Intermediate Filament Protein Vimentin Research Articles

Isolation of SDS-stable complexes of the intermediate filament protein vimentin with repetitive, mobile, nuclear matrix attachment region, and mitochondrial DNA sequence elements from cultured mouse and human fibroblasts.

Crosslinkage of vimentin to DNA in mouse L929 cells by formaldehyde and isolation of SDS-stable DNA-vimentin complexes from normal L929 cells and mouse and human embryo fibroblasts indicated close spatial relations between these components in the intact cell. The adducts, obtained by immunoprecipitation with anti-vimentin antibody, contained substantial quantities, not only of repetitive and mobile sequence elements such as centromeric satellite DNA, telomere DNA, microsatellites and minisatellites, long and short interspersed nucleotide elements, and retroposons, but also of mitochondrial (mt) DNA. Because the SDS-stable complexes could be isolated with distinctly higher yields from oxidatively stressed, senescent fibroblasts and were dissociated by boiling, they possibly arose from accidental condensation reactions mediated by unsaturated and dialdehydes, products of free radical-induced lipid peroxidation. They can therefore be considered vestiges of a general interaction of vimentin with cellular DNA. The sequence patterns of their DNA fragments were similar to those of extrachromosomal circular and linear DNA, including retroviral elements, markers and enhancers of genomic instability that also occur in the cytoplasm and are able to transport vimentin into the nucleus. Many of the fragments were also remarkably similar to AT-rich nuclear matrix attachment regions (MARs) in that they contained, in addition to various mobile elements, a palette of typical MAR motifs. With its tendency to multimerize and to interact with single-stranded and supercoiled DNA, vimentin thus behaves like a nuclear matrix protein and may as such participate in a variety of nuclear matrix-associated processes such as replication, recombination, repair, and transcription of DNA. These activities seem to be extendible to the mitochondrial compartment, as vimentin was also crosslinked to mtDNA, preferentially to its D-loop and hypervariable main control region. These sites are prone to point and deletion mutations and, like nuclear MARs, are associated with the cyto-karyomatrix. Moreover, as a developmentally regulated and tissue-specific cyto-karyomatrix protein, vimentin may contribute to the organization of chromatin, including centromeric and telomeric heterochromatin at the nuclear periphery, with all its consequences for genomic activities during embryogenesis and in adulthood of vertebrates. However, because of its high affinity for hypervariable, recombinogenic DNA sequences, vimentin is proposed to play a major role in both the preservation and the evolution of the nuclear and mitochondrial genome.

Course of glial immunoreactivity for vimentin, tenascin and alpha1-antichymotrypsin after traumatic injury to human brain.

In a total of 104 individuals who had sustained traumatic brain injury (TBI), the course of glial immunoreactivity was investigated at the injured cortical area during the first 30 weeks after the trauma, in order to provide data for a forensic wound age estimation. Glial cells were stained with antibodies against the intermediate filament protein vimentin, the extracellular matrix protein tenascin and the serine protease inhibitor alpha1-antichymotrypsin (alpha1-ACT). Injury-induced glial staining reactions could be observed, at the earliest, after a post-infliction interval of 3.1 h for alpha1-ACT, 22 h for vimentin and 7 days for tenascin.

Mitotic Reorganization of the Intermediate Filament Protein Nestin Involves Phosphorylation by cdc2 Kinase

The intermediate filament protein nestin is expressed during early stages of development in the central nervous system and in muscle tissues. Nestin expression is associated with morphologically dynamic cells, such as dividing and migrating cells. However, little is known about regulation of nestin during these cellular processes. We have characterized the phosphorylation-based regulation of nestin during different stages of the cell cycle in a neuronal progenitor cell line, ST15A. Confocal microscopy of nestin organization and (32)P in vivo labeling studies show that the mitotic reorganization of nestin is accompanied by elevated phosphorylation of nestin. The phosphorylation-induced alterations in nestin organization during mitosis in ST15A cells are associated with partial disassembly of nestin filaments. Comparative in vitro and in vivo phosphorylation studies identified cdc2 as the primary mitotic kinase and Thr(316) as a cdc2-specific phosphorylation site on nestin. We generated a phosphospecific nestin antibody recognizing the phosphorylated form of this site. By using this antibody we observed that nestin shows constitutive phosphorylation at Thr(316), which is increased during mitosis. This study shows that nestin is reorganized during mitosis and that cdc2-mediated phosphorylation is an important regulator of nestin organization and dynamics during mitosis.

Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis

Caspases are key mediators of apoptosis. Using a novel expression cloning strategy we recently developed to identify cDNAs encoding caspase substrates, we isolated the intermediate filament protein vimentin as a caspase substrate. Vimentin is preferentially cleaved by multiple caspases at distinct sites in vitro, including Asp85 by caspases-3 and -7 and Asp259 by caspase-6, to yield multiple proteolytic fragments. Vimentin is rapidly proteolyzed by multiple caspases into similar sized fragments during apoptosis induced by many stimuli. Caspase cleavage of vimentin disrupts its cytoplasmic network of intermediate filaments and coincides temporally with nuclear fragmentation. Moreover, caspase proteolysis of vimentin at Asp85 generates a pro-apoptotic amino-terminal fragment whose ability to induce apoptosis is dependent on caspases. Taken together, our findings suggest that caspase proteolysis of vimentin promotes apoptosis by dismantling intermediate filaments and by amplifying the cell death signal via a pro-apoptotic cleavage product.

Uridine Phosphorylase Association with Vimentin: INTRACELLULAR DISTRIBUTION AND LOCALIZATION

Uridine phosphorylase (UPase), a key enzyme in the pyrimidine salvage pathway, is associated with the intermediate filament protein vimentin, in NIH 3T3 fibroblasts and colon 26 cells. Affinity chromatography was utilized to purify UPase from colon 26 and NIH 3T3 cells using the uridine phosphorylase inhibitor 5'-amino benzylacyclouridine linked to an agarose matrix. Vimentin copurification with UPase was confirmed using both Western blot analysis and MALDI-MS methods. Separation of cytosolic proteins using gel filtration chromatography yields a high molecular weight complex containing UPase and vimentin. Purified recombinant UPase and recombinant vimentin were shown to bind in vitro with an affinity of 120 pm and a stoichiometry of 1:2. Immunofluorescence techniques confirm that UPase is associated with vimentin in both NIH 3T3 and colon 26 cells and that depolymerization of the microtubule system using nocodazole results in UPase remaining associated with the collapsed intermediate filament, vimentin. Our data demonstrate that UPase is associated with both the soluble and insoluble pools of vimentin. Approximately 60-70% of the total UPase exists in the cytosol as a soluble protein. Sequential extraction of NIH 3T3 or colon 26 cells liberates an additional 30-40% UPase activity associated with a detergent extractable fraction. All pools of UPase have been shown to possess enzymatic activity. We demonstrate for the first time that UPase is associated with vimentin and the existence of an enzymatically active cytoskeleton-associated UPase.

Divide-and-conquer crystallographic approach towards an atomic structure of intermediate filaments

Intermediate filaments (IFs) represent an essential component of the cytoskeleton in higher eukaryotic cells. The elementary building block of the IF architecture is an elongated dimer with its dominant central part being a parallel double-stranded α-helical coiled coil. Filament formation proceeds via a specific multi-step association of the dimers into the unit-length filaments, which subsequently anneal longitudinally and finally radially compact into mature filaments. To tackle the challenge of a crystallographic structure determination, we have produced and characterised 17 overlapping soluble fragments of human IF protein vimentin. For six fragments ranging in length between 39 and 84 amino acid residues, conditions yielding macroscopic crystals could be established and X-ray diffraction data were collected to the highest resolution limit between 1.4 and 3 Å. We expect that solving the crystal structures of these and further fragments will eventually allow us to patch together a molecular model for the full-length vimentin dimer. This divide-and-conquer approach will be subsequently extended to determining the crystal structures of a number of complexes formed by appropriate vimentin fragments, and will eventually allow us to establish the three-dimensional architecture of complete filaments at atomic resolution.

