Ординатура / Офтальмология / Английские материалы / Development of the Ocular Lens_Lovicu, Lee Robinson_2004

Figure 7.1. (See color plate XI.) Detection of filensin and CP49 within the embryonic lens vesicle. Panels (a–d) show sections of the primary lens vesicle from five-week-old human embryos stained with keratin antibody (a), vimentin antibody 3052 (b), CP49 antibody 2981 (c), and filensin rabbit polyclonal antibody fpa-R (d) using horseradish peroxidase and diaminobenzidine as the colour developer. The arrow in (a) indicates the trace keratin reactivity within the anterior lens cells. Both CP49 reactivity (c) and filensin reactivity (d) are confined to the lens vesicle and show no obvious cross-reactivity with the surrounding ectoderm. Filensin and CP49 reactivity is detected in the lens epithelium at this stage of development. At week 7 of development, there is intense CP49 reactivity, which is particularly strong at the posterior pole of the lens vesicle. There is also significant CP49 staining within the anterior lens epithelial cells. Filensin shows a similar distribution (see Fig. 2 in Ireland et al., 2000). Scale bars: 50 µm. We thank Michael Kasper (University of Dresden) for these images.

was also found to drive CP49 expression in this system, but it was not inhibited by UO126. These data therefore indicate that there are multiple signal transduction pathways that can drive the expression of CP49. Indeed, it is possible to uncouple cell differentiation from the expression of CP49 using cyclic AMP analogues (Ireland et al., 1997).

It is worth mentioning at this point that there are species differences in the expression of intermediate filament proteins in the lens. For instance, in birds and in particular the chicken, the CP49 gene gives rise to two transcripts that are expressed in the lens, CP49 and CP49ins (Wallace et al., 1998). No equivalent to the CP49ins has yet been identified in mammalian lenses. Also in the chicken, the protein synemin was identified as a lens cytoskeletal component (Granger and Lazarides, 1984) that can coassemble with vimentin (Bilak et al., 1998). Equally, nestin, another intermediate filament protein that can coassemble with vimentin (Steinert et al., 1999), has been identified in the mammalian lens, and its expression pattern has been documented during lens development (Yang et al., 2000). Other intermediate filament proteins, such as desmin, are expressed in rodent lens epithelial cells in response to TGFß during anterior subcapsular formation (McAvoy et al., 1998; Lovicu et al., 2002), whilst in some strains of mice, GFAP is constitutively expressed in lens epithelial cells (Boyer et al., 1990; Hatfield et al., 1985), but these unexpected expression patterns have to be confirmed for other species. In conclusion, the expression pattern for intermediate filament proteins changes with respect to stage of development and differentiation and according to the species being studied.

The potential importance of such species differences is very relevant to CP49 and filensin, as it has been recently reported in the mouse that filensin and CP49 are expressed first in the more differentiated fibre cells, concentrated at the anterior ends of the fibres (Blankenship

Figure 7.3. Vimentin reactivity is rapidly reduced during lens fiber cell maturation in the developed human lens. Panels a and b are single-labelled confocal microsope images of a section from a two-year-old human lens stained with vimentin monoclonal antibody V9. Panel a shows an area of the anterior lens epithelium (arrow), which is vimentin positive. Panel b shows a region of the posterior lens, demonstrating the abrupt reduction in vimentin reactivity in the fiber cells. The arrow indicates the outer edge of the section. Lens fiber cell age increases from right to left.

not change its distribution during lens fibre differentiation. Some 200–300 µm from the lens capsule in the juvenile bovine lens, the CP49-filensin network undergoes a dramatic redistribution as part of the differentiation programme (Sandilands et al., 1995a), and such a redistribution has been confirmed in the human lens (Fig. 7.3). The mechanism for this dramatic change in the subcellular distribution of CP49-filensin could be linked to the complex proteolytic processing that filensin undergoes during lens fibre cell differentiation (Sandilands et al., 1995b). Alternatively, phosphorylation could be involved, as seen with other intermediate filament proteins (Inagaki et al., 1996), for phosphorylation is at least partly responsible for the membrane association and insolubilisation of CP49 (Ireland et al., 1993). The redistribution of CP49-filensin coincides with the loss of nuclei and mitochondria in the differentiating lens fibre cells (Bassnett, 1997; Bassnett and Beebe, 1992; Kuwabara and Imaizumi, 1974; Sandilands et al., 1995a). There has not been a detailed enough characterisation of the beaded filament protein phosphorylation to link specific phosphorylation events with changes in filament distribution. Nevertheless, the discussion above details ample reasons to suggest that the filensin-CP49 network contributes fundamentally to lens fibre cell differentiation.

