The nuclear lamina is a karyoskeletal structure at the nucleoplasmic surface of the inner nuclear membrane. Its assembly state is regulated by phosphorylation of the intermediate filament type lamin proteins. Strong evidence has been obtained for a causal link between phosphorylation of lamins by the p34cdc2 protein kinase and disassembly of the nuclear lamina during mitosis. In contrast, no information is currently available on the role of lamin phosphorylation during interphase of the cell cycle. Here, we have identified four protein kinase C phosphorylation sites in purified chicken lamin B2 as serines 400, 404, 410, and 411. In vivo, the tryptic peptide containing serines 400 and 404 is phosphorylated throughout interphase, whereas serines 410 and 411 become phosphorylated specifically in response to activation of protein kinase C by phorbol ester. Prompted by the close proximity of serines 410/411 to the nuclear localization signal of lamin B2, we have studied the influence of phosphorylation of these residues on nuclear transport. Using an in vitro assay, we show that phosphorylation of lamin B2 by protein kinase C strongly inhibits transport to the nucleus. Moreover, phorbol ester treatment of intact cells leads to a substantial reduction of the rate of nuclear import of newly synthesized lamin B2 in vivo. These findings have implications for the dynamic structure of the nuclear lamina, and they suggest that the modulation of nuclear transport rates by cytoplasmic phosphorylation may represent a general mechanism for regulating nuclear activities.

The nuclear lamina is the karyoskeletal structure, intimately associated with the nuclear envelope, that is widespread among the diverse types of eukaryotic cells. A family of proteins, termed lamins, has been shown to be a prominent component of this lamina, and various members of this family are differentially expressed in different cell types. In mammals, three major lamins (A, B, C) have been identified, and in all cells so far examined lamin B is constitutively expressed while lamins A and C are not, suggesting that lamin B is sufficient to form a functional lamina. Because of this key importance of lamin B, cDNA clones encoding mammalian lamin B were isolated by screening murine cDNA libraries, representing F9 teratocarcinoma cells and fetal liver, with the corresponding cDNA probe of lamin LI of Xenopus laevis. The nucleotide sequence of the murine lamin B mRNA (approximately 2.9 kb) was determined. The deduced amino acid sequence of the encoded polypeptide (587 amino acids; mol. wt. 66760) is highly homologous to X. laevis lamin LI (72.9% identical residues) but displays lower similarity to A-type lamins (53.8% identical amino acid residues with human lamin A). Lamin B also conforms to the general molecular organization principle of the members of the intermediate filament (IF) protein family, i.e., an extended alpha-helical rod domain that is interrupted by two non alpha-helical linkers and flanked by non-alpha-helical head (amino-terminal) and tail (carboxy-terminal) domains. The tail domain, which does not reveal a hydrophobic region of considerable length, contains a typical karyophilic signal sequence and an uninterrupted stretch of eight negatively charged amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

When microinjected into the cytoplasm of 3T3 cells, biotinylated human lamin A rapidly enters the nucleus and gradually becomes incorporated into the nuclear lamina region as determined by immunofluorescence. The incorporation of the microinjected material takes several hours and progresses through a series of morphologically identifiable stages. Within minutes after microinjection, lamin A is found in spots distributed throughout the nucleus, except in nucleolar regions. Over a time course of up to 6 h, these spots appear to decrease in size and number as the biotinylated lamin A becomes associated with the endogenous nuclear lamina. Eventually, the typical nuclear rim staining pattern normally revealed by immunofluorescence with nuclear lamin antibodies is seen with antibiotin. This latter rim staining property is passed on to daughter cells following mitosis. These results indicate that the microinjected biotinylated nuclear lamin A retains those properties required for its integration into the lamina, as well as those necessary for the disassembly and subsequent reassembly of the nuclear lamina during cell division. The initial rapid accumulation into foci and the subsequent slower incorporation into the nuclear lamina appear to be analogous to the stages of incorporation following the microinjection of cytoskeletal intermediate filament proteins such as vimentin and keratin (Vikstrom, K., G. G. Borisy, and R. D. Goldman. 1989. Proc. Natl. Acad. Sci. USA. 86:549-553; Miller, R. K., K. Vikstrom, and R. D. Goldman. 1991. J. Cell Biol. 113:843-855). Foci are also observed in some uninjected cells using nuclear lamin antibodies, indicating that these features are a genuine component of nuclear substructure. Evidence is presented that shows the appearance of these nuclear structures is cell cycle dependent.