Fully functional, naturally occurring and C-terminally truncated variant human immunodeficiency virus (HIV) Vif does not bind to HIV Gag but influences intermediate filament structure.

A variant human immunodeficiency virus type 1 (HIV-1) vif gene, vifA45-2, which encodes a protein lacking 19 amino acids at the C terminus but which is fully functional in supporting HIV replication in non-permissive cells has been described previously. By employing newly generated anti-VifA45 serum, further properties of VifA45 and its full-length counterpart, VifA45open, in comparison to Vif from HIV strain BH10 are reported in permissive HeLa and COS-7 cells. The results obtained using confocal microscopic localization studies and in vitro binding assays do not support a requirement for the direct interaction of HIV Gag with Vif. Furthermore and in contrast to previous conclusions, detergent solubility analyses do not demonstrate a role for the C terminus of Vif in mediating localization to the fraction containing cellular membrane proteins. Localization of Vif from HIV strain BH10 to perinuclear aggregates in a small fraction (about 10%) of transfected HeLa cells has been previously reported. The intermediate filament protein vimentin colocalizes to these structures. In contrast, VifA45 and VifA45open form perinuclear aggregates in nearly all transfected HeLa cells; vimentin as well as the cytoskeletal-bridging protein plectin, but not the microtubular protein tubulin, become relocalized to these structures. Interestingly, in COS-7 cells, all of the functional Vif proteins tested (Vif from strain BH10, VifA45 and VifA45open) predominantly localize in the cytoplasm but still induce dramatic aggregation of vimentin and plectin, i.e. in these cells the respective Vif proteins are influencing intermediate filament structure in the absence of colocalization.

Involvement of histone H4 gene transcription factor 1 in downregulation of vimentin gene expression during skeletal muscle differentiation

Upon in vitro myogenesis, the intermediate filament protein vimentin is replaced by desmin, the switch in gene expression occurring essentially at the transcriptional level. Trying to elucidate the molecular mechanisms of this genetic control, we show here that the vimentin promoter is specifically recognized and activated by a protein most probably identical to H4TF-1, and that this factor is present in proliferating myoblasts but disappears upon fusion of these cells into multinucleated myotubes. Our results suggest that H4TF-1 is a differentiation stage-specific factor involved in the downregulation of vimentin gene expression during myogenesis.

Transient transfections and heterokaryons as tools for the analysis of keratin IF dynamics.

All eukaryotic cells contain three distinct cytoskeletal networks: microtubules, microfilaments, and intermediate filaments (IF). In epithelial cells in vivo, the IF cytoskeleton is composed of heteropolymers of type I (acidic) and type II (neutral-basic) keratins, whose expression is carefully controlled in a cell type- and differentiation-specific manner (, , ). In addition, many epithelial cells in culture also induce the synthesis of the IF protein vimentin. In contrast to the other cytoskeletal proteins, which have been shown to be highly dynamic (, , , , ), IF in general, and keratins in particular, have long been considered the most stable and consequently the least dynamic components of the cell cytoskeleton (,). The majority of the dynamic studies on subcellular structures have been performed by microinjecting the proteins under study after their in vitro labeling. This approach, however, is very difficult with keratins due to the high insolubility shown by these proteins over a wide range of physiological conditions. In addition, this technique could lead to alteration in the dynamic process due to the large amount of protein injected into the cells () and because the incorporation of new molecules into IF structures also differs between preexisting and newly assembling filaments (). As an alternative to microinjection, transfections can be used. However, in standard transient transfection experiments, expression of the transgene takes place during a broad range of time (12–96 h), and it is impossible to know the precise moment at which the expression started in any particular cell, thus making conventional transfection techniques unfavorable for dynamic studies. However, the standard transfection technique is very useful for investigating the behavior of mutated genes (, , ).

