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.

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.

During mitosis, several types of intermediate-sized filaments (IFs) undergo an extensive remodelling in response to phosphorylation by cdc 2 and other protein kinases. However, unlike the nuclear lamins, the cytoplasmic IFs do not seem to follow a fixed disassembly stereotype and often retain their physical continuity without depolymerizing into soluble subunits. To investigate potential interactions between mitotically modified IFs and other cellular structures, we have examined prometaphase-arrested cells expressing the IF protein vimentin. We demonstrate here that vimentin filaments associate in situ and co-fractionate with a distinct population of mitotic vesicles. These vesicles carry on their surfaces nuclear lamin B, the inner nuclear membrane protein p58, and wheat germ agglutinin (WGA)-binding proteins. Consistent with a tight interaction between the IFs and the mitotic membranes, vimentin, nuclear lamin B, and a 180-kD WGA-binding protein are co-isolated when whole mitotic homogenates are incubated with anti-vimentin or anti-lamin B antibodies immobilized on magnetic beads. The vimentin-associated vesicles are essentially depleted of ER, Golgi and endosomal membrane proteins. The interaction of vimentin with lamin B-carrying membranes depends on phosphorylation and is weakened by dephosphorylation during nuclear reassembly in vitro. These observations reveal a novel interaction between IFs and cellular membranes and further suggest that the vimentin filaments may serve as a transient docking site for inner nuclear membrane vesicles during mitosis.

Previous studies have shown that neuronal cells in culture can switch neurotransmitters when grown in the presence of different target cells. To examine whether this plasticity extends to structural proteins, we cocultured hippocampal neurons and pituitary-derived neuroendocrine (AtT20) cells with astrocytes, kidney epithelial cells, or skeletal muscle cells. As a marker of phenotypic change we used the cytoskeletal protein peripherin, a type III intermediate filament (IF) subunit which is not expressed in hippocampal neurons and AtT20 cells. We show here that soluble factor(s) secreted specifically from skeletal muscle cells can induce the expression and de novo assembly of peripherin in a subset of post-mitotic neurons. We further demonstrate that one of these factors is the Leukemia Inhibitory Factor/Cholinergic Neuronal Differentiation Factor. The environmentally regulated expression of peripherin implies a remarkable degree of plasticity in the cytoskeletal organization of postmitotic CNS cells and provides a noninvasive model system to examine the de novo assembly of IF proteins under in vivo conditions.

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.

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.

To identify sites of self-association in type III intermediate filament (IF) proteins, we have taken an "anti-idiotypic antibody" approach. A mAb (anti-Ct), recognizing a similar feature near the end of the rod domain of vimentin, desmin, and peripherin (epsilon site or epsilon epitope), was characterized. Anti-idiotypic antibodies, generated by immunizing rabbits with purified anti-Ct, recognize a site (presumably "complementary" to the epsilon epitope) common among vimentin, desmin, and peripherin (beta site or beta epitope). The beta epitope is represented in a synthetic peptide (PII) modeled after the 30 COOH-terminal residues of peripherin, as seen by comparative immunoblotting assays. Consistent with the idea of an association between the epsilon and the beta site, PII binds in vitro to intact IF proteins and fragments containing the epsilon epitope, but not to IF proteins that do not react with anti-Ct. Microinjection experiments conducted in vivo and filament reconstitution assays carried out in vitro further demonstrate that "uncoupling" of this site-specific association (by competition with PII or anti-Ct) interferes with normal IF architecture, resulting in the formation of filaments and filament bundles with diameters much greater than that of the normal IFs. These thick fibers are very similar to the ones observed previously when a derivative of desmin missing 27 COOH-terminal residues was assembled in vitro (Kaufmann, E., K. Weber, and N. Geisler. 1985. J. Mol. Biol. 185:733-742). As a molecular explanation, we propose here that the epsilon and the beta sites of type III IF proteins are "complementary" and associate during filament assembly. As a result of this association, we further postulate the formation of a surface-exposed "loop" or "hairpin" structure that may sterically prevent inappropriate filament-filament aggregation and regulate filament thickness.

