Academic literature on the topic 'Filensin'

Filensin is a 100/110 kDa membrane-associated protein found in lens fiber cells. Previous studies have shown that this protein polymerizes in vitro and binds strongly to vimentin and to another 47 kDa lens membrane protein. Using cosedimentation assays, flotation assays and immunoelectron microscopy, we have examined the properties of purified filensin and measured its binding to lens membranes. Filensin behaves as a ureaextractable, hydrophilic protein which does not partition with Triton X-114 and is not affected by 1 M hydroxylamine at alkaline pH, an agent known to release fatty-acylated proteins from the membrane. Immunoblotting of urea-extracted lens membranes with two different affinity-purified antibodies reveals that, unlike intact filensin, a COOH-terminal filensin degradation product (51 kDa) remains tightly associated with the membranes. Purified filensin binds directly to urea-stripped lens membranes, but not to protein-free vesicles reconstituted from total lens lipids. The binding of filensin is not significantly influenced by the purified 47 kDa protein. Interestingly, the filensin-binding capacity of urea-extracted membranes is increased at least two-fold after trypsin treatment, which removes entirely the 51 kDa peptide from the membranes and presumably unmasks additional filensin-acceptor sites. Consistent with this, filensin binds to trypsinized and non-trypsinized membranes with similar affinities (2 × 10(−7) and 4 × 10(−7) M, respectively). Treatment of the membranes with thrombin, which also eliminates the 51 kDa peptide, does not increase their binding capacity, apparently because filensin-acceptor sites are also destroyed during proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Filensin, a 100 kDa, membrane-associated, cytoskeletal protein, is uniquely expressed in the lens fiber cell (Merdes, A., Brunkener, M., Horstmann, H., and Georgatos, S. D. (1991) J. Cell Biol. 115, 397–410). I cloned and sequenced a full-length chicken lens cDNA encoding filensin, also known as CP95 (Ireland, M. and Maisel, H. (1989) Lens and Eye Toxicity Research 6, 623–638). The deduced amino acid sequence of 657 residues contained an internal 280 residue heptad repeat domain with sequence similarities to the rod domain of intermediate filament proteins. The putative filensin rod domain could be divided into three alpha-helical segments (1A, 1B and 2) separated by short, non-helical linkers. The sequence of the amino-terminal end of the filensin rod domain contained the highly conserved intermediate filament segment 1A motif (Conway, J. F. and Parry, D. A. D. (1988) Int. J. Biol. Macromol. 10, 79–98). Allowing conservative amino acid substitutions, the sequence of the carboxy-terminal end of the filensin rod domain was similar to that of the highly conserved intermediate filament rod carboxy terminus. The alpha-helical segments of the shorter filensin rod domain aligned with the corresponding segments of intermediate filament proteins by allowing a gap of four heptad repeats in the amino-terminal half of filensin segment 2. Filensin rod segment 2 contained the characteristic stutter in heptad repeat phasing, nine heptads from the end of the intermediate filament rod. The overall sequence identity between the rod domains of filensin and individual intermediate filament proteins was 20 to 25%, approximately the level of sequence identity observed between intermediate filament proteins of different types. The open reading frame of chicken filensin predicted a 657 amino acid protein with molecular mass of 76 kDa. Embryonic chicken filensin migrated in SDS-PAGE as a triplet of 102, 105 and 109 kDa, while rooster filensin migrated as a 105 and 109 kDa doublet. Antibodies to filensin labeled lens fiber cells but not lens epithelial cells. By immunofluorescence methods filensin was localized to the fiber cell plasma membranes, including the ends of elongated fiber cells.

Filensin and phakinin constitute the subunits of a heteropolymeric, lens-specific intermediate filament (IF) system known as the beaded-chain filaments (BFs). Since the rod of filensin is four heptads shorter than the rods of all other IF proteins, we decided to examine the specific contribution of this protein in filament assembly. For these purposes, we constructed chimeric proteins in which regions of filensin were exchanged with the equivalent ones of vimentin, a self-polymerizing IF protein. Our in vitro studies show that the filensin rod domain does not allow homopolymeric filament elongation. However, the filensin rod is necessary for co-polymerization of filensin with phakinin and seems to counteract the inherent tendency of the latter protein to homopolymerize into large, laterally associated filament bundles. Apart from the rod domain, the presence of an authentic or substituted tail domain in filensin is also essential for co-assembly with the naturally tail-less phakinin and formation of extended filaments in vitro. Finally, transfection experiments in CHO and MCF-7 cells show that the rod domain of filensin plays an important role in de novo filament formation and distribution. The same type of analysis further suggests that the end-domains of filensin interact with cell-specific, assembly-modulating factors.