Estrogen modulates norepinephrine-induced accumulation of adenosine cyclic monophosphate in a subpopulation of immortalized luteinizing hormone-releasing hormone secreting neurons from the mouse hypothalamus

A subpopulation of luteinizing hormone-releasing hormone (LHRH)-producing cells that express the intermediate filament protein vimentin and the neuronal marker neurofilament 145, but not neurofilament 200 nor glial fibrillary acidic protein, has been isolated from GT1–7 cultures. These cells express the mRNA encoding estrogen receptor alpha (ERα) and respond to physiological concentrations of 17β-estradiol (E 2) by reducing the accumulation of cyclic adenosine monophosphate induced by norepinephrine, but not that induced by direct activation of adenylate cyclase. These results indicate that the activity of LHRH-producing neurons may be directly modulated by estrogen. In addition, they are suggestive of an estrogen-dependent desensitization of the β-adrenoceptor in these cells.

Contractile proteins in pericytes at the blood-brain and blood-retinal barriers.

Evidence from a variety of sources suggests that pericytes have contractile properties and may therefore function in the regulation of capillary blood flow. However, it has been suggested that contractility is not a ubiquitous function of pericytes, and that pericytes surrounding true capillaries apparently lack the machinery for contraction. The present study used a variety of techniques to investigate the expression of contractile proteins in the pericytes of the CNS. The results of immunocytochemistry on cryosections of brain and retina, retinal whole-mounts and immunoblotting of isolated brain capillaries indicate strong expression of the smooth muscle isoform of actin (alpha-SM actin) in a significant number of mid-capillary pericytes. Immunogold labelling at the ultrastructural level showed that alpha-SM actin expression in capillaries was exclusive to pericytes, and endothelial cells were negative. Compared to alpha-SM actin, non-muscle myosin was present in lower concentrations. By contrast, smooth muscle myosin isoforms, were absent. Pericytes were strongly positive for the intermediate filament protein vimentin, but lacked desmin which was consistently found in vascular smooth muscle cells. These results add support for a contractile role in pericytes of the CNS microvasculature, similar to that of vascular smooth muscle cells.

Intermediate filament proteins define different glial subpopulations.

In certain species, specialized glial cells delineate cell domains in the central nervous system and assist in the elongation of axons by providing mechanical and chemical barriers. We showed previously, that the glial intermediate filament proteins vimentin and glial fibrillary acidic protein are extensively coexpressed in radial glia in the developing hindbrain, and that subsequently, the two proteins define distinct rhombomere domains: vimentin is localized in radial glia at the rhombomere boundaries and glial fibrillary acidic protein expression is restricted to the rhombomere centers (Yoshida and Colman [2000] J. Comp. Neurol. 424:47-57). The present study reveals that vimentin and glial fibrillary acidic protein continue to display distinct expression domains throughout the developing Xenopus central nervous system. Although the precise function of the two intermediate filaments in glial cells has yet to be revealed, the observations presented here suggests that glial intermediate filament proteins demarcate different populations of glial cells during nervous system development and that the existence of different glial populations may define glial boundaries.

Perinuclear Localization and Insulin Responsiveness of GLUT4 Requires Cytoskeletal Integrity in 3T3-L1 Adipocytes