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145-3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40-70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical-basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37 degrees C or in n-octyl-beta-D-glycoside at 4 degrees C (representative of GPI-anchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na+-K+ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.

In the presence of ATP, recA protein forms a presynaptic complex with single-stranded DNA that is an obligatory intermediate in homologous pairing. Presynaptic complexes of recA protein and circular single strands that are active in forming joint molecules can be isolated by gel filtration. These isolated active complexes are nucleoprotein filaments with the following characteristics: (i) a contour length that is at least 1.5 times that of the corresponding duplex DNA molecule, (ii) an ordered structure visualized by negative staining as a striated filament with a repeat distance of 9.0 nm and a width of 9.3 nm, (iii) approximately 8 molecules of recA protein and 20 nucleotide residues per striation. The widened spacing between bases in the nucleoprotein filament means that the initial matching of complementary sequences must involve intertwining of the filament and duplex DNA, unwinding of the latter, or some combination of both to equalize the spacing between nascent base pairs. These experiments support the concept that recA protein first forms a filament with single-stranded DNA, which in turn binds to duplex DNA to mediate both homologous pairing and subsequent strand exchange.

Seventy-four out of 113 sera from patients with infectious hepatitis, chickenpox, measles and mumps reacted with both smooth muscle and cytoplasmic filaments in cultured fibroblasts and neuroblastoma. Five out of eighty-five control sera also reacted in this way. That the cytoplasmic structures are intermediate filaments was suggested by their rearrangement into coils of perinuclear filaments in colchicine- or vinblastine-treated fibroblasts, but not in cytochalasin B-treated cells. The idenity of these structures was confirmed by the demonstration that the same structures reacted with the post-viral sera and a rabbit and human anti-intermediate filament antibody. Immunoabsorption studies showed that twenty-seven out of thirty-two positive sera were neutralised by skeletin, the intermediate filament protein from smooth muscle. In all but one of the sera, the antibody was IgM. Antibody titres fell in the second specimen in eleven out of fourteen pairs of acute and convalescent sera. The association between viral infections and autoantibodies suggest that production of antibodies suggests that production of antibody to intermediate filaments may be initiated by viruses.

The adult brain contains a small population of central nervous system (CNS) cells in the subependyma which, like embryonic CNS progenitor cells, express the intermediate filament nestin. In this report, the differentiation capacity in vivo of these cells was analysed following a standardized trauma. Before the trauma, the subependymal cells expressed nestin but not the astrocytic and neuronal differentiation markers glial fibrillary acidic protein (GFAP) and neurofilament respectively. In response to injury, the majority of the subependymal cells coexpressed nestin and GFAP, but never nestin and neurofilament. Furthermore, cells coexpressing nestin and GFAP were found progressively further away from the subependyma and closer to the lesion at later time points after the injury, indicating that these cells migrate towards the lesion. Nestin was in addition re-expressed in reactive astrocytes near the lesion and in non-reactive astrocytes very far from the lesion throughout the ipsilateral cortex. In conclusion, our data indicate that the nestin-positive subependymal cells are an in vivo source for the generation of new astrocytes but not neurons after injury, and that nestin re-expression in astrocytes following traumatic stimuli can be used as a sensitive marker for astroglial activation.

OBJECTIVE

To determine the function of the lens fiber cell-specific cytoskeletal protein, filensin, in lens biology.

METHODS

Targeted genomic deletion was used to delete exon 1 and the transcriptional start site of the filensin gene. Resultant chimeric animals were bred to homozygosity for the mutant allele. These animals were outbred to mice bearing the wild-type CP49 alleles to eliminate the mutant CP49 gene carried by the 129 strain of mice. Animals homozygous for the mutated filensin gene and wild-type CP49 gene were compared with wild-type and heterozygous animals by Northern and Western blot analyses, light and electron microscopy, and slit lamp microscopy.

RESULTS

Disruption of the filensin gene successfully blocked production of filensin mRNA, reduced levels of filensin's assembly partner CP49, and prevented the assembly of beaded filaments. Despite the absence of beaded filaments, lenses did not show obvious changes in fetal development, nor in the differentiation of epithelial cells into mature fiber cells, as judged by light microscopic analysis. Filensin knockouts began to show evidence of light-scattering by 2 months and worsened with age. Heterozygous animals exhibited an intermediate phenotype, showing a reduction in filensin transcript and moderate light-scattering at 5 months.

