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From the Cataract Research Center, Washington University School of Medicine, St. Louis, Missouri.
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Abstract |
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METHODS. Fixed or living lenses were sliced with a tissue slicer, permeabilized or extracted with detergents, stained with antibodies or fluorescent-labeled phalloidin, and viewed with a confocal microscope. The distribution of N-cadherin in elongating and mature fiber cells was determined by Western blot analysis. Reverse transcription–polymerase chain reaction (RT-PCR) was used to determine the distribution of vinculin and paxillin transcripts.
RESULTS. Staining for N-cadherin and band 4.1 protein decreased soon after fiber cells detached from the capsule. Detergent extraction of lens sections and Western blots of dissected lens regions showed that much of this decrease in staining was due to epitope masking. Vinculin immunoreactivity was barely detectable on the lateral membranes of elongating fiber cells but increased markedly once these cells reached their maximum length and formed the sutures. Staining for paxillin was also low in elongating fiber cells but increased late in fiber cell differentiation, just before the cells destroyed their membrane-bound organelles. Spectrin and ankyrin immunoreactivity did not change when fiber cells reached the sutures. Staining for F-actin increased transiently in cells that had just reached the sutures. Messenger RNAs for vinculin and paxillin were more abundant in maturing than in elongating fiber cells.
CONCLUSIONS. The adhesion complexes of lens fiber cells change in organization and composition soon after these cells finish elongating and detach from the capsule. Increased staining for vinculin and paxillin defines distinct stages of fiber cell differentiation that are intermediate between the completion of cell elongation and the time when lens fiber cells degrade their membrane-bound organelles. Remodeling adhesion complexes during fiber cell maturation may assure the stability of fiber–fiber associations, once these cells are no longer transcriptionally active.
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Introduction |
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Cell–cell adhesions may stabilize the packing arrangement of lens fibers. They may also be important for holding cells together during accommodation, when substantial bending and shearing forces must be generated between the cells. Several investigators have suggested that the cytoskeleton plays an active or passive role in accommodation.8 9 10 To function in this manner, cytoskeletal components must be connected, directly or indirectly, to the junctions that hold fiber cells together.
Previous studies have identified a number of proteins in lens fibers that function as cell-cell adhesion molecules or that serve as links between cytoskeletal elements and the components of cell junctions. One of these is N-cadherin, a transmembrane protein that forms calcium-dependent homophilic associations between neighboring cells.11 12 13 Also present in lens fibers are proteins, such as vinculin14 and plakoglobin,15 that are likely to connect adhesive junctions to the cytoskeleton. These molecules are most abundant along the shorter sides of elongating fiber cells.14 15 Several proteins that were first discovered in the submembrane cytoskeleton of red blood cells have also been identified in the lens. These include spectrin16 17 18 (also called fodrin), ankyrin,19 band 4.1 protein,16 band 4.9 protein,20 and band 3 protein.19 Based on their functions in erythrocytes, these molecules may be important in stabilizing lens fiber cell plasma membranes or in maintaining cell shape. Finally, proteins involved in modulating the assembly, function, and stability of the actin cytoskeleton, including tropomodulin, tropomyosin, caldesmon, and myosin, have recently been described in the lens.18 21 22
Actin filaments are known to interact, directly or indirectly, with most of the proteins we have mentioned. Direct association of lens actin with spectrin has been demonstrated.17 Proteins that link the plasma membrane–adhesion complex with other cytoskeletal elements, such as microtubules and intermediate filaments, have not been identified in the lens, although such links may be important in lens fiber cell formation or stability.
The present study was designed to determine whether proteins that contribute to structure and function of the cytoskeleton–cell adhesion complex change during fiber cell elongation and maturation. We used immunofluorescence to localize several of these proteins in chicken embryo fiber cells. Immunostaining for some proteins changed abruptly during fiber cell maturation. These changes defined two distinct phases of fiber cell differentiation that occurred after fiber cell elongation and before the elimination of intracellular membrane-bound organelles.
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Methods |
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In some cases, unfixed lens slices (400 or 500 µm) underwent detergent extraction for 3 hours at 4°C in a buffer modified from that described by FitzGerald23 (0.5% Nonidet P [NP]-40, 100 mM KCl, 5 mM MgCl2, 1 mM disodium EDTA, protease inhibitor cocktail [Roche Molecular Biochemicals, Indianapolis, IN] and 2 mM 2-mercaptoethanol). After extraction, slices were stained as described.