Previous studies have shown that turkey erythrocyte lamin B is anchored to the nuclear envelope via a 58-kD integral membrane protein termed p58 or lamin B receptor (Worman H. J., J. Yuan, G. Blobel, and S. D. Georgatos. 1988. Proc. Natl. Acad. Sci. USA. 85:8531-8534). We now identify a p58 analogue in the yeast Saccharomyces cerevisiae. Turkey erythrocyte lamin B binds to yeast urea-extracted nuclear envelopes with high affinity, associating predominantly with a 58-kD polypeptide. This yeast polypeptide is recognized by polyclonal antibodies against turkey p58, partitions entirely with the nuclear fraction, remains membrane bound after urea extraction of the nuclear envelopes, and is structurally similar to turkey p58 by peptide mapping criteria. Using polyclonal antibodies against turkey erythrocyte lamins A and B, we also identify two yeast lamin forms. The yeast lamin B analogue has a molecular mass of 66 kD and is structurally related to erythrocyte lamin B. Moreover, the yeast lamin B analogue partitions exclusively with the nuclear envelope fraction, is quantitatively removed from the envelopes by urea extraction, and binds to turkey lamin A and vimentin. As many higher eukaryotic lamin B forms, the yeast analogue is chemically heterogeneous comprising two serologically related species with different charge characteristics. Antibodies against turkey lamin A detect a 74-kD yeast protein, slightly larger than the turkey lamin A. It is more abundant than the yeast lamin B analogue and partitions between a soluble cytoplasmic fraction and a nuclear envelope fraction. The yeast lamin A analogue can be extracted from the nuclear envelope by urea, shows structural similarity to turkey and rat lamin A, and binds to isolated turkey lamin B. These data indicate that analogues of typical nuclear lamina components (lamins A and B, as well as lamin B receptor) are present in yeast and behave as their vertebrate counterparts.

In vitro binding studies with isolated bovine lens vimentin and avian erythrocyte membranes reveal the existence of two functionally distinct sets of intermediate filament attachment sites. One population of such receptors is located along the nuclear envelope and comprises polypeptides recognizing the carboxy-terminal tail domain of vimentin. Vimentin associates with these nuclear surface receptors in a cooperative manner and forms extensive 10-nm filaments in a concentration-dependent fashion. Conversely, the plasma membrane contains binding sites that interact in a noncooperative, saturable fashion with vimentin, recognizing its amino-terminal head domain. The functional dichotomy of the vimentin-binding sites under in vitro conditions may reflect a vectorial assembly process whereby 10-nm filaments, although structurally apolar, acquire polar features brought about by the differential attachment to specific receptors arranged along the plasma membrane and the nuclear envelope.

We found that urea extraction of turkey erythrocyte nuclear envelopes abolished their ability to bind exogenous 125I-vimentin, while, at the same time, it removed the nuclear lamins from the membranes. After purification of the lamins from such urea extracts, a specific binding between isolated vimentin and lamin B, or a lamin A + B hetero-oligomer, was detected by affinity chromatography. Similar analysis revealed that the 6.6-kD vimentin tail piece was involved in this interaction. By other approaches (quantitative immunoprecipitation, rate zonal sedimentation, turbidometric assays) a substoichiometric lamin B-vimentin binding was determined under in vitro conditions. It was also observed that anti-lamin B antibodies but not other sera (anti-lamin A, anti-ankyrin, preimmune) were able to block 70% of the binding of 125I-vimentin to native, vimentin-depleted, nuclear envelopes. These data, which were confirmed by using rat liver nuclear lamins, indicate that intermediate filaments may be anchored directly to the nuclear lamina, providing a continuous network connecting the plasma membrane skeleton with the karyoskeleton of eukaryotic cells.

We have characterized the association of the intermediate filament protein, vimentin, with the plasma membrane, using radioiodinated lens vimentin and various preparations of human erythrocyte membrane vesicles. Inside-out membrane vesicles (IOVs), depleted of spectrin and actin, bind I125-vimentin in a saturable manner unlike resealed, right-side-out membranes which bind negligible amounts of vimentin in an unsaturable fashion. The binding of vimentin to IOVs is abolished by trypsin or acid treatment of the vesicles. Extraction of protein 4.1 or reconstitution of the membranes with purified spectrin do not basically affect the association. However, removal of ankyrin (band 2.1) significantly lowers the binding. Upon reconstitution of depleted vesicles with purified ankyrin, the vimentin binding function is restored. If ankyrin is added in excess the binding of vimentin to IOVs is quantitatively inhibited, whereas protein 4.1, the cytoplasmic fragment of band 3, band 6, band 4.5 (catalase), or bovine serum albumin do not influence it. Preincubation of the IOVs with a polyclonal anti-ankyrin antibody blocks 90% of the binding. Preimmune sera and antibodies against spectrin, protein 4.1, glycophorin A, and band 3 exhibit no effect. On the basis of these data, we propose that vimentin is able to associate specifically with the erythrocyte membrane skeleton and that ankyrin constitutes its major attachment site.

Fragments of vimentin, generated by chemical or enzymatic cleavages, were analyzed for their capacity to bind to human inverted erythrocyte membrane vesicles. Only peptides comprising the amino-terminal head domain of vimentin molecules were competent in associating with the membranes. In vitro studies also demonstrated that isolated ankyrin (the major vimentin acceptor site on the membrane) binds to an oligomeric species of vimentin and prevents the formation of characteristic 10-nm filaments. These data, taken together with the observation that the NH2-terminal end of vimentin is implicated in the polymerization process (Traub, P., and C. Vorgias, J. Cell Sci., 1983, 63:43-67), imply that intermediate filaments may contact the membrane in an end-on fashion, using the exposed head domains of their terminal subunits.