Specialized cytoskeletons play many fascinating roles, including mechanical integrity and wound-healing in epidermal cells, cell polarity in simple epithelia, contraction in muscle cells, hearing and balance in the inner ear cells, axonal transport in neurons, and neuromuscular junction formation between muscle cells and motor neurons. These varied functions are dependent upon cytoplasmic networks of actin microfilaments (6 nm), intermediate filaments (10 nm) and microtubules (23 nm), and their many associated proteins. In this chapter, I review what is known about the cytoskeletons of intermediate filaments and their associated proteins. I focus largely on epidermal cells, which devote most of their protein-synthesizing machinery to producing an extensive intermediate filament network composed of keratin. Recent studies have shown that many of the devastating human disorders that arise from degeneration of this cell type have as their underlying basis either defects in the genes encoding keratins or abnormalities in keratin IF networks. I discuss what we know about the functions of IFs, and how the link to genetic disease has enhanced this understanding.

BACKGROUND

Intracytoplasmic inclusion bodies (ICI) have been identified in ciliated bronchial epithelium of Kawasaki disease (KD) patients using a synthetic antibody derived from acute KD arterial IgA plasma cells; ICI may derive from the KD etiologic agent.

METHODS

Acute KD bronchial epithelium was subjected to immunofluorescence for ICI and cytokeratin, high-throughput sequencing, and transmission electron microscopy (TEM). Interferon pathway gene expression profiling was performed on KD lung.

RESULTS

An intermediate filament cytokeratin "cage" was not observed around KD ICI, making it unlikely that ICI are overproduced or misfolded human protein aggregates. Many interferon-stimulated genes were detected in the bronchial epithelium, and significant modulation of the interferon response pathway was observed in the lung tissue of KD patients. No known virus was identified by sequencing. Aggregates of virus-like particles (VLP) were detected by TEM in all 3 acute KD patients from whom nonembedded formalin-fixed lung tissue was available.

CONCLUSIONS

KD ICI are most likely virus induced; bronchial cells with ICI contain VLP that share morphologic features among several different RNA viral families. Expedited autopsies and tissue fixation from acute KD fatalities are urgently needed to more clearly ascertain the VLP. These findings are compatible with the hypothesis that the infectious etiologic agent of KD may be a "new" RNA virus.

The role of the different epithelial compartments during degeneration and regeneration of the rat prostate is examined on basis of intermediate filament protein (IFP) expression pattern. With the monoclonal antibodies RCK 103 and RGE 53, directed against specific keratins, it was possible to differentiate between the basal (RCK 103+) and luminal (RGE 53+) cells of the prostatic epithelium. After testosterone deprivation, by orchiectomy, an extensive and rapid cell loss was observed which appeared to affect mainly the luminal cells. In the process of prostate regeneration, induced by testosterone administration, using silastic implants, the luminal compartment rapidly regained its normal thickness. A heterogeneous population of morphologically luminal cells was observed showing keratin expression patterns intermediate between basal and luminal cells. These findings support the idea of a relationship between basal and luminal cells as being members of the same lineage of differentiation.

Certain glia cells, notably astrocytes and tumor cells derived therefrom, express simultaneously two types of proteins of intermediate-sized filaments, vimentin and glia filament protein (GFP). We have used an established human glioma (astrocytoma) cell culture line (U 333 CG/343 MG) in which both proteins are seen in partly overlapping fibrillar structures by immunofluorescence microscopy, to examine the possible existence of heteropolymer filaments of these two proteins by using reversible oxidative cross-linking facilitated by the 1,10-phenanthroline-cupric ion complex. Dimeric cross-link products are characterized by one-dimensional and two-dimensional gel electrophoresis under non-reducing and reducing conditions as well as by peptide mapping. The relatively large proportions of heterodimers of vimentin and GFP obtained in cytoskeletal filaments cross-linked in this way, demonstrate the frequency of heteropolymer filaments in this cell as well as the frequency of face-to-face 'pairs' of GFP and vimentin in such filaments. Together with our related observations on heteropolymer filaments between vimentin and desmin in some smooth muscle cells [Quinlan, R. A. and Franke, W. W. (1982) Proc. Natl Acad. Sci. USA, 79, 3452-3456], we discuss this as evidence for common principles of molecular arrangements of vimentin, GFP and desmin, at least in the cysteine-containing surface domains. The results are also discussed in relation to cytoskeletal changes during glial differentiation.

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 alpha-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 A. 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.