CONCLUSIONS

The lens fiber cell-specific intermediate filament protein filensin is essential for beaded filament assembly. However, although beaded filaments are not needed for normal lens fetal development or fiber cell differentiation, they appear to be necessary for the long-term maintenance of optical clarity. The mechanism by which the absence of filensin and the beaded filament affects optical clarity has yet to be defined.

Desmosomes are cell junctions that act as sites of strong intercellular adhesion and also serve to anchor the intermediate filament (IF) cytoskeleton to the plasma membrane of a variety of cell types. Previous studies demonstrated that the COOH terminus of the desmosomal plaque protein, desmoplakin (DP), is required for the association of DP with IF networks in cultured cells and that this domain interacts directly with type II epidermal keratin polypeptides in vitro. However, these studies left open the question of how desmosomes might anchor other IF types known to associate with these junctions. In this report we used yeast two-hybrid and in vitro dot blot assays to further examine the requirements for direct interactions between desmoplakin and various IF types. Our results confirm the ability of the DP COOH terminus (DPCT) to interact with at least two regions of the head domain of the type II epidermal keratin K1 and also demonstrate that DPCT can interact with the type III IF family members, vimentin and desmin, as well as simple epithelial keratins. Unlike the situation for type II epidermal keratins, the interaction between DPCT and simple epithelial keratins appears to depend on heterodimerization of the type I and II keratin polypeptides, since both are required to detect an interaction. Furthermore, although the interaction between DPCT and K1 requires the keratin head domain, deletion of this domain from the simple epithelial keratins does not compromise interaction with DPCT. The interaction between DPCT and type III or simple epithelial keratins also appeared to be less robust than that between DPCT and K1. In the case of K8/K18, however, the interaction as assessed by yeast two-hybrid assays increased 9-fold when a serine located in a protein kinase A consensus phosphorylation site 23 residues from the end of DP was altered to a glycine. Taken together, these data indicate that DP interacts directly with different IF types in specific ways.

We examined regions of human lamins A and C involved in binding to surfaces of mitotic chromosomes. An Escherichia coli expression system was used to produce full-length lamin A and lamin C, and truncated lamins retaining the central alpha-helical rod domain (residues 34-388) but lacking various amounts of the amino-terminal 'head' and carboxy-terminal 'tail' domains. We found that lamin A, lamin C and lamin fragments lacking the head domain and tail sequences distal to residue 431 efficiently assembled into paracrystals and strongly associated with mitotic chromosomes. Furthermore, the lamin rod domain also associated with chromosomes, although efficient chromosome coating required the pH 5-6 conditions needed to assemble the rod into higher order structures. Biochemical assays showed that chromosomes substantially reduced the critical concentration for assembly of lamin polypeptides into pelletable structures. Association of the lamin rod with chromosomes was abolished by pretrypsinization of chromosomes, and was not seen for vimentin (which possesses a similar rod domain). These data demonstrate that the alpha-helical rod of lamins A and C contains a specific chromosome binding site. Hence, the central rod domain of intermediate filament proteins can be involved in interactions with other cellular structures as well as in filament assembly.

Perikaryal collections of intermediate filaments have been described in the anterior horn motoneurons of patients with amyotrophic lateral sclerosis (ALS), but these inclusions have generally been considered rare and mainly associated with the familial form of ALS. Using the monoclonal antibody NF2F11, which recognizes phosphorylated neurofilament epitopes, we showed that focal collections of neurofilaments in anterior horn motoneurons were a characteristic finding in sporadic as well as in familial ALS; they were present in seven of nine ALS patients, but in none of nine control spinal cords. These neurofilamentous collections are not cross-reactive with antibodies directed against paired helical filaments and the microtubule associated protein tau. In addition, diffuse staining for phosphorylated neurofilament epitopes in chromatolytic anterior horn perikarya was significantly more frequent in ALS patients than in controls.