Primary antibodies and the dilutions used were mouse monoclonal
anti-N-cadherin (anti-A-CAM, clone GC-4; Sigma), 1:200; rat monoclonal
anti-N-cadherin (hybridoma supernatant NC-2), 1:200; mouse
monoclonal anti-vinculin (clone hVIN-1; Sigma) 1:200; rabbit
anti-ankyrin (chicken erythrocyte; Calbiochem–Novabiochem, La Jolla,
CA), 1:200; mouse monoclonal anti-paxillin (clone Z035; Zymed, South
San Francisco, CA), 1:200; rabbit anti-
-actinin (Sigma), 1:100;
rabbit anti-spectrin (chicken erythrocyte; Sigma), 1:200; mouse
monoclonal anti-protein 4.1 (chicken erythrocyte; East-Acres
Biologicals, Southbridge, MA), 1:200; and mouse monoclonal anti-talin
(Sigma), 1:200. Secondary antibodies were lissamine rhodamine-labeled
goat anti-mouse or anti-rabbit IgG or fluorescein-labeled goat anti-rat
IgG (Jackson ImmunoResearch, West Grove, PA), 1:200. Rhodamine
phalloidin (1 µg/ml; Sigma) was used to stain actin filaments. In
most cases the fluorescent nucleic acid stain (TOTO-1 iodide, 1:10,000;
Molecular Probes, Eugene OR) was added along with the secondary
antibody to stain the nuclei of the lens fiber cells.
Western blot analysis was performed by standard methods. Cortical (elongating) and nuclear (elongated) fiber cells were dissected from thick lens slices (500 µm) that had been extracted with detergent, as described. Protein concentrations in extracts were determined by a protein assay kit (DC; Bio-Rad, Hercules, CA), using bovine serum albumin as a standard. Extracts were separated on 7.5% polyacrylamide gels. Transfer of similar amounts of protein was confirmed by staining the blots with ponceau S. N-cadherin was detected with the same monoclonal antibody used for immunostaining (1:250), peroxidase-labeled anti-mouse secondary antibody (1:5000) and a luminol chemiluminescence detection kit (Santa Cruz Biotechnology, Santa Cruz, CA).
To determine the relative expression of vinculin and paxillin mRNAs in elongating and maturing fiber cells, freshly dissected E15 lenses were sliced as described earlier, and elongating lens fibers were dissected from fully elongated lens fibers. Total RNA was extracted from these tissues, and reverse transcription–polymerase chain reaction (RT-PCR) was performed on equal amounts of total RNA using standard methods. PCR primers were designed to amplify chicken vinculin and paxillin cDNAs. Two 5' primers and one 3' primer were used to generate two vinculin PCR products in separate reactions (forward primer 1, 5'-AAC TGC TAA TAA AAC TAC TGT G-3'; forward primer 2, 5'-CTG TGC AGA CAA CAG AAG ACC AG; reverse primer, 5' CTG AGG CTG AAA GGC TTC TC). The paxillin primer sequences were: forward 5'-TGC TTT CTC AAA CTC TTC TG-3' and reverse, 5'-CAC ACA GGC AGA ACC CTA CA-3'. Primers for chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as internal controls for the amount of cDNA present in each reaction (forward 5'-TCA ATG GGC ACG CCA TCA CT-3' and reverse 5'-AAG AGC AGG GGC TCC AAC AA-3'). Vinculin and paxillin PCR reactions were run for 29 cycles and GAPDH reactions for 15 cycles (94°C 15 seconds, 56°C 20 seconds, 72°C 2 minutes). PCR products were run on agarose gels and stained with ethidium bromide to reveal the relative abundance of the transcripts.
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Results |
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A monoclonal antibody to vinculin, a protein found in cell-cell and cell–substrate junctions, revealed a pattern of staining in the fiber cells that was, in some ways, reciprocal to that seen with antibodies to N-cadherin (Fig. 2C) . Staining for vinculin was present on the basal and lateral membranes of lens epithelial cells and in the apical adherens complexes of epithelial and fiber cells (Figs 2C 3A) . Staining for vinculin was minimal along the lateral membranes of elongating fiber cells, but was prominent at the basal surface of these cells where they attached to the capsule (Fig. 3A ). Staining for vinculin at the basal surface of elongating fiber cells decreased as these cells approached the sutures (Fig. 3B) . Staining for vinculin increased greatly along the lateral membranes of E17 fiber cells soon after they detached from the capsule (Figs. 2C 3B) . This was the same region of the lens in which staining for N-cadherin decreased. Staining for vinculin was most prominent in the region of the sutures (Fig. 3B) and persisted after the fiber cells degraded their organelles. Similar to N-cadherin and band 4.1 protein, staining for vinculin was strongest along the short sides of the fibers in which this protein was expressed (Fig. 2C , inset).