In the youngest fibre cells of adult or juvenile human lenses, filensin and CP49 appear in a predominantly membrane-associated distribution. It has been proposed that filensin and

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CP49 compose a specialised fibre cell plasma membrane-associated cytoskeleton within the lens (Georgatos et al., 1994). Whilst this is probably true of the most recently differentiating fibre cells, the increased cytoplasmic staining observed in older fibres suggests that filensin and CP49 also compose a substantial cytoplasmic filament network (Sandilands et al., 1995b). Transgenic studies have revealed that the overexpression (Capetanaki et al., 1989) or the inappropriate expression of intermediate filaments within the lens (Dunia et al., 1990) leads to fibre cell plasma membrane disruption and the generation of cataract. In addition, such lenses show the ineffective removal of fibre cell organelles as well as the nucleus. The expression of mutated forms of CP49 also lead to cataract in the human (Conley et al., 2000; Jakobs et al., 2000). Conversely, the loss of vimentin does not induce cataracts in knockout mice (Colucci-Guyon et al., 1994), suggesting that an increased level or inappropriate expression of intermediate filament protein or the expression of mutated intermediate filament proteins is more damaging to the lens. Based on this, it is difficult to single out one specific function for intermediate filaments in the lens other than the generic structural role proposed for other tissues (Lazarides, 1980).

7.2.1.3. Intermediate Filament–Associated Proteins (IFAPs)

The points of attachment and the interactions available to the lenticular cytoskeleton will be key to any structural role. It is often the cytoskeletal-associated proteins that provide the physical links, and there is a growing interest in those proteins that link more than one element of the cytoskeleton, as these proteins are thought to be important in coordinating the cytoskeleton as a whole.

One protein that has been shown to associate with intermediate filaments in the lens is plectin (Weitzer and Wiche, 1987). The patterns for intermediate filament proteins and plectin overlap in lens fibre cells (Sandilands et al., 1995a), as does the pattern of a protein called intermediate filament–associated protein 300 (IFAP300; Lieska et al., 1991; Yang et al., 1985), which has recently been identified as plectin (Clubb et al., 2000). Interestingly, plectin introduces cross-links between intermediate filaments and microtubules (Svitkina et al., 1996) and also to actin (Andra et al., 1998). Plectin’s role as a linker protein became clear when patients with both muscular dystrophy and Epidermolysis Bullosa Simplex were discovered to have plectin mutations (Smith et al., 1996), demonstrating how important these types of linker protein are to cytoskeletal function (Andra et al., 1997).

Plectin is also important for linking intermediate filament proteins to integrin complexes in hemidesmosomes. There are no hemidesmosomes in the lens, but the integrin complex α6β4, which is found in hemidesmosomes, is expressed in the lens, at least in the chick, and is localised to the plasma membranes of the fibre cells (Walker and Menko, 1999; see also chap. 10). It is important to note that the expression of this α6β4 integrin complex is not restricted to those basal membranes in contact with the posterior membrane (i.e., where there is no laminin), so the integrin complex here is certainly performing a different role in the lens than in other cell types (Walker and Menko, 1999). The location of this integrin complex would still permit signalling as well as anchorage of the cytoplasmic intermediate filaments via plectin. However, no cataract phenotype has so far been reported for patients carrying the mutant plectin or in plectin knockout mice (Andra et al., 1997), as might be expected for such an abundant plasma membrane component that has an important role in linking intermediate filaments, actin, and microtubule cytoskeletons. Possible explanations include the existence of tissue-specific splice variants (Fuchs et al., 1999) or perhaps compensation by other lens-specific proteins. This point has yet to be fully addressed, but plectin and the

α6β4 integrin complex are strong candidates for the role of facilitating intermediate filament attachment to the plasma membrane.

The associated proteins are a major factor in determining how, for instance, intermediate filaments attach to other cytoskeletal elements, the plasma membrane, or other cellular components. Candidates include ankyrin (Georgatos and Marchesi, 1985) and spectrin (Macioce et al., 1999), both of which are present in the lens. Ankyrin and spectrin are better known as key components of the actin-based red blood cell cytoskeletal–plasma membrane complex. The other components of this complex, such as adducin (Kaiser et al., 1989), band 3 (Allen et al., 1987), band 4.1 (Granger and Lazarides, 1985), band 4.9 (Faquin et al., 1988), caldesmon (Bassnett et al., 1999), and tropomyosin and tropomodulin (Woo and Fowler, 1994), are also present in the lens. Indeed, electron microscopy studies show how the actin and intermediate filaments lie in close proximity to the plasma membranes of the lens fibre cells, concentrated along the shorter faces of these hexagonal cells (Lo et al., 1997). Proteins that enable cross-talk between these different cytoskeletal elements promote their integration, which is as functionally important to fibre cells as to other cell types.

The arrangement of intermediate filaments just below the plasma membrane resembles that of a filament mat (Franke et al., 1987). The protein plakoglobin, another IFAP, is heavily enriched in this region of the cell, and in fact the lens was used as a useful starting point for the purification of this protein from a non-keratinising tissue (Franke et al., 1987). Plakoglobin interacts with N-cadherin (A-CAM; Angst et al., 2001), the major cadherin in the lens (Volk and Geiger, 1986a) and a key component of the actin-containing adherens junctions at the lens plasma membrane, which is required for lens cell differentiation (FerreiraCornwell et al., 2000). In keratinising epithelia, plakoglobin links intermediate filaments to the membrane desmoglein-desmocollin complex via desmoplakin in the desmosome, but these have not been found so far in the lens (e.g., Koch et al., 1992; Ramaekers et al., 1980). Plakophilin 1 and 2 have been found in cultured lens cells (Heid et al., 1994; Mertens et al., 1999) though in the apparent absence of desmosomes, this suggests a novel type of adherens-type junction in the lens, as has recently been identified in the retina (Paffenholz et al., 1999). The attachment of intermediate filaments to the plasma membrane is therefore still an open question, and, as detailed above, there are many alternatives.

7.3.Microtubule Networks in the Lens

7.3.1.General Properties of Microtubules

Microtubules are essential cytoskeletal elements required for chromosome movement during meiosis and mitosis, for cytokinesis, for intracellular transport, and for the positioning of intracellular organelles. Microtubules are dynamic structures comprising α- and β-tubulin heterodimers that add only to the ends of the microtubules. The rate of this addition is different at the two ends of the microtubules, as these polymers have polarity, unlike intermediate filaments, for instance. Microtubule polarity and subunit dynamics are central to the functional properties of microtubules, and GTP hydrolysis establishes the dynamic properties of this filament system.

Microtubule organisation in cells is dictated by the location of microtubule-organising centres (MTOCs), such as the centrosome, which binds one specific end, termed the (−) end, whilst the distal end is designated the (+) end. Microtubule arrays, therefore, also have polarity, which means that the movement of organelles, vesicles, and other cargoes in cells

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can be vectorially organised. Specific motor proteins, such as kinesin, move to the (+) end, whilst other motor proteins, such as dynein, move to the (−) end.

Cells generally contain at least two microtubule populations, one a very dynamic population and the other a more stable population (Schulze and Kirschner, 1987). At present, the functional significance of the two populations is unclear, although the biochemical reasons for the increased stability are beginning to emerge (Infante et al., 2000). Nevertheless, the stable population can be distinguished from the dynamic using antibodies against post-translational modifications of tubulin which only occur when tubulin is assembled into microtubules. For instance, both acetylation and detyrosination are modifications that occur on the stable population of microtubules, and these markers can be used to define this subset in cells and tissues (Infante et al., 2000; Schulze and Kirschner, 1987).

In tissues, the organisation of microtubules can be quite different from that described for cells in culture (Mogensen et al., 2000). In particular, the MTOCs in cells from some tissues are peripherally located, in contrast to the perinuclear centrosomes described in most tissue culture cells (Joshi, 1994), and consequently microtubule networks originate in quite distinct non-periniuclear regions of the cell. Cultured cells can be induced to polarise the MTOCs (microtubule organisation reflects this polarity), with the MTOCs localised at the apical plasma membrane (Meads and Schroer, 1995). This arrangement of microtubules in polarised epithelia seems to depend, at least in part, upon the intermediate filaments (Ameen et al., 2001). In elongated cells like neurones, the microtubules are released into the axon (Baas, 1998) and organised to permit axonal function by facilitating vectorial transport within the axon (Miki et al., 2001; Zhao et al., 2001). What then is the situation in lens fibre cells?

7.3.2. Microtubules and Their Role in the Lens

The high energy required for microtubule dynamics has important implications for the lens. The normal process of differentiation removes mitochondria, and the resulting fibre cells then rely upon glycolysis to supply ATP. Perhaps as a consequence of this, microtubules are infrequently observed in denucleated fibre cells and have not been seen in the lens nucleus. Their contrbutions to lens function is therefore most significant in the lens epithelium and early fibre cells.

In the lens, cell differentiation involves a dramatic change in cell shape as well as the repositioning and elimination of a number of cell organelles (see Bassnett, 1992, 1997), and microtubules are likely to be involved in these numerous changes. Microtubules are present in differentiating fibre cells, as documented by electron microscopy studies (Beebe and Cerrelli, 1989; Kuwabara, 1968; Pearce and Zwaan, 1970), and in cultured lens epithelial cells, as demonstrated by indirect immunofluorescence labelling (Lonchampt et al., 1976). The motor proteins, kinesin and dynein, are present in the lens (Lo and Wen, 1999), suggesting that vectorial transport of cargoes occurs within lens fibre cells. Some of these cargoes will be other cytoskeletal proteins. For instance, the soluble precursors of intermediate filaments are transported in a kinesin-dependent manner on microtubules (Prahlad et al., 1998).

Despite these potential functions for microtubules in the lens, their role in lens fibre cell differentiation is disputed. Using a microtubule depolymerising drug, colchicine, initial research indicated that microtubules were necessary for the dramatic cell elongation that occurs during lens fibre cell differentiation in the chick embryo (Piatigorsky, 1975). The interpretation of this research was subsequently questioned (Beebe et al., 1979), and to this day the essential role of microtubules is open to debate. In other cell systems, we know that

their role (see Neff et al., 1983) and the role of their associated motor proteins are vital to cell function (Miki et al., 2001; Zhao et al., 2001), as they are in the Drosophila eye, as shown by studies with glued, a cytoplasmic dynein (Fan and Ready, 1997). These data have altered the question from “Are microtubules important in the lens?” to “What do microtubules do in the lens?” The answer to this latter question is reflected in the organisation and distribution of microtubules and MTOCs.

7.3.3. MTOCs in the Lens

In lens epithelial cells, the centrosome is the major MTOC. The microtubules radiate from this structure, which is located close to the cell nucleus (Lonchampt et al., 1976; Prescott et al., 1991). In this respect, there is nothing unusual about the microtubule organisation in lens epithelial cells, as there is a stellate arrangement of the microtubules originating at the centrosome (Millar et al., 1997). This organisation, however, changes noticeably at the lens equator, when the more posterior epithelial cells and newly emerging fibre cells become aligned relative to one another. Coincidently, the microtubules also adopt a more regular organisation (Millar et al., 1997). The epithelial cell nuclei also undergo shape changes, and at this time there is significant changes in gene expression. It is tempting to speculate that microtubules help facilitate these rearrangements, but there is currently no evidence in support of this.

One surprising aspect of microtubule organisation in the lens is the apparent lack of a centrosome or another distinct microtubule-organising site during lens fibre cell differentiation. The epithelial cells in the post-germinative zone, closest to the first fibre cells, do not contain a γ -tubulin- or pericentrin-staining centrosome, but still have an intact microtubule array (Millar et al., 1997). This lack of a discrete MTOC structure is not unique. For example, the microtubule network in the supporting epithelial cells of the cochlea are not centrosome orientated (Mogensen et al., 2000), albeit the cells still contain centrosomes. The microtubules in nerve axons also lack γ -tubulin staining at their (−) ends (Baas, 1998), like those in lens fibre cells (Millar et al., 1997); however, a γ -tubulin–positive centrosome is still present in the cell body of these neurons. Other cell types lacking defined centrosomes include ciliated epithelial cells and retinal photoreceptor cells, but these have

γ-tubulin associated with other MTOCs, such as basal bodies (Joshi, 1994). The surprise in the lens was the apparent disappearance of the centrosome from the lens fibre cells and the more posterior epithelial cells, at least as seen by the failure to stain these cells with the centrosome-specific markers γ -tubulin and pericentrin (Millar et al., 1997). Our observations suggest that the more mature fibre cells contacting the epithelium do regain a

γ-tubulin- and pericentrin-positive MTOC located at their apical ends near the epithelium (unpublished results).

In these types of situation, a nucleation, release, and capture model of the centrosome has been proposed (Henderson et al., 1995), and evidence is accumulating in support of novel microtubule (−) end complexes involved in the stabilisation of microtubules (Mogensen et al., 2000). Perhaps the lens will be another example of such a system.

7.3.4. Microtubule Organisation in the Lens

In the lenses of mouse, rat, rabbit, and cow, the microtubule distribution in the lens epithelial cells (Fig. 7.4) is very similar to that previously reported for the frog lens (Prescott et al., 1991). Like the epithelial cells, the lens fibre cells also contain both stable microtubule

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Figure 7.4. (See color plate XII.) Microtubule organisation in the lens. Microtubules (blue) are organised from a perinuclear centrosome (red) in the epithelium, while fiber cells have an MTOC at their apical end. This nucleates microtubules that run the length of the fibres. Microtubules are associated with the MTOC in the fibre cells; unmodified microtubules are in green and posttranslationally modified microtubules are in red.

populations (containing acetylated, detyrosinated tubulin) and dynamic microtubule populations. The stable microtubules, at least in the rat lens fibres, lie closer to the centre of the cell and run continuously from one end of the cell to the other. Other species show some differences from this organisation. For instance, the stable microtubules of the rabbit lens fibres are more helically arranged than those of the rat lens fibres.

The function of these two different microtubule populations has not been clearly defined, but some differences are beginning to emerge. For instance, kinesin preferentially associates with detyrosinated tubulin (Liao and Gundersen, 1998), and this population of microtubules

is the more important, at least in fibroblasts, for intermediate filament redistributions (Gurland and Gundersen, 1995; Liao and Gundersen, 1998). Kinesin is also required for this process (Prahlad et al., 1998). The more dynamic population of microtubules is associated with the plasma membrane, whilst the stable population is more centrally localised in both epithelium and fibre cells. Earlier electron microscopy studies of embryonic chick lenses (Pearce and Zwaan, 1970) and human lenses (Kuwabara, 1968) showed that microtubules were predominantly orientated parallel to the long axis of the fibre cells. It is now clear that these represent the stable microtubules, whilst the dynamic microtubules are at an angle to the fibre cell long axis (Millar et al., 1997). These microtubules would not have been quite so obvious in the electron microscopy sections for a variety of reasons, not least the difficulty of rapidly fixing the tissue before microtubule disassembly occurred (Millar et al., 1997). As many of the microtubules are at the fibre cell plasma membrane, roles in membrane subcompartmentalisation (Gruijters et al., 1987), organelle positioning (Bassnett, 1995), and the redistribution of actin and intermediate filament networks (Sandilands et al., 1995a) are likely. Indeed, in the context of the other cytoskeletal elements, microtubules are important for the redistribution of intermediate filaments (Prahlad et al., 1998) and are required for the maintenance of adherens junctions (Waterman-Storer et al., 2000). Intermediate filaments also contribute to the establishment of cell polarity and microtubule organisation (Ameen et al., 2001), and the tumour-suppressor gene product APC (adenomatous polposis coli) provides a link between cell adhesion, microtubule stability, and cell differentiation (Aberle et al., 1996; Zumbrunn et al., 2001). There is thus extensive cross-talk between the microtubule, intermediate filament, and actin networks (Correia et al., 1999; Kaverina et al., 1998; Waterman-Storer et al., 2000), particularly in regard to common associated proteins (reviewed in Herrmann and Aebi, 2000) and the correlation between important events during lens development and differentiation and dramatic changes in the organisation of the cytoskeleton.

7.4. Actin in the Lens

Since the unequivocal identification of actin in cultured lens cells (Lonchampt et al., 1976) and the intact lens (Kibbelaar et al., 1980; Kibbelaar et al., 1979), the characterisation of the actin complex in the lens has progressed steadily. Although not yet tested experimentally, a role for actin in lens accommodation has been proposed (Rafferty and Goossens, 1978). However, there has been an accumulation of data correlating actin and its associated proteins with critical events during lens development and differentiation (see Beebe and Cerrelli, 1989; Ferreira-Cornwell et al., 2000; Lo et al., 2000).

During early development, the lens is formed from a vesicle that is pinched off from the surface head ectoderm (see chap. 1). It is hard to envisage how this process can be completed in the absence of a functional actin cytoskeleton given the fundamental role that the actin–adherens junction complex plays in morphogenic events during development. It is also important to remember that the lens continues to grow throughout life and that the differentiation process of the lens fibre cells requires movement of the fibre cells concomitantly across the posterior lens capsule and over the anterior lens epithelial cells. Of necessity, this means that the cell-cell junctions and cell-matrix junctions at the epithelial–fibre cell interface and at the fibre cell–capsule interface have to be dynamic, as do the associated cytoskeletal structures.

Defects in the actin machinery – defects in the structural arrangement and in the signalling pathways – are the cause of a wide spectrum of inherited human diseases, from haemolytic