The GLUT4 glucose transporter resides mostly in perinuclear membranes in unstimulated 3T3-L1 adipocytes and is acutely translocated to the cell surface in response to insulin. Using a novel method to purify intracellular GLUT4-enriched membranes, we identified by mass spectrometry the intermediate filament protein vimentin and the microtubule protein alpha-tubulin as components of these membranes. Immunoelectron microscopy of the GLUT4-containing membranes also revealed their association with these cytoskeletal proteins. Disruption of intermediate filaments and microtubules in 3T3-L1 adipocytes by microinjection of a vimentin-derived peptide of the helix initiation 1A domain caused marked dispersion of perinuclear GLUT4 to peripheral regions of the cells. Inhibition of the microtubule-based motor dynein by brief cytoplasmic acidification of cultured adipocytes also dispersed perinuclear GLUT4 and inhibited insulin-stimulated GLUT4 translocation to the cell surface. Insulin sensitivity was restored as GLUT4 was again concentrated near the nucleus upon recovery of cells in physiological buffer. These data suggest that GLUT4 trafficking to perinuclear membranes of cultured adipocytes is directed by dynein and is required for optimal GLUT4 regulation by insulin.

Properties of the Nonhelical End Domains of Vimentin Suggest a Role in Maintaining Intermediate Filament Network Structure

To investigate the functional role of the nonhelical domains of the intermediate filament (IF) protein vimentin, we carried out transient transfection of constructs encoding fusion proteins of these domains with enhanced green fluorescent protein (EGFP). Expression of these fusion proteins did not have any effect on the endogenous IF networks of transfected cells. However, the head domain–EGFP fusion protein localized almost exclusively to the nucleus. This localization could be disrupted in a reversible fashion by chilling cells. Furthermore, the head domain was capable of targeting to the nucleus a strictly cytoplasmic protein, pyruvate kinase. Thus, the vimentin head domain contains information that specifically directs proteins into the nucleus. In contrast, the nonhelical tail domain of vimentin, when expressed as a fusion protein with EGFP, was retained in the cytoplasm. Cytoplasmic retention of tail domain-containing fusion proteins appeared to be dependent on the integrity of the microtubule network. Our results are consistent with a proposal that the nonhelical end domains of vimentin are involved in maintaining an extended IF network by exerting oppositely directed forces along the filaments. The head domains exert a nuclear-directed force while the tail domains extend the IF network toward the cell periphery via a microtubule-dependent mechanism.

The Raf-1 kinase associates with vimentin kinases and regulates the structure of vimentin filaments.

Using immobilized GST-Raf-1 as bait, we have isolated the intermediate filament protein vimentin as a Raf-1-associated protein. Vimentin coimmunoprecipitated and colocalized with Raf-1 in fibroblasts. Vimentin was not a Raf-1 substrate, but was phosphorylated by Raf-1-associated vimentin kinases. We provide evidence for at least two Raf-1-associated vimentin kinases and identified one as casein kinase 2. They are regulated by Raf-1, since the activation status of Raf-1 correlated with the phosphorylation of vimentin. Vimentin phosphorylation by Raf-1 preparations interfered with its polymerization in vitro. A subset of tryptic vimentin phosphopeptides induced by Raf-1 in vitro matched the vimentin phosphopeptides isolated from v-raf-transfected cells labeled with orthophosphoric acid, indicating that Raf-1 also induces vimentin phosphorylation in intact cells. In NIH 3T3 fibroblasts, the selective activation of an estrogen-regulated Raf-1 mutant induced a rearrangement and depolymerization of the reticular vimentin scaffold similar to the changes elicited by serum treatment. The rearrangement of the vimentin network occurred independently of the MEK/ERK pathway. These data identify a new branch point in Raf-1 signaling, which links Raf-1 to changes in the cytoskeletal architecture.

