R.A. QUINLAN, A. SANDI LANDS, The eye lens J.E. PROCTER, A.R. PRESCOTT, A.M. HUTCHESON, R. DAHM, cytoskeleton C. GRIBBON, P. WALLACE, LM. CARTER Abstract During lens cell differentiation there are a number of very characteristic morphological changes that occur. These include a 50- to 100- fold increase in cell length as the equatorial lens epithelial cells differentiate into fibre cells and the loss of the cellular organelles such as mitochondria, nuclei, Golgi apparatus and endoplasmic reticulum. Coincident with these changes are dramatic alterations in the organisation of the lens fibre cell cytoskeleton and in particular the lens-specific intermediate filament network comprising CP49 and filensin. Cell shape and cell polarisation as well as tissue integrity are all processes that depend upon the cytoskeleton and are therefore important to the lens. The unique aspects of the lenticular cytoskeleton are the subject of this review. RA Quinlan A. Sandi lands Key words CP49, Cytoskeleton, Differentiation, !.E. Procter Filensin, Intermediate filaments, Lens A.R. Prescott A.M. Hutcheson R. Dahm C Gribbon P. Wallace J.M. Carter The lenticular cytoskeleton Department of Biochemistry In common with other cells, lens cells possess Medical Sciences Institute microfilaments, microtubules and intermediate The University Dundee, Scotland, UK filaments (Fig. 1). It is expected that they also perform equivalent functions in the lens as Dr RAQuinlan � found in other cell types. Thus, the actin Department of Biochemistry The University cytoskeleton, through the actions of actin­ Dundee DD1 5EH binding proteins and motor proteins, will Scotland, UK facilitate changes in cell shape, strengthen Tel: +44 (0)1382344752 cell-cell contacts and cell-extracellular matrix Fax: +44 (0)1382201603 interactions, and define plasma membrane e-mail: compartments. Likewise the microtubule [email protected] cytoskeleton will direct intracellular transport RAQ and JM.C processes as well as contributing to the acknowledge the financial distribution of organelles. Intermediate support of the Wellcome filaments are major structural elements of cells Trust J.E.P. and R.D. are supported by MRC and helping them to resist physical stresses, which BBSRC-CASE studentships in the context of the primate lens will include respectively. The support of lens accommodation. Of all these cytoskeletal Pharmacia-Upjohn as the systems it is the intermediate filaments of the industrial sponsor of the Fig. 1. Cultured human lens epithelial cells labelled BBSRC -CASE studentship is eye lens that are the most distinct and for which with an antibody to a-tubulin (A), phalloidin to actin acknowledged. CG. is a direct link with cataract has recently been (B) and an antibody to vimentin (C). Scale bars represent supported by a Fight for suggested. 10/Lm. Sight studentship Eye (1999) 13, 409-416 © 1999 Royal College of Ophthalmologists 409 Table 1. The intermediate filament protein family Intermediate filament type Protein name Molecular weight (x 10-3) Cell/tissue location I Keratin 9-20 40-64 Epithelial cells II Keratin 1-8 52-68 Epithelial cells III Vimentin 55 Mesenchymal cells Desmin 53 Muscle cells GFAP 51 Glia and astrocytes Peripherin 54 Neuronal cells IV Neurofilament proteins: NF-L/NF-Ma /NF-H" 70-160 Neurons a-internexin" 56 Neurons Nestina 240 Neuroepithelial stem cells and muscle V Nuclear lamins: A-type 55-72 Differentiated cells B-type 55-68 All cells "The apparent mobility of these proteins by SDS-PAGE is quite different from the calculated molecular weights determined from the amino acid sequence. Intermediate filaments of the eye lens Diseases caused by intermediate filaments and their associated proteins Our own studies have concentrated on the intermediate filament proteins found in the lens, which have led to a Intermediate filaments are some of the most resilient number of important discoveries. Firstly, through the structures present in the cell?O A number of inherited cloning and sequencing of the two lens-specific proteins, diseases have been discovered which are due to CP49I-6 and filensin/-12 the homology of these proteins mutations in intermediate filament proteins or their to the intermediate filament protein family was associated proteins.21 These include skin blistering established. Secondly, CP49 and filensin were found in a diseases (reviewed in21), muscular dystrophy,22 complex with two other major lens proteins, aA- and aB­ myopathies,23,24 corneal dystrophies2s and also lens cataract.18,26 A defective intermediate filament network crystallin}3,14 which are protein chaperones and which severely compromises the structural integrity of the cell are essential for lens transparency. This complex of as well as the tissue comprising these cells.27 