We have studied the molecular properties of a 100-kD protein, termed filensin, which we have isolated from porcine lens membranes. Filensin represents a membrane-associated element, resistant to salt and nonionic detergent treatment, and extractable only by alkali or high concentrations of urea. By indirect immunofluorescence and immunoelectron microscopy, this protein can be localized at the periphery of the lens fiber cells. Immunochemical analysis suggests that filensin originates from a larger 110-kD component which is abundantly expressed in lens but not in other tissues. Purified filensin polymerizes in a salt-dependent fashion and forms irregular fibrils (integral of 10 nm in diameter) when reconstituted into buffers of physiological ionic strength and neutral pH. Radiolabeled filensin binds specifically to lens vimentin under isotonic conditions, as demonstrated by affinity chromatography and ligand-blotting assays. By the latter approach, filensin also reacts with a 47-kD peripheral membrane protein of the lens cells. Purified filensin binds to PI, a synthetic peptide modelled after a segment of the COOH-terminal domain of peripherin (a type III intermediate filament protein highly homologous to vimentin), but not to various other peptides including the NH2-terminal headpiece of vimentin and derivatives of its middle (rod) domain. The filensin-PI binding is inhibited by purified lamin B, which is known to interact in vitro with PI (Djabali, K., M.-M. Portier, F. Gros, G. Blobel, and S. D. Georgatos. 1991. Cell. 64:109-121). Finally, limited proteolysis indicates that the filensin-vimentin interaction involves a 30-kD segment of the filensin molecule. Based on these observations, we postulate that the lens fiber cells express a polymerization-competent protein which is tightly associated with the plasma membrane and has the potential to serve as an anchorage site for vimentin intermediate filaments.

The cDNA coding for calf filensin, a membrane-associated protein of the lens fiber cells, has been cloned and sequenced. The predicted 755-amino acid-long open reading frame shows primary and secondary structure similarity to intermediate filament (IF) proteins. Filensin can be divided into an NH2-terminal domain (head) of 38 amino acids, a middle domain (rod) of 279 amino acids, and a COOH-terminal domain (tail) of 438 amino acids. The head domain contains a di-arginine/aromatic amino acid motif which is also found in the head domains of various intermediate filament proteins and includes a potential protein kinase A phosphorylation site. By multiple alignment to all known IF protein sequences, the filensin rod, which is the shortest among IF proteins, can be subdivided into three subdomains (coils 1a, 1b, and 2). A 29 amino acid truncation in the coil 2 region accounts for the smaller size of this domain. The filensin tail contains 6 1/2 tandem repeats which match analogous motifs of mammalian neurofilament M and H proteins. We suggest that filensin is a novel IF protein which does not conform to any of the previously described classes. Purified filensin fails to form regular filaments in vitro (Merdes, A., M. Brunkener, H. Horstmann, and S. D. Georgatos. 1991. J. Cell Biol. 115:397-410), probably due to the missing segment in the coil 2 region. Participation of filensin in a filamentous network in vivo may be facilitated by an assembly partner.