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In contrast to the staining patterns described, some proteins appeared to be more uniformly distributed in fiber cells at different stages of maturation. For example, staining for both spectrin and ankyrin was strong in epithelial cells and, after the initial phase of elongation, appeared to be of equal intensity in elongating and maturing fiber cells (Figs. 2E 2F) . In contrast to the other membrane-associated proteins examined, staining for spectrin and ankyrin was uniformly distributed around all lateral fiber cell membranes (insets in Figs. 2E 2F ).
All the molecules examined in this study interact directly or indirectly with the actin cytoskeleton. We, therefore, stained lenses with rhodamine-labeled phalloidin to determine whether the distribution of F-actin changed after fiber cells reached the sutures. Figure 4A shows that strong staining for F-actin was present at the apical and basal ends of the epithelial and elongating fiber cells. Lower levels of staining were seen along the lateral membranes of all fiber cells. A region of increased staining was consistently seen in fiber cells just after they reached the sutures (Figs. 4A 4B) .
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To distinguish between these possibilities, we immunostained lenses from embryos at E7 and E8. If the first alternative were correct, the fiber cells of these lenses should stain for only vinculin and paxillin. If the second alternative were correct, the youngest lenses should express only N-cadherin and band 4.1 protein in their fibers. Vinculin and paxillin should appear later in the center of the lens in a small region that increases in size as the lens grows.
Figures 5A and 5B show sections through lenses at E7 and E8, respectively, stained with antibodies to N-cadherin. At E7 all fibers stained, whereas at E8 fibers in the center of the lens did not react with the antibody. Similar staining was seen with antibody to band 4.1 protein (data not shown). At E7, light staining for vinculin was present in the central fiber cells that had detached from the capsule (Fig. 5C) . By E8, a large area of central fibers showed strong immunostaining for vinculin (Fig. 5D) . Staining for paxillin was uniformly very low in the lens fiber membranes at these ages (data not shown). Because the fibers formed early in lens development remain in the center of the lens, the central fibers that did not stain strongly for vinculin at E7 were the same fibers that showed marked staining at E8. Because primary fiber cells first detach from the posterior capsule at E6,24 increased vinculin expression was seen soon after fiber cells detached from the capsule in E7, E8, and E17 lenses. These data demonstrate that, as lens fiber cells mature, their cell-cell adhesion complexes become different from those found in elongating fibers.
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To test this possibility, lens slices were subjected to detergent extraction before fixation to see whether this treatment would alter the staining patterns in the lens. Lens slices underwent exraction at 4°C with a buffer containing 0.5% NP-40 that was formulated to preserve the lens cytoskeleton and prevent proteolysis.23 In fixed unextracted lens slices, staining for N-cadherin decreased when fiber cells reached the sutures (Figs. 2A 6A ). After detergent extraction for 3 hours, most of the crystallins were removed from the lens slices, indicating that most cells had been fully permeabilized (Fig. 6B) . In immunostained, detergent-extracted lenses N-cadherin immunoreactivity again decreased in fibers after they reached the sutures (Fig. 6C) . However, staining for N-cadherin was more evident in the deeper lens fibers of detergent extracted lenses than in lenses that had not undergone detergent extraction (compare Figs. 6A and 6 ). Similar results were obtained for lenses stained with antibodies against band 4.1 protein (data not shown). These observations suggested that the epitopes recognized by antibodies against band 4.1 and N-cadherin were masked in fully elongated lens fibers, compared with elongating fibers. In contrast, the pattern of antibody staining for vinculin (Fig. 6D) and paxillin (not shown) was similar in extracted and unextracted lens slices.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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However, previous studies and the results presented in this article show that discrete changes occur during fiber cell differentiation. In earlier studies we found that nuclei, mitochondria,2 3 and the endoplasmic reticulum4 are abruptly degraded late in the process of fiber formation, well after the fiber cells complete elongation. In addition, in chicken embryo lenses, the membranes of adjacent fiber cells fuse just before they degrade their organelles.5 The present data show that, at earlier stages, soon after the fiber cells finished elongating, cell adhesion complexes were extensively remodeled. This restructuring involved a reduction of N-cadherin and band 4.1 immunoreactivity and a simultaneous increase in staining for vinculin. Later, at approximately the time when fiber cells fused and just before their organelles were degraded, paxillin immunoreactivity increased markedly.