The genetic basis of a number of inherited cardiovascular diseases has been elucidated over the last few years, including the long QT syndromes, hypertrophic cardiomyopathy and dilated cardiomyopathy. While genetic heterogeneity has been demonstrated in most of these diseases, a pattern has emerged, specifically that genes encoding proteins with similar functions or involved in the same pathway are responsible for a particular disease or syndrome. Based on this observation we proposed the "final common pathway" hypothesis. In the case of the arrhythmogenic disorders, the long QT syndromes and Brugada syndrome, mutations have been described in a number of ion channel proteins, including cardiac potassium (KVLQT1, HERG and minK) and sodium (SCN5A) channels. Thus, using the "final common pathway" hypothesis we have proposed these diseases to be "ion channelopathies". Hypertrophic cardiomyopathy appears to be a disease of the sarcomere ("sarcomyopathy") since all the disease-causing mutations have been identified in the gene encoding many of the sarcomeric proteins, including beta-myosin heavy chain, alpha-tropomyosin, troponin I and troponin T, as well as in actin, close to the beta-myosin heavy chain binding site. The genes responsible for familial dilated cardiomyopathy have been less well characterized. For X-linked dilated cardiomyopathy, mutations in the dystrophin and G4.5 genes have been reported. In addition, mutations in actin (close to the dystrophin binding domain) and desmin, a component of the intermediate filaments, have been reported. However, the genes at a further 6 loci associated with autosomal dominant dilated cardiomyopathy (associated with conduction disease in 2 cases) remain unidentified. Due to the mutations in dystrophin, actin and desmin, we have proposed that dilated cardiomyopathy is a "cytoskeletalopathy", and we are currently investigating the involvement of these genes in patients.

In animal cells the nuclear lamina, which consists of lamins and lamin-associated proteins, serves several functions: it provides a structural scaffold for the nuclear envelope and tethers proteins and heterochromatin to the nuclear periphery. In yeast, proteins and large heterochromatic domains including telomeres are also peripherally localized, but there is no evidence that yeast have lamins or a fibrous nuclear envelope scaffold. Nonetheless, we found that the Lem2 and Man1 proteins of the fission yeast Schizosaccharomyces pombe, evolutionarily distant relatives of the Lap2/Emerin/Man1 (LEM) sub-family of animal cell lamin-associated proteins, perform fundamental functions of the animal cell lamina. These integral inner nuclear membrane localized proteins, with nuclear localized DNA binding Helix-Extension-Helix (HEH) domains, impact nuclear envelope structure and integrity, are essential for the enrichment of telomeres at the nuclear periphery and by means of their HEH domains anchor chromatin, most likely transcriptionally repressed heterochromatin, to the nuclear periphery. These data indicate that the core functions of the nuclear lamina are conserved between fungi and animal cells and can be performed in fission yeast, without lamins or other intermediate filament proteins.

The inner face of the nuclear envelope of metazoan cells is covered by a thin lamina consisting of a one-layered network of intermediate filaments interconnecting with a complex set of transmembrane proteins and chromatin associating factors. The constituent proteins, the lamins, have recently gained tremendous recognition, because mutations in the lamin A gene, LMNA, are the cause of a complex group of at least 10 different diseases in human, including the Hutchinson-Gilford progeria syndrome. The analysis of these disease entities has made it clear that besides cytoskeletal functions, the lamina has an important role in the "behaviour" of the genome and is, probably as a consequence of this function, intimately involved in cell fate decisions. Furthermore, these functions are related to the involvement of lamins in organizing the position and functional state of interphase chromosomes as well as to the occurrence of lamins and lamina-associated proteins within the nucleoplasm. However, the structural features of these lamins and the nature of the factors that assist them in genome organization present an exciting challenge to modern biochemistry and cell biology.

cDNA clones for nuclear pore complex glycoprotein p62 of two distantly related species, mouse and Xenopus laevis, were isolated. Antibodies raised against recombinant murine p62 react on protein blots with p62 of both species and decorate pore complexes. Analysis of the predicted protein sequence indicates that vertebrate p62 is organized into two structurally different regions. The entire carboxy-terminal half (86.7% identical amino acids) and the amino-terminal 56 amino acids (62.5% identity) have been highly conserved during evolution. The amino-terminal half contains several penta amino acid repeats and is able to form beta-sheets, whereas the carboxy-terminal half is predominantly organized in alpha-helical structures in part with heptad repeats typical for intermediate filament proteins. p62 of mouse and Xenopus is glycosylated by N-acetylglucosamine additions in the amino-terminal half. The region containing these potential glycosylation sites has been identified.

Hyperphosphorylated microtubule-associated protein tau is the major proteinaceous component of the paired helical and straight filaments which constitute a defining neuropathological characteristic of Alzheimer's disease and a number of other neurodegenerative disorders. We have recently shown that full-length recombinant tau assembles into Alzheimer-like filaments upon incubation with heparin. Heparin also promotes phosphorylation of tau by a number of protein kinases, prevents tau from binding to taxol-stabilized microtubules, and produces rapid disassembly of microtubules assembled from tau and tubulin. Here, we have used the above parameters to study the interactions between tau protein and a number of naturally occurring and synthetic glycosaminoglycans. We show that the magnitude of the glycosaminoglycan effects is proportional to their degree of sulfation. Thus, the strongly sulfated glycosaminoglycans dextran sulfate, pentosan polysulfate, and heparin were the most potent, whereas the non-sulfated dextran and hyaluronic acid were without effect. The moderately sulfated glycosaminoglycans heparan sulfate, chondroitin sulfate, and dermatan sulfate had intermediate effects, whereas keratan sulfate had little or no effect. These in vitro interactions between tau protein and sulfated glycosaminoglycans reproduced the known characteristics of paired helical filament-tau from Alzheimer's disease brain. Sulfated glycosaminoglycans are present in nerve cells in Alzheimer's disease brain in the early stages of neurofibrillary degeneration, suggesting that their interactions with tau may constitute a central event in the development of the neuronal pathology of Alzheimer's disease.

Plakoglobin is a protein closely related to beta-catenin that links desmosomal cadherins to intermediate filaments. Plakoglobin can also substitute for beta-catenin in adherens junctions, providing a connection between E-cadherin and alpha-catenin. Association of beta-catenin with E-cadherin and alpha-catenin is regulated by phosphorylation of specific tyrosine residues; modification of beta-catenin Tyr654 and Tyr142 decreases binding to E-cadherin and alpha-catenin, respectively. We show here that plakoglobin can also be phosphorylated on tyrosine residues, but unlike beta-catenin, this modification is not always associated with disrupted association with junctional components. Protein tyrosine kinases present distinct specificities on beta-catenin and plakoglobin, and phosphorylation of beta-catenin-equivalent Tyr residues of plakoglobin affects its interaction with components of desmosomes or adherens junctions differently. For instance, Src, which mainly phosphorylates Tyr86 in beta-catenin, modifies Tyr643 in plakoglobin, decreasing the interaction with E-cadherin and alpha-catenin and increasing the interaction with the alpha-catenin-equivalent protein in desmosomes, desmoplakin. The tyrosine kinase Fer, which modifies beta-catenin Tyr142, lessening its association with alpha-catenin, phosphorylates plakoglobin Tyr549 and exerts the contrary effect: it raises the binding of plakoglobin to alpha-catenin. These results suggest that tyrosine kinases like Src or Fer modulate desmosomes and adherens junctions differently. Our results also indicate that phosphorylation of Tyr549 and the increased binding of plakoglobin to components of adherens junctions can contribute to the upregulation of the transcriptional activity of the beta-catenin-Tcf-4 complex observed in many epithelial tumor cells.

CLIP-170 is a microtubule-binding protein isolated from HeLa cells that is involved in the interaction of endosomes with microtubules. The basic N-terminal domain of CLIP-170 binds to microtubules in vitro. To characterize further the functional domains of this cytoplasmic linker protein, we have transiently expressed intact and mutant forms of CLIP-170 in mammalian cells (HeLa and Vero cells) and show that the tandem repeat present in the N-terminal domain is essential for its binding to microtubules in vivo as previously found in vitro. With increasing levels of expression of CLIP-170, the sites with which the peripheral ends of microtubules interact enlarge, eventually forming large patches, which finally lead to the apparent bundling of microtubules. These patches do not form when the C-terminal domain is absent from the transfected protein. Modification of the microtubule-binding region, particularly of the tandem repeat motif, modulates the binding of CLIP-170 to microtubules. Overexpressed CLIP-170 appears neither to interact with nor to influence the organization of the intermediate filaments, and collapsing the network of intermediate filaments with microinjected antibodies against vimentin has no effect on the distribution of CLIP-170. These data suggest that CLIP-170 has at least two functional domains in vivo, an N-terminal microtubule-binding domain, and a C-terminal domain that is involved in the anchoring of microtubules to peripheral cytoplasmic structures.