In previous studies we have characterized a lens-specific intermediate filament (IF) protein, termed filensin. Filensin does not self-assemble into regular IFs but is known to associate with another 47-kD lens-specific protein which has been suggested to represent its assembly partner. To address this possibility, we cloned and sequenced the cDNA coding for the bovine 47-kD protein which we have termed phakinin (from the greek phi alpha kappa omicron sigma = phakos = lens). The predicted sequence comprises 406 amino acids and shows significant similarity (31.3% identity over 358 residues) to type I cytokeratins. Phakinin possesses a 95-residue, non-helical domain (head) and a 311 amino acid long alpha-helical domain punctuated with heptad repeats (rod). Similar to cytokeratin 19, phakinin lacks a COOH-terminal tail domain and it therefore represents the second known example of a naturally tailless IF protein. Confocal microscopy on frozen lens sections reveals that phakinin colocalizes with filensin and is distributed along the periphery of the lens fiber cells. Quantitative immunoblotting with whole lens fiber cell preparations and fractions of washed lens membranes suggest that the natural stoichiometry of phakinin to filensin is approximately 3:1. Under in vitro conditions, phakinin self-assembles into metastable filamentous structures which tend to aggregate into thick bundles. However, mixing of phakinin and filensin at an optimal ratio of 3:1 yields stable 10-nm filaments which have a smooth surface and are ultrastructurally indistinguishable from "mainstream" IFs. Immunolabeling with specific antibodies shows that these filaments represent phakinin/filensin heteropolymers. Despite its homology to the cytokeratins, phakinin does not coassemble with acidic (type I), or basic (type II) cytokeratins. From these data we conclude that filensin and phakinin are obligate heteropolymers which constitute a new membrane-associated, lens-specific filament system related to, but distinct from the known classes of IFs.

The fiber cells of the eye lens possess a unique cytoskeletal system known as the "beaded-chain filaments" (BFs). BFs consist of filensin and phakinin, two recently characterized intermediate filament (IF) proteins. To examine the organization and the assembly of these heteropolymeric IFs, we have performed a series of in vitro polymerization studies and transfection experiments. Filaments assembled from purified filensin and phakinin exhibit the characteristic 19-21-nm periodicity seen in many types of IFs upon low angle rotary shadowing. However, quantitative mass-per-length (MPL) measurements indicate that filensin/phakinin filaments comprise two distinct and dissociable components: a core filament and a peripheral filament moiety. Consistent with a nonuniform organization, visualization of unfixed and unstained specimens by scanning transmission electron microscopy (STEM) reveals the the existence of a central filament which is decorated by regularly spaced 12-15-nm-diam beads. Our data suggest that the filamentous core is composed of phakinin, which exhibits a tendency to self-assemble into filament bundles, whereas the beads contain filensin/phakinin hetero-oligomers. Filensin and phakinin copolymerize and form filamentous structures when expressed transiently in cultured cells. Experiments in IF-free SW13 cells reveal that coassembly of the lens-specific proteins in vivo does not require a preexisting IF system. In epithelial MCF-7 cells de novo forming filaments appear to grow from distinct foci and organize as thick, fibrous laminae which line the plasma membrane and the nuclear envelope. However, filament assembly in CHO and SV40-transformed lens-epithelial cells (both of which are fibroblast-like) yields radial networks which codistribute with the endogenous vimentin IFs. These observations document that the filaments formed by lens-specific IF proteins are structurally distinct from ordinary cytoplasmic IFs. Furthermore, the results suggest that the spatial arrangement of filensin/phakinin filaments in vivo is subject to regulation by host-specific factors. These factors may involve cytoskeletal networks (e.g., vimentin IFs) and/or specific sites associated with the cellular membranes.

The cells of the eye lens contain the type III intermediate filament protein vimentin, as well as two other intermediate filament proteins, CP49 and filensin. These two proteins appear to be unique to the differentiated lens fibre cell. Immunoblotting and confocal microscopy were used to describe changes which occur in these three intermediate filament proteins and the networks they form during fibre cell differentiation and maturation. The vimentin network was present in both epithelial cells and some fibre cells. Fibre cells were vimentin positive up to a specific point 2–3 mm in from the lens capsule where the vimentin signal was drastically reduced. The CP49/filensin network was not present in the undifferentiated epithelial cells but emerged in the differentiating fibre cells. This latter network exhibited a principally plasma membrane localization in younger fibre cells but became more cytoplasmic in older fibre cells. This change also occurred at a distinct point in fibre cell differentiation, much earlier than the observed loss of the vimentin network. The subcellular changes in the distributions of these cytoskeletal networks were correlated to the loss of the fibre cell nucleus, another feature of fibre cell differentiation. No correlation was found to changes in the vimentin network but nuclear loss did coincide with changes in the CP49/filensin network. Concomitant with nuclear pyknosis, there were also changes in the nuclear lamina as well as infringement of the nuclear compartment by CP49, as shown by confocal microscopy. This study demonstrates vimentin and the CP49/filensin network to be independent in the lens but both networks undergo dramatic changes in subcellular distribution during the differentiation/maturation of the fibre cell. Only changes in the CP49/filensin network can be correlated to nuclear loss. Thus in the lens, unlike mammalian erythropoiesis which is also characterized by nuclear loss, the vimentin network does not appear linked to nuclear retention.