We propose that fiber cells pass through specific programmed stages during their differentiation. Although these stages may not be marked by gross morphologic alterations, they are accompanied by significant changes in the composition of the fiber cell membrane proteins and can be discerned by the presence or absence of characteristic biochemical markers.
Based on previous observations and those reported in this article, the life of a fiber cell can be divided into at least four distinct stages. Fiber cells first elongate from columnar progenitor cells at the lens equator. During the period of elongation, their lateral membranes appear relatively smooth, there are distinct adherens junctions near their apical and basal ends and along their lateral membranes, and they contact the capsule and the epithelium at their basal and apical surfaces, respectively. When elongation is complete, fiber cells lose contact with the capsule and the epithelium. Distinct basal and apical adherens complexes can no longer be visualized by antibody staining. At the same time fiber cells appear to restructure their lateral membrane complexes. Staining for N-cadherin and band 4.1 decreases and staining for vinculin increases. These changes are accompanied by an increase in the folding of the lateral membranes of the fiber cells.4 These membrane surfaces eventually become extensively interdigitated, presumably more firmly locking the fibers to their neighbors.6 7 An increase in paxillin immunoreactivity just before fibers degrade their membrane-bound organelles marks a third phase of fiber cell maturation. At approximately the same stage of maturation, the fiber cell lateral membranes fuse, creating large pores between adjacent cells.5 This event links the cytoplasm of cells capable of synthesizing mRNA and protein to the cytoplasm of cells that have lost these synthetic capabilities. The final stage in the life of a fiber cell begins with the loss of its intracellular, membrane-bound organelles.
The value of this proposed staging system is that reference to easily identifiable landmarks that are common to all lenses (such as the presence or absence of organelles and whether fibers have reached the sutures) should allow for direct comparisons between the events that characterize fiber cell maturation in the lenses of different species. In embryonic chicken lenses a fiber cell passes through these stages in a matter of days. For example, a cell that enters the elongation stage on E12 matures and loses its organelles by E17. It will be interesting to determine whether the relative timing of the different stages remains constant throughout embryonic and postnatal life.
The Function of N-Cadherin, Band 4.1 Protein, Vinculin, and
Paxillin in Fiber Cell Differentiation
Although staining for N-cadherin and band 4.1 protein followed a
similar pattern during fiber cell differentiation, these proteins are
known to serve distinct functions in other cell types. N-cadherin is a
transmembrane protein that links a cell to its neighbors by associating
with the extracellular domains of N-cadherin molecules on adjacent
cells. The cytoplasmic domains of cadherins are anchored to the
cytoskeleton by a complex of proteins that includes -catenin,
ß-catenin, p120ctn, and
vinculin.25
Based on its distribution, the major function
of N-cadherin in lens fiber cells is likely to be holding cells
together along their lateral membranes and stabilizing apical and basal
cell-cell adherens junctions. The presence of N-cadherin–containing
junctions along the length of the lateral cell membranes of lens fiber
cells may also be responsible for the small extracellular space between
these cells, a factor that is believed to be important for lens
transparency.
Band 4.1 protein links one or more transmembrane proteins to the actin cytoskeleton in nearly all cell types.26 In erythrocytes, where the function of band 4.1 has been studied most thoroughly, band 4.1 connects the transmembrane proteins, band 3 and glycophorin C, to the actin-spectrin complex. Interaction of band 4.1 with calmodulin can alter the association of the actin cytoskeleton with the membrane, thereby affecting cell shape and the elasticity of the cell surface. Therefore, band 4.1 may help to stabilize the submembrane cytoskeleton of lens fiber cells. Band 3 has been identified in lens fiber cells and is likely to be one of the binding partners for band 4.1.19 It is not known whether band 4.1 interacts with other lens transmembrane proteins. Interestingly, the antibody used in the present study did not detect band 4.1 in lens epithelial cells, suggesting that band 4.1 is not expressed in epithelial cells or that a different isoform of this protein is expressed as epithelial cells differentiate into fiber cells.