Calyculin A-induced Vimentin Phosphorylation Sequesters 14-3-3 and Displaces Other 14-3-3 Partners in Vivo

14-3-3 proteins bind their targets through a specific serine/threonine-phosphorylated motif present on the target protein. This binding is a crucial step in the phosphorylation-dependent regulation of various key proteins involved in signal transduction and cell cycle control. We report that treatment of COS-7 cells with the phosphatase inhibitor calyculin A induces association of 14-3-3 with a 55-kDa protein, identified as the intermediate filament protein vimentin. Association of vimentin with 14-3-3 depends on vimentin phosphorylation and requires the phosphopeptide-binding domain of 14-3-3. The region necessary for binding to 14-3-3 is confined to the vimentin amino-terminal head domain (amino acids 1-96). Monomeric forms of 14-3-3 do not bind vimentin in vivo or in vitro, indicating that a stable complex requires the binding of a 14-3-3 dimer to two sites on a single vimentin polypeptide. The calyculin A-induced association of vimentin with 14-3-3 in vivo results in the displacement of most other 14-3-3 partners, including the protooncogene Raf, which nevertheless remain capable of binding 14-3-3 in vitro. Concomitant with 14-3-3 displacement, calyculin A treatment blocks Raf activation by EGF; however, this inhibition is completely overcome by 14-3-3 overexpression in vivo or by the addition of prokaryotic recombinant 14-3-3 in vitro. Thus, phosphovimentin, by sequestering 14-3-3 and limiting its availability to other target proteins can affect intracellular signaling processes that require 14-3-3.

Regulation of the rat oligodendroglia cell line OLN‐93 by antisense transfection of butyrylcholinesterase

Butyrylcholinesterase (BChE) is a glial cell marker with unknown function. For neuroepithelial cells, BChE has been shown to regulate cell division and expression of the postmitotic marker acetylcholinesterase (AChE), while similar studies are lacking for glial cells. By transducing an antisense-5'BChE cDNA expression vector via calcium phosphate precipitation, we have analyzed the effect of BChE inhibition on proliferation and differentiation of rat oligodendroglia-derived OLN-93 cells. OLN-93 cells were chosen because they are highly proliferative, while expressing markers of differentiated oligodendrocytes (Richter-Landsberg and Heinrich, 1996). First, we established that OLN-93 cells do express BChE protein, albeit chiefly in an inactive state, and that BChE was decreased by antisense-5'BChE transfection. Cell proliferation was also strongly diminished, protein kinase C (PKCalpha) was upregulated, and expression of cytoskeletal and cell surface proteins was altered. In particular, immunoreactivities of the intermediate filament proteins vimentin and the cell adhesion protein F11 were detected, indicating that BChE-inhibited OLN-93 cells have shifted toward an astrocytic phenotype. These data support a role of the glia marker BChE in CNS glial cell proliferation and differentiation, achieved via a nonenzymatic mechanism. The possible biomedical impact of BChE protein, e.g., on CNS nerve regeneration, is briefly discussed.

Altered flow-induced arterial remodeling in vimentin-deficient mice.

The endothelial cytoskeleton plays a key role in arterial responses to acute changes in shear stress. We evaluated whether the intermediate filament protein vimentin is involved in the structural responses of arteries to chronic changes in blood flow (BF). In wild-type mice (V+/+) and in vimentin-deficient mice (V-/-), the left common carotid artery (LCA) was ligated near its bifurcation, and 4 weeks later, the structures of the occluded and of the contralateral arteries were evaluated and compared with the structures of arteries from sham-operated mice. Body weight and mean carotid artery BF did not differ between the strains, but LCA and right carotid artery (RCA) diameter (737+/-14 microm [LCA] and 723+/-14 microm [RCA] for V-/- versus 808+/-20 microm [LCA] and 796+/-20 microm [RCA] for V+/+) and medial cross-sectional area (CSAm) were significantly smaller in V-/- (21+/-1 and 22+/-2 x 10(3) microm(2) for LCA and RCA, respectively) than in V+/+ (28+/-2 and 28+/-3 x 10(3) microm(2) for LCA and RCA, respectively). In V+/+, LCA ligation eliminated BF in the occluded vessel (before ligation, 0. 35+/-0.02 mL/min) and increased BF from 0.34+/-0.02 to 0.68+/-0.04 mL/min in the RCA. In V-/-, the BF change in the occluded LCA was comparable (from 0.38+/-0.05 mL/min to zero-flow rates), but the BF increase in the RCA was less pronounced (from 0.33+/-0.02 to 0. 50+/-0.05 mL/min). In the occluded LCA of V+/+, arterial diameter was markedly reduced (-162 microm), and CSAm was significantly increased (5 x 10(3) microm(2)), whereas in the high-flow RCA of V+/+, carotid artery diameter and CSAm were not significantly modified. In the occluded LCA of V-/-, arterial diameter was reduced to a lesser extent (-77 microm) and CSAm was increased to a larger extent (10 x 10(3) microm(2)) than in V+/+. In contrast to V+/+, the high-flow RCA of V-/- displayed a significant increase in diameter (52 microm) and a significant increase in CSAm (5 x 10(3) microm(2)). These observations provide the first direct evidence for a role of the cytoskeleton in flow-induced arterial remodeling. Furthermore, they dissociate (1) between acute and chronic arterial responses to altered BF, (2) between alterations of lumen diameter and wall mass during arterial remodeling, and (3) between developmental and imposed flow-induced arterial remodeling.

Dose-dependent linkage, assembly inhibition and disassembly of vimentin and cytokeratin 5/14 filaments through plectin's intermediate filament-binding domain.

Plectin, the largest and most versatile member of the cytolinker/plakin family of proteins characterized to date, has a tripartite structure comprising a central 200 nm-long (&agr;)-helical rod domain flanked by large globular domains. The C-terminal domain comprises a short tail region preceded by six highly conserved repeats (each 28-39 kDa), one of which (repeat 5) contains plectin's intermediate filament (IF)-binding site. We used recombinant and native proteins to assess the effects of plectin repeat 5-binding to IF proteins of different types. Quantitative Eu(3+)-based overlay assays showed that plectin's repeat 5 domain bound to type III IF proteins (vimentin) with preference over type I and II cytokeratins 5 and 14. The ability of both types of IF proteins to self-assemble into filaments in vitro was impaired by plectin's repeat 5 domain in a concentration-dependent manner, as revealed by negative staining and rotary shadowing electron microscopy. This effect was much more pronounced in the case of vimentin compared to cytokeratins 5/14. Preassembled filaments of both types became more and more crosslinked upon incubation with increasing concentrations of plectin repeat 5. However, at high proportions of plectin to IF proteins, disassembly of filaments occurred. Again, vimentin filaments proved considerably more sensitive towards disassembly than those composed of cytokeratins 5 and 14. In general, IFs formed from recombinant proteins were found to be slightly more responsive towards plectin influences than their native counterparts. A dose-dependent plectin-inflicted collapse and putative disruption of IFs was also observed in vivo after ectopic expression of vimentin and plectin's repeat 5 domain in cotransfected vimentin-deficient SW13 (vim(-)) cells. Our results suggest an involvement of plectin not only in crosslinking and stabilization of cytoskeletal IF networks, but also in regulation of their dynamics.

Analysis of UVA-Related Alterations upon Aging of Eye Lens Proteins by Mini Two-Dimensional Polyacrylamide Gel Electrophoresis

This study is a first approach to identify UVA-related alterations in situ of bovine eye lens proteins from the water-soluble and urea-soluble fractions upon aging. The fractions were obtained from irradiated long-term organ culture lenses and analyzed by mini two-dimensional gel electrophoresis. This micropreparative method followed by computer analysis allows high resolution and separation of microgram quantities of proteins and to detect spots which arose as a consequence of irradiation. To facilitate the analysis we first separated the water-soluble fraction into the major crystallin classes by gel filtration. Moreover, we immunoblotted the gel of the urea-soluble fraction with a specific antibody against the intermediate filament protein vimentin. Upon irradiation of young and adult lenses, αA-crystallin and vimentin showed obvious modifications. During aging the susceptibility to irradiation increased when vimentin started to degrade, whereas deamidation of αA-crystallin seems to occur.