The cytoskeleton is a complex of detergent-insoluble components of the cytoplasm playing critical roles in cell motility, shape generation, and mechanical properties of a cell. Fibrillar polymers-actin filaments, microtubules, and intermediate filaments- are major constituents of the cytoskeleton, which constantly change their organization during cellular activities. The actin cytoskeleton is especially polymorphic, as actin filaments can form multiple higher order assemblies performing different functions. Structural information about cytoskeleton organization is critical for understanding its functions and mechanisms underlying various forms of cellular activity. Because of the nanometer-scale thickness of cytoskeletal fibers, electron microscopy (EM) is a key tool to determine the structure of the cytoskeleton.This article describes application of rotary shadowing (or metal replica) EM for visualization of the cytoskeleton. The procedure is applicable to thin cultured cells growing on glass coverslips and consists of detergent extraction of cells to expose their cytoskeleton, chemical fixation to provide stability, ethanol dehydration and critical point drying to preserve three-dimensionality, rotary shadowing with platinum to create contrast, and carbon coating to stabilize replicas. This technique provides easily interpretable three-dimensional images, in which individual cytoskeletal fibers are clearly resolved, and individual proteins can be identified by immunogold labeling. More importantly, replica EM is easily compatible with live cell imaging, so that one can correlate the dynamics of a cell or its components, e.g., expressed fluorescent proteins, with high resolution structural organization of the cytoskeleton in the same cell.

Intermediate filament networks in the cytoplasm and nucleus are critical for the mechanical integrity of metazoan cells. However, the mechanism of crosslinking in these networks and the origins of their mechanical properties are not understood. Here, we study the elastic behavior of in vitro networks of the intermediate filament protein vimentin. Rheological experiments reveal that vimentin networks stiffen with increasing concentrations of Ca(2+) and Mg(2+), showing that divalent cations act as crosslinkers. We quantitatively describe the elastic response of vimentin networks over five decades of applied stress using a theory that treats the divalent cations as crosslinkers: at low stress, the behavior is entropic in origin, and increasing stress pulls out thermal fluctuations from single filaments, giving rise to a nonlinear response; at high stress, enthalpic stretching of individual filaments significantly modifies the nonlinearity. We investigate the elastic properties of networks formed by a series of protein variants with stepwise tail truncations and find that the last 11 amino acids of the C-terminal tail domain mediate crosslinking by divalent ions. We determined the single-filament persistence length, l(P) approximately 0.5 mum, and Young's modulus, Y approximately 9 MPa; both are consistent with literature values. Our results provide insight into a crosslinking mechanism for vimentin networks and suggest that divalent ions may help regulate the cytoskeletal structure and mechanical properties of cells.

Astrocytes play many pivotal roles in the adult brain, including their reaction to injury. A hallmark of astrocytes is the contact of their endfeet with the basement membrane surrounding blood vessels, but still relatively little is known about the signaling mediated at the contact site. Here, we examine the role of beta1-integrin at this interface by its conditional deletion using different Cre lines. Thereby, the protein was reduced only at postnatal stages either in both glia and neurons or specifically only in neurons. Strikingly, only the former resulted in reactive gliosis, with the hallmarks of reactive astrocytes comprising astrocyte hypertrophy and up-regulation of the intermediate filaments GFAP and vimentin as well as pericellular components, such as Tenascin-C and the DSD-1 proteoglycan. In addition, we also observed to a certain degree a non-cell autonomous activation of microglial cells after conditional beta1-integrin deletion. However, these reactive astrocytes did not divide, suggesting that the loss of beta1-integrin-mediated signaling is not sufficient to elicit proliferation of these cells as observed after brain injury. Interestingly, this partial reactive gliosis appeared in the absence of cell death and blood brain barrier disturbances. As these effects did not appear after neuron-specific deletion of beta1-integrin, we conclude that beta1-integrin-mediated signaling in astrocytes is required to promote their acquisition of a mature, nonreactive state. Alterations in beta1-integrin-mediated signaling may hence be implicated in eliciting specific aspects of reactive gliosis after injury.

Cell adhesion and spreading are regulated by complex interactions involving the cytoskeleton and extracellular matrix proteins. We examined the interaction of the intermediate filament protein vimentin with the actin cross-linking protein filamin A in regulation of spreading in HEK-293 and 3T3 cells. Filamin A and vimentin-expressing cells were well spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, cells treated with small interfering RNA (siRNA) to knock down filamin A or vimentin were poorly spread; both of these cell populations exhibited >50% reductions of cell adhesion, cell surface beta1 integrin expression, and beta1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions, whereas knockdown of filamin and/or vimentin inhibited the formation of cell extensions. Reduced vimentin phosphorylation, cell spreading, and beta1 integrin surface expression, and activation were phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase C-epsilon. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins, we found an association between filamin A and vimentin. Filamin A also associated with protein kinase C-epsilon, which was enriched in cell extensions. These data indicate that filamin A associates with vimentin and to protein kinase C-epsilon, thereby enabling vimentin phosphorylation, which is important for beta1 integrin activation and cell spreading on collagen.

OBJECTIVE

To determine the roles of glial fibrillary acidic protein (GFAP) and vimentin in Müller cell reactivity.

METHODS

Retinal detachments were created in mice deficient for GFAP and vimentin (GFAP(-/-)vim(-/-)) and age-matched wild-type (wt) mice. The reactivity of the retina was studied by immunofluorescence and electron microscopy.

RESULTS

Müller cell morphology was different and glutamine synthetase immunoreactivity was reduