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

exists as seven different isoforms in mammalian cells. In non-muscle cells, including lens cells (Schmitt et al., 1990), the complement of actin isoforms includes β- and γ -non- muscle actin. Only in some forms of cataract does this change, as with the expression of α-smooth muscle (αsm) actin (Schmitt et al., 1990) in subcapsular cataract. In fact, it was proposed that αsm-actin could be a marker for this cataract type (Hales et al., 1994). The exposure of isolated rat lenses to TGFβ induced the expression of αsm-actin (Hales et al., 1994; Lovicu et al., 2002). Lens epithelial cells in culture also express αsm-actin (Nagamoto et al., 2000), indicating the sensitivity of lens epithelial cells to altered growth environments and the importance of the cytoskeleton in the adaptive response of the lens and lens cells. Indeed, studies on Drosophila eye development demonstrate the importance of TGFβ family members for the eye, as ectopic expression of decapentaplegic (dpp) along the anterior margin of the eye disc induces new discs (Pignoni and Zipursky, 1997). Although the role of dpp in eye development is still being established (Chen et al., 1999), links have been proposed with the actin cytsoskeleton via band 4.1 proteins. This is because merlin (schwannomin; Claudio et al., 1997) is thought to mediate the effects of dpp on cell proliferation and differentiation in the eye (McCartney et al., 2000). These data collectively suggest that misexpression of TGFβ is at least an additional risk factor in cataractogenesis and that the regulation of actin is important during lens development and differentiation.

Support for this hypothesis came initially from drug studies. Cytochalasins have been used to explore actin function in the lens (Beebe and Cerrelli, 1989; Mousa and Trevithick, 1977), and these studies showed that lens fibre cell elongation could be prevented by these drugs. Interestingly, cytochalasin D prevented K+ efflux, and this was highlighted as a reason for the prevention of cell elongation (Beebe and Cerrelli, 1989). The actin cytoskeleton is vital to establishing the plasma membrane compartments (Garner and Kong, 1999; Yeaman et al., 1999), and so it is easy now to understand how compromising the actin cytoskeleton may alter ion transport.

7.4.2. Actin Membrane Complexes in the Polarised Cells of the Lens

The actin–plasma membrane complex acts as a major contributor to cell polarity in epithelial cells by directing the sorting of adhesion complexes, pumps, and ion channels. Cell polarity is fundamental to cell function, and thus there are similar expectations of the lenticular actin cytoskeleton. The first comparative accounts of the actin cytoskeleton in mammalian lenses described a three-dimensional network (Lonchampt et al., 1976; Perry et al., 1981; Rafferty and Goossens, 1978), later described as a polygonal lattice (Rafferty and Scholz, 1984), that was concentrated at either side of the epithelial–fibre cell interface. The surfaces of this interface represent the apical ends of the epithelial and fibre cells, respectively. The actin concentrated in these regions is linked via adhesion junctions, as has recently been elegantly demonstrated (Lo et al., 2000). This study on the embryonic chick lens established the association of actin networks with zonulae adherens junctions throughout the plain of the epithelial and fiber cell apical domains. Indeed, it was proposed that during development the zonulae adherens form a continuous belt linking the epithelial cells and fibre cells in the equatorial region, where the epithelial cells are differentiated into fiber cells. Fasciae adherens were also seen between the apical ends of the epithelial and fibre cells (Lo et al., 2000). The fasciae adherens are not as robust as the zonulae adherens, for the epithelial–fibre cell interface is easily ruptured when the lens mass is removed, at least in the mouse (Liou, 1990) and cow (unpublished observation). Interestingly, this is not the case for the basal ends of the lens fibre cell, which are linked to the posterior lens capsule

186 Roy Quinlan and Alan Prescott

by another actin-enriched complex termed the basal membrane complex (BMC; Bassnett et al., 1999). Removal of the capsule from chick lenses ruptures the lens fibre cells at this point, leaving behind the cell-ECM complex. This interaction is calcium dependent and allows very effective purification of the complex (Bassnett et al., 1999; see also chap. 9).

The junctions themselves comprise a familiar list of actin-binding proteins and adherent junction components. These include α-actinin (Lo et al., 1997); α-, β-, and γ -catenin (Ferreira-Cornwell et al., 2000); N-cadherin (Volk and Geiger, 1986b; Watanabe et al., 1992); B-cadherin (Murphy-Erdosh et al., 1994); L-CAM (Thiery et al., 1984); band 4.1; paxillin (Beebe et al., 2001); plakoglobin (Franke et al., 1987); spectrin; and vinculin. The cell-cell adherens junctions themselves are considered unusual by some and have been given the name “cortex adhaerens” in recognition of the fact that contact extends over virtually the whole surface (Schmidt et al., 1994). These junctions are indeed unusual in as much as the α6β4 integrin complex (Walker and Menko, 1999) and a β1 complex (Bassnett et al., 1999) with α6 (Menko and Philip, 1995) are localised to the same plasma membrane regions in the lens fibre cells. It is worth noting the potential of the different cadherins to localise to different subdomains in the plasma membrane (Leong et al., 2000). The cortex adhaerens is therefore not a homogenous mixture of adherent junction proteins. As has emerged recently, the expression of some of these named components changes dramatically during lens development and lens cell differentiation and also according to the subcellular location of the junctions (Bassnett et al., 1999; Beebe et al., 2001; Leong et al., 2000; Menko and Philip, 1995; Walker and Menko, 1999). This means that the adherens junctions probably perform very discrete roles depending on their local environment.

The most extensive studies on these changes in adherens junction components have been done in the chick (Bassnett et al., 1999; Beebe et al., 2001; Leong et al., 2000). As with the other elements of the cytoskeleton, there are dramatic changes in the expression of the adherens junction components. For instance, band 4.1 is not present in lens epithelial cells but is expressed in the newly differentiated fibre cells, where it appears concentrated along the four short faces of these hexagonal cells. This relative increase in expression begins to wane once the fibre cells have detached from the posterior capsule at a later stage in differentiation. This particular example illustrates the complexity of the changes involved, but it is difficult at this moment to relate such expression patterns to function. In another example, the actin-binding protein tropomodulin is concentrated at the apical and basal ends of the newly differentiated fibre cells (Lee et al., 2000) in an unusual structure that does not completely overlap the actin filaments in this region of the lens (Fischer et al., 2000). Tropomodulin, like tropomyosin, is not proteolysed during lens fibre cell differentiation, and this is thought to contribute to the stability of actin in the lens nucleus (Lee et al., 2000). Evidence from the bovine lens suggests that there are at least two distinct actin complexes associated with the plasma membrane, one containing tropomodulin and the other spectrin (Woo et al., 2000). The functional significance of this duality remains unknown, but the arrangement is interesting, as it is quite different from actin organisation in the erythrocyte plasma membrane. Such data do, however, define in more detail the finer points of lens fibre cell differentiation, which is important if we are to correlate structure with function.

From these studies, it should be clear that the adherens junctions and the associated actin network are dynamic, responding to changes in the local environment and to external signals. This point is made more poignant by the observed link between Pax6, N-cadherin expression, and eye defects. It has been shown that Pax6 levels determine the expression of N-cadherin in the lens placode and consequently the successful formation of a lens (van Raamsdonk and Tilghman, 2000). The Sey phenotype in the mouse appears to arise from

inadequate adhesive properties due to a loss of cadherin function in these cells (Collinson et al., 2000; Quinn et al., 1996). The targeted expression of Pax6(5A) to the fibre cells induces cataract and up-regulates the level of integrins (Duncan et al., 2000), components of the cell-cell junctions in the fibre cells (Walker and Menko, 1999). The loss of Prox1 expression by a knockout strategy also results in the inappropriate expression of E-cadherin, the loss of cell polarisation, and the failure of the lens cells to elongate properly during lens vesicle closure (Wigle et al., 1999). These data highlight the importance of the cell junctions and the associated cytoskeletal machinery to lens development and illustrate the link between transcription factors, the cytoskeleton, and the processes of development and differentiation.

7.5. Conclusion

The lens cytoskeleton stands at the apex of all the important pathways that regulate lens cell development and differentiation. The cytoskeleton and associated structures are the verbs in the language used to make and maintain lens cells. They organise compartments, assist and even direct the transport and sorting of other cellular compartments, and provide the structural support required for lens accommodation and transparency. The cytoskeleton is a complex arrangement of filaments that provide a physical scaffold for facilitating protein interactions required in key cellular events, such as signal transduction and protein repair and maintenance. Medical and functional genetics have provided the evidence that the cytoskeleton is essential to the lens, and it is now up to us to fill in the details.

Note added in proof: Details of targeted (Alizadeh et al., 2002; Sandilands et al., 2003) and a natural CP49 knockout (Alizadeh et al., 2004) as well as a filensin knockout (Alizadeh et al., 2003) have been published. Some of these data demonstrate the great importance of the CP49 intermediate filament network to the optical properties of the lens (Sandilands et al., 2003) and also show the interdependence of the vimentin and the CP49-filensin networks (Sandilands et al., 2004). Details of the unique membrane complex on lens plasma membranes have also recently been published (Straub et al., 2003).

Part Three

Lens Development and Growth

192 Anne E. Griep and Pumin Zhang

Figure 8.1. The cell cycle. The mammalian cell cycle is divided into four phases, G1, S, G2, and M, progression through which is catalyzed by cyclin-dependent kinases activated by different cyclins at different phases during the cycle. Cells can also enter a nondividing quiescent state (G0).

Cell division cycles are the fundamental means that an organism uses to grow. In most somatic cells, one cell cycle is divided into four phases: G1, S, G2, and M. Cells synthesize their genome in the S phase (DNA synthesis), then segregate the duplicated genome into two daughter cells in mitosis (the M phase). DNA synthesis and mitosis are separated by two gap phases, G1 and G2 (Fig. 8.1). In addition, cells can enter a G0, or quiescent, state. Proliferation of eukaryotic cells is regulated primarily at two points in the cell cycle, in G1 prior to entry into the S phase and in G2 prior to entry into mitosis. Whether to commit to a round of cell division or exit the cell cycle is decided at a point in G1 referred to as the “restriction point” in mammalian cells (Draetta, 1994; Dulic et al., 1992; Ohtsubo and Roberts, 1993; Pardee, 1989; Quelle et al., 1993) or “START” in yeast (Koch and Nasmyth, 1994). In fibroblasts, passage through the restriction point depends critically on the signals that are received through mitogen-activated pathways, but once this point is passed, cells are committed to the S phase and the remainder of the cycle in a mitogen-independent manner (Pardee, 1989). In somatic tissues, passage through the restriction point is thought to be the primary event controlling cell proliferation.

Progression through the cell cycle is regulated by the action of two factors acting in concert, the cyclin-dependent kinases (Cdks) and the cyclins (see Table 8.1 for a summary). The Cdks are serine-threonine protein kinases that are activated by association with their cognate cyclin (see Fig. 8.1 and Table 8.1). Once activated by the cyclin, the Cdk is responsible for phosphorylating specific targets. There are five Cdks (Cdk1, 2, 3, 4, and 6) and four classes of cyclins (cyclin A, B, D, and E) that have been shown to regulate cell cycle progression. Other Cdks and their cyclin partners are involved in regulating other biological processes, such as transcription. Search of the near-complete human genome sequences identified three new cyclins but no new Cdks (Murray and Marks, 2001). Two of the new cyclins are likely to be involved in transcription regulation, and the third has weak homology to B-type cyclins (Murray and Marks, 2001). The levels of Cdks are generally constant throughout the cell cycle, while the levels of their cyclin partners fluctuate periodically during the cycle – hence their name, cyclins (Evans et al., 1983). Biochemical and genetic data from several systems have demonstrated that cyclins promote cell cycle transitions via their ability to associate with and activate their cognate Cdks (Coleman and Dunphy, 1994; Draetta, 1994; Hunter and Pines, 1994; King et al., 1994; Koch and Nasmyth, 1994; Meyerson et al., 1992; Nurse, 1994; Sherr, 1994). D-type and E-type cyclins function in the G1 phase of the

Table 8.1. Cyclin-Dependent Kinases and Their Cyclin Partners

cell cycle (Baldin et al., 1993; Draetta, 1994; Dulic et al., 1992; Hunter and Pines, 1994; Koff et al., 1992; Matsushime et al., 1991; Meyerson et al., 1992; Sherr, 1994), and overexpression of cyclin Dl or cyclin E shortens G1, accelerating entry into the S phase (Ohtsubo et al., 1995; Quelle et al., 1993; Resnitsky et al., 1995; Resnitsky and Reed, 1995). D-type cyclins associate with Cdk4 and Cdk6 kinases, while cyclin E associates with Cdk2 (Sherr, 1993). In addition, a close homolog of Cdk2, Cdk3, is also thought to play a unique role in the G1-S transition (van den Heuvel and Harlow, 1993). However, Cdk3 is not essential in mice, as several laboratory strains encode a truncated form of the protein (Ye et al., 2001). Cyclin A binds Cdk2 and Cdc2 (Cdk1), another cyclin-dependant kinase that has a primary function in G2 and mitosis and is required for both the S phase and the G2-M transition (Girard et al., 1991; Pagano et al., 1992; Zindy et al., 1992), while cyclin B–Cdc2 complexes appear to be specific for control of mitotic entry.

What are the critical substrates for the cyclin-Cdk complexes in regulating cell cycle progression? One family of target substrates for these regulators is the retinoblastoma family of pocket proteins, pRb, p107, and p130 (Mulligan and Jacks, 1998; Nevins, 1998). The founding member of this family is pRb, the product of the retinoblastoma tumor susceptibility gene, whose mutational inactivation is the hallmark of the childhood retinal tumor retinoblastoma (Cavenee et al., 1983; Dryja et al., 1984; Friend et al., 1986; Fung et al., 1987; Lee et al., 1987). Since the cloning of RB in 1986 (Friend et al., 1986; Lee et al., 1987), there has been much effort to understand its function. These studies have led to the concept that pRb, the gene product of RB, is the master brake of the cell cycle, the molecular factor controlling the restriction point (Lundberg and Weinberg, 1999) in G1 phase of the cell cycle.

In cycling cells, pRb is unphosphorylated at the conclusion of mitosis. As the cell progresses through G1 (Fig. 8.2), pRb becomes phosphorylated by D-type cyclins in association with Cdk4 or 6 (Ewen et al., 1993; Hinds et al., 1992; Kato et al., 1993; Sherr, 1994; Weinberg, 1995). In this hypophosphorylated state, pRb binds to members of the E2F transcription factor family (preferentially E2Fs 1–3), masking the transactivation domain of the E2F but not interfering with the ability of E2F to bind to its DP partner (Dyson, 1998; Mulligan and Jacks, 1998; Nevins, 1998). Thus, when bound to E2Fs, the pRb-E2F-DP complex prevents the expression of target genes (Fig. 8.3). These E2F target genes encode enzymes for DNA synthesis and other cell cycle regulators such as cyclin E, DHFR (dihydrofolate reductase), cdc2, B-myb, c-myc, and N-myc (Dyson, 1998; Mulligan and Jacks, 1998; Nevins, 1998). Recent evidence has, however, modified the classical view that pRb suppresses E2F target genes only through masking E2F’s activation domain (Dyson, 1998). It is recognized now that pRb also actively represses promoters of E2F target genes via its ability to recruit histone deacetylase (HDAC; Fig. 8.3; Brehm et al., 1998; Luo et al., 1998; Magnaghi-Jaulin et al., 1998). Histone deacetylation is thought to facilitate the formation of nucleosomes and therefore hinder access to promoters by transcription factors (Grunstein, 1997).