For nearly three decades cytoplasmic intermediate filaments (IFs) have been described as 10 nm thick, unbranched ropes radiating from the cell nucleus and extending to the plasma membrane. This stereotype is now being challenged by the discovery and molecular characterization of the beaded filaments (BFs), a novel class of IFs composed of the lens-specific proteins filensin and phakinin. In contrast to ‘mainstream’ IFs, BFs have a distinctly nodular appearance and form a meshwork underneath the plasma membrane of the lens fiber cells. In vitro assembly studies, expression of filensin and phakinin in cultured cells, and analysis of the corresponding genes reveal that these proteins have evolved from two different subfamilies of IF proteins, thus yielding a unique structure. The new information provides a basis for understanding how the various forms of tissue-specific IF proteins might have developed adopting to the constraints of a specialized environment.

The lens of the eye is a vital tissue in the visual system, responsible for the collection and focusing of light on to the retina. Comprised of epithelial cells at differing stages of differentiation, the transparency of the lens is dependent on the highly ordered crystalline structure of lens proteins. The lens consists of several proteins including crystallins (α, β, γ) that make up 90% of the soluble protein, and the lens cytoskeletal proteins. Cytoskeletal proteins contribute only a fraction of the total lens protein, but are thought to play an important role in the establishment and maintenance of transparency. Calpain-induced degradation of these proteins may be involved in the development of cataracts. This has been an area of research at Lincoln University where a flock of sheep genetically predisposed to cataract maintained as a cataract development model. The aim of this research was to investigate the distribution of cytoskeletal proteins in the lens, and to examine the effects of calpain proteolysis on these proteins, with the goal of establishing the role of the lens cytoskeletal proteins in the ovine cataract model. A combination of techniques was used including immunohistochemistry, which required the development of a specific protocol for ovine lenses. Cytoskeletal proteins were identified using immunohistochemistry in lens tissue sections and exhibited characteristic distributions. Actin displayed preferential distribution in the short sides of the fibre cells in the cortex of the lens but was absent in the lens nucleus, while spectrin in the cortex and nucleus was associated with the fibre cell membrane. Filensin was observed in the outer cortex of lens sections associated with the fibre cell membrane and cytoplasm, although the pattern of localisation was indistinct due to the abundance of filensin breakdown products. Vimentin displayed membrane and cytoplasmic association in the outer cortex that diminished toward the lens nucleus, with membrane associated vimentin only persisting in the deeper regions of the cortex and nucleus. Additionally, the effect of novel calpain inhibitors (Cat0059 and Cat811) in preventing proteolysis of lens cytoskeletal protein was investigated and compared with calpain inhibitors developed elsewhere (SJA6017). The inhibitors were tested at between 10 and 0.1 μM (100 nM). All inhibitors were effective at 10 μM. SJA6017 provided significant protection to vimentin at 1 μM. Cat0059 was found to protect spectrin and filensin at 1 μM, but not vimentin, while inhibitor Cat811 was found to protect spectrin only. SJA6017 added to assays at 100 nM offered significant protection to spectrin, and Cat0059 was found to protect filensin and spectrin to a significant degree at 100 nM, indicating the novel inhibitors were comparable to those developed elsewhere in terms of their effectiveness. Taken together, the evidence presented in this thesis shows the cytoskeletal proteins as crucial elements in the lens. Their pervasive presence coupled with evidence that lens cytoskeletal proteins are sensitive to calpain-induced proteolysis that is inhibited with novel calpain inhibitors suggests that the lens cytoskeletal proteins may be useful targets in cataract prevention for future research.