The distribution of vinculin and paxillin described in this article suggests that these proteins may be important in several aspects of fiber cell function. Vinculin is a well-known component of cadherin-containing cell-cell and integrin-mediated cell–substrate complexes, where it plays an important role in regulating the interaction of these complexes with the actin cytoskeleton.27 Vinculin localized to the apical adherens junctions of lens epithelial and fiber cells, the basal ends of fiber cells at the posterior capsule, and in abundance along the lateral membranes of fiber cells after these cells reached the sutures. The localization of vinculin at the basal ends of elongating fiber cells observed in this study is likely to be involved in anchoring these cells to the posterior capsule by integrin-mediated adhesion.28 However, the strong staining for vinculin along the lateral membranes of fiber cells that had completed elongation was unexpected. Talin, another component of integrin-mediated cell–substratum complexes, was found at the basal ends, but not along the lateral surfaces of fiber cells (data not shown). This observation suggests that vinculin participates in at least three kinds of adhesion complexes in the lens: apical cell-cell adherens junctions, basal cell–substrate adhesions, and extended cell-cell adhesions along the lateral surfaces of mature fiber cells. It is possible that vinculin-containing adhesion complexes along the lateral surfaces of mature lens fiber cells are important in maintaining lens structure during accommodation and aging. Consistent with this view, staining for F-actin increased when lens fiber cells reached the sutures. Increased staining for vinculin and F-actin may also be related to the increase in membrane interdigitations seen in mature fiber cells.4 6 7
Paxillin is a multifunctional component of cell–substrate adhesion complexes that binds to several proteins, including vinculin.29 The observation that staining for paxillin increased along the lateral membranes of fiber cells at approximately the time they were fusing with their neighbors and just before they lost their organelles is intriguing. Additional studies are required to indicate whether paxillin plays a role in either of these processes. The distinct expression patterns of vinculin and paxillin suggests that these proteins play different roles in mature lens fiber cells.
Changes in N-Cadherin and Band 4.1 Immunoreactivity after Detergent
Extraction
Detergent extraction largely reversed the decrease in
immunostaining for N-cadherin and band 4.1 seen in deeper fiber cells.
The antibodies to N-cadherin used in this study were specific for
extracellular epitopes. For this reason we originally thought that the
decrease in immunostaining might have been due to changes in the
extracellular space in fully elongated fiber cells. However, band 4.1
protein is an intracellular protein. Therefore, the decrease in
antibody staining for these proteins reflected a more general
alteration in their accessibility to antibodies. It was also striking
that staining for ankyrin and spectrin did not decrease in a similar
manner, which suggests that the masking of N-cadherin and band 4.1 was
relatively specific. Additional studies are needed to reveal the
biochemical or structural basis for the decrease in staining for
N-cadherin and band 4.1 during fiber cell maturation.
The modest decline in N-cadherin detected by Western blot analysis is consistent with that seen in detergent-extracted lens slices. A decrease in N-cadherin levels was described previously in the mature fibers of adult human lenses.30
Regulation of Vinculin and Paxillin Expression during Fiber Cell
Maturation
The increase in vinculin and paxillin immunostaining during fiber
cell maturation is likely to be related to the increases that we
detected in the transcripts for these proteins. The relative increase
in these mRNAs may result from increased transcription. However, we
cannot exclude the possibility that the rate of transcription of these
mRNAs is unaltered but that they accumulate because they are degraded
more slowly in fully elongated fiber cells than in elongating fiber
cells. Additional studies are required to distinguish between these
alternatives. In support of the first possibility, we recently
identified two mRNAs that are first transcribed soon after fiber cells
detach from the capsule (Vasiliev and Beebe, manuscript in
preparation). This finding demonstrates that transcriptional activation
can occur after fiber cells have fully elongated.
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Acknowledgements |
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Footnotes |
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Submitted for publication June 13, 2000; revised November 1, 2000; accepted November 30, 2000.
Commercial relationships policy: N.
Corresponding author: David C. Beebe, Cataract Research Center, Department Ophthalmology and Visual Sciences, Washington University School of Medicine, Campus Box 8096, 660 South Euclid Avenue, St. Louis, MO 63110. beebe{at}vision.wustl.edu
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