For chaperones and intermediate filaments forms a specific instance, the first diseases attributed to mutations in structure with a very distinctive morphology called the intermediate filament proteins were the skin blistering beaded filament. It is found only in the lens.IS Thirdly, diseases. Here, mutations in the keratins, the the mechanism of assembly for CP49 proteins and intermediate filament proteins found in epithelial cells, filensin is apparently different from that observed for caused collapse of the filament network in the epidermal other intermediate filament proteins, which has cells. The barrier function of the epidermis is lost which, challenged several of the central dogmas concerning depending on the mutation, can present a range of intermediate filament assembly.14,16,17 Lastly, the phenotypes from skin blistering, in response to mild discovery that mutations in aB-crystallin can induce the stress, to the shedding of large areas of epidermis in the collapse of intermediate filament networksI8 shows most severe cases. More recently, mutations in the intermediate and beaded filaments to be important muscle-specific intermediate filament protein, desmin, physiological targets for the chaperone function of aB­ have been found to cause desmin related myopathy crystallinI9 and suggests the beaded filament is a key (DRM23,24). This disease can also be caused by a mutation structural element required for lens transparency. in aB-crystallin,18 demonstrating the importance of non a-helical non a-helical N-terrninal C-tenninal ROD DOMAIN ---------f domain domain HELIX I LIllI HELIX II MOTIF FORMS A PARALLEL AND IN REGISTER COILED-COIL WITH ASSEMBLY PART VIA THESE o:-HELCAL DOMAINS ESSENTIAL FOR ER CONTRIBUTES FILAMENT TO FILAMENT ASSEMBLY AS EMBLY Fig. 2. Schematic representation of the structural features common to the intermediate protein family. 410 associated proteins to intermediate filament function. which are essential for assembly (Fig. 2). These are the Patients with DRM caused by aB-crystallin mutations LNDR- and TYRKLLEGE-motifs found at the N- and C­ also present with cataract, suggesting an important role terminal ends of the central rod domain.36,37 for intermediate filaments in lens transparency. In mouse Intermediate filaments themselves are apolar models of cataractogenesis, disruption of the normal polymers, unlike actin filaments and microtubules which expression pattern and levels of intermediate filament are polar. Soluble intermediate filament subunits can proteins occurs in the lenses of these mice?8-31 therefore exchange directly with the filament along its length. Tubulin and actin are restricted to exchange events at the ends of microtubules and microfilaments, Intermediate filament family respectively.38,39 There are over 60 different gene products attributed to the intermediate filament protein family on the basis of Intermediate filament proteins present in the lens their primary amino acid sequence similarities, assembly In the adult vertebrate lens there are at least three characteristics and gene structure (Table 1). These different intermediate filament proteins. These include proteins are arranged into five major classes. The type I vimentin, CP49 and filensin.37 The lens has often been and II proteins comprise the keratins, which cover those used as a source for the purification of vimentin, as it was intermediate filament proteins found in epithelia. The considered for a long time to be the only intermediate type III proteins are GFAP, desmin, peripherin and filament protein present in the mature lens.4o Early in vimentin, which are specific for astrocytes, muscle, development, though, keratins are expressed in the peripheral nerves and cells of mesenchymal origin, primary lens fibre cells, although these are then 10St.41 respectively. The type IV proteins are the neurofilament Vimentin itself is found in the epithelial cells and the proteins, including a-internexin, which are all found secondary fibre cells but here the expression is restricted largely in neurons. Lastly type V are the nuclear lamins, to the younger fibre cells.42,43 There is a distinct transition which are found in all cells that possess a nucleus. More during lens fibre cell differentiation when vimentin is recently, the discovery of additional vertebrate apparently lost from the secondary fibre cells. This point intermediate filament proteins such as CP49 (see below), is far deeper in the lens beyond the stage where the cell filensin (see below), synemin32 and paranemin,33 which organelles are 10st.43.44 The lens-specific proteins, CP49 do not fit easily into either one of the established protein and filensin, are found at all stages of lens fibre cell types, may mean that this categorisation