distribution along individual pole-to-pole fibres. This may be correlated cither to the large distance of individual junctions from the nucleus (nuclei of elongating fibres are preferentially located in the lens equatorial plane) and/or to the particular microenvironment at the poles.

Growth and redistribution of junctional plaques could occur in several ways: small junctions could assemble from de novo synthesized and translocated MP70 in the equatorial plane of the lens, disperse by diffusion towards the poles and fuse with each other to form larger junctions. Alternatively, MP70 synthesis and translocation could occur along the full fibre length and junctional plaques might be assembled at their final positions.

An important feature of the lens is that fully elongated, mature fibres discard their nuclei (Kuwabara, 1975), and protein synthesis becomes negligible deeper in the lens (Wannemacher & Spector, 1968). Junctional plaques dissociate by fragmentation in these enucleated fibres and subsequently MP70 is cleaved to MP38 (Kistler & Bullivant, 1987). Freeze-fracture electron microscopy shows that some small junctions, possibly composed of MP38, persist in the central lens region. This is consistent with the finding that nuclear fibres are electrically coupled (Rae, 1979).

The most striking result of junctional mapping, in our view, is the unique alignment of junctional plaques in the middle of the broad sides of the fibres. Similarly, single rows of small junctions have been observed on the narrow sides of the fibres by freeze-fracture electron microscopy (Lo & Harding, 1986). It is not known whether initially randomized junctions line up by diffusion or whether they are assembled in these positions. While the very large junctional plaques could be kept in place simply by the limited width of the broad sides, the smaller junctions must be positioned by a different mechanism. Hence, it appears that integral membrane proteins other than MP70, peripheral membrane proteins or cytoskeletal components participate in this unique positioning of fibre junctions.

We are grateful to the workers at the Auckland Municipal Abattoir for their cooperation in obtaining sheep lens. This work was supported by grants from the Auckland Medical Research Foundation (to W. T. M. Gruijters) and from the New Zealand Medical Research Council (to J. Kistler and S. Bullivant).

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Figs 49. Lens inner bow region (depth 1 mm). Anti-MP70 mapping along a single fibre bundle reveals a positiondependent distribution of junctions on the broad sides, and small junctions generally lined up in the middle of the narrow sides. × 1400.