Loss of the astrocyte-specific intermediate filament protein, glial fibrillary acidic protein (GFAP) results in an increased susceptibility to ischemic insult, enhanced hippocampal LTP, and decreased cerebellar long-term depression (LTD). Because glutamate receptor activation plays a key role in cell death and cellular plasticity responses, we wanted to determine if alterations in glial glutamate transport could contribute to the GFAP null phenotype. To address functional changes in glutamate transport, we measured glutamate uptake in cortical, cerebellar, and hippocampal synaptosomal preparations from age-matched adult wild type and GFAP null mice and demonstrated a 25-30% reduction in the V(max) for d-aspartate uptake in the cortex and hippocampus of GFAP null animals. Western blot analysis of cortical synaptosomal fractions from wild type and GFAP null animals demonstrated that loss of GFAP results in decreases in both astrocytic (EAAT1) and neuronal (EAAT3) glutamate transporter subtypes. Immunohistochemical analysis demonstrated a region-specific modification of neuronal glutamate transporter, EAAT3 trafficking in the GFAP null phenotype. Analysis of primary cortical astrocyte cultures prepared from GFAP null and wild type mice demonstrated that loss of GFAP results in an inability to traffic the glial glutamate transporter, EAAT2, to the surface of the cell following protein kinase A (PKA) stimulation by dibutyryl cAMP. Taken together, these results suggest that the intermediate filament protein, GFAP plays a key role in modulating astrocytic and neuronal glutamate transporter trafficking and function.

Intermediate filaments (IFs), in addition to microtubules and microfilaments, are one of the three major components of the cytoskeleton in eukaryotic cells, playing a vital role in mechanotransduction and in providing mechanical stability to cells. Despite the importance of IF mechanics for cell biology and cell mechanics, the structural basis for their mechanical properties remains unknown. Specifically, our understanding of fundamental filament properties, such as the basis for their great extensibility, stiffening properties, and their exceptional mechanical resilience remains limited. This has prevented us from answering fundamental structure-function relationship questions related to the biomechanical role of intermediate filaments, which is crucial to link structure and function in the protein material's biological context. Here we utilize an atomistic-level model of the human vimentin dimer and tetramer to study their response to mechanical tensile stress, and describe a detailed analysis of the mechanical properties and associated deformation mechanisms. We observe a transition from alpha-helices to beta-sheets with subsequent interdimer sliding under mechanical deformation, which has been inferred previously from experimental results. By upscaling our results we report, for the first time, a quantitative comparison to experimental results of IF nanomechanics, showing good agreement. Through the identification of links between structures and deformation mechanisms at distinct hierarchical levels, we show that the multi-scale structure of IFs is crucial for their characteristic mechanical properties, in particular their ability to undergo severe deformation of approximately 300% strain without breaking, facilitated by a cascaded activation of a distinct deformation mechanisms operating at different levels. This process enables IFs to combine disparate properties such as mechanosensitivity, strength and deformability. Our results enable a new paradigm in studying biological and mechanical properties of IFs from an atomistic perspective, and lay the foundation to understanding how properties of individual protein molecules can have profound effects at larger length-scales.

The intermediate filament protein nestin is characterized by its specific expression during the development of neuronal and myogenic tissues. We identify nestin as a novel in vivo target for cdk5 and p35 kinase, a critical signaling determinant in development. Two cdk5-specific phosphorylation sites on nestin, Thr-1495 and Thr-316, were established, the latter of which was used as a marker for cdk5-specific phosphorylation in vivo. Ectopic expression of cdk5 and p35 in central nervous system progenitor cells and in myogenic precursor cells induced elevated phosphorylation and reorganization of nestin. The kinetics of nestin expression corresponded to elevated expression and activation of cdk5 during differentiation of myoblast cell cultures and during regeneration of skeletal muscle. In the myoblasts, a disassembly-linked phosphorylation of Thr-316 indicated active phosphorylation of nestin by cdk5. Moreover, cdk5 occurred in physical association with nestin. Inhibition of cdk5 activity-either by transfection with dominant-negative cdk5 or by using a specific cdk5 inhibitor-blocked myoblast differentiation and phosphorylation of nestin at Thr-316, and this inhibition markedly disturbed the organization of nestin. Interestingly, the interaction between p35, the cdk5 activator, and nestin appeared to be regulated by cdk5. In differentiating myoblasts, p35 was not complexed with nestin phosphorylated at Thr-316, and inhibition of cdk5 activity during differentiation induced a marked association of p35 with nestin. These results demonstrate that there is a continuous turnover of cdk5 and p35 activity on a scaffold formed by nestin. This association is likely to affect the organization and operation of both cdk5 and nestin during development.

The origin of introns and their role (if any) in gene expression, in the evolution of the genome, and in the generation of new expressed sequences are issues that are understood poorly, if at all. Multigene families provide a favorable opportunity for examining the evolutionary history of introns because it is possible to identify changes in intron placement and content since the divergence of family members from a common ancestral sequence. Here we report the complete sequence of the gene encoding the 68-kilodalton (kDa) neurofilament protein; the gene is a member of the intermediate filament multigene family that diverged over 600 million years ago. Five other members of this family (desmin, vimentin, glial fibrillary acidic protein, and type I and type II keratins) are encoded by genes with six or more introns at homologous positions. To our surprise, the number and placement of introns in the 68-kDa neurofilament protein gene were completely anomalous, with only three introns, none of which corresponded in position to introns in any characterized intermediate filament gene. This finding was all the more unexpected because comparative amino acid sequence data suggest a closer relationship of the 68-kDa neurofilament protein to desmin, vimentin, and glial fibrillary acidic protein than between any of these three proteins and the keratins. It appears likely that an mRNA-mediated transposition event was involved in the evolution of the 68-kDa neurofilament protein gene and that subsequent events led to the acquisition of at least two of the three introns present in the contemporary sequence.

As a first step towards isolation of autophagic sequestering membranes (phagophores), we have purified autophagosomes from rat hepatocytes. Lysosomes were selectively destroyed by osmotic rupture, achieved by incubation of hepatocyte homogenates with the cathepsin C substrate glycyl-phenylalanyl-naphthylamide (GPN). Mitochondria and peroxisomes were removed by Nycodenz gradient centrifugation, and cytosol, microsomes and other organelles by rate sedimentation through metrizamide cushions. The purified autophagosomes were bordered by dual or multiple concentric membranes, suggesting that autophagic sequestration might be performed either by single autophagic cisternae or by cisternal stacks. Okadaic acid, a protein phosphatase inhibitor, disrupted the hepatocytic cytokeratin network and inhibited autophagy completely in intact hepatocytes, perhaps suggesting that autophagy might be dependent on intact intermediate filaments. Vinblastine and cytochalasin D, which specifically disrupted microtubules and microfilaments, respectively, had relatively little (25-30%) inhibitory effect on autophagic sequestration. In a cryo-ultrastructural study, the various autophagic-lysosomal vacuoles were immunogold-labelled, using the cytosolic enzyme superoxide dismutase as an autophagic marker, Lgp120 as a lysosomal membrane marker, and bovine serum albumin as an endocytic marker. Vinblastine (50 microM) was found to inhibit both autophagic and endocytic flux into the lysosomes, with a consequent reduction in lysosomal size. Asparagine (20 mM) caused swelling of the lysosomes, probably as a result of the ammonia formation that could be observed at this high asparagine concentration. Autophagosomes and amphisomes (autophagic-endocytic, prelysosomal vacuoles) accumulated in asparagine-treated cells, reflecting an inhibition of autophagic flux that might be a consequence of lysosomal dysfunction.

Cell adhesion, spreading and migration on extracellular matrices are regulated by complex processes that involve the cytoskeleton and a large array of adhesion receptors, including the β1 integrin. Filamin A is a large, multi-domain, homodimeric actin binding protein that contributes to the mechanical stability of cells and interacts with several proteins that regulate cell adhesion including β1 integrin and several protein kinases. Here we review current data on the structure, mechanical properties and intracellular signaling functions of filamin that regulate cell adhesion. We also consider new data showing that interactions of filamin A with intermediate filaments and protein kinase C enable tight regulation of β1 integrin function and consequently early events in cell adhesion and migration on extracellular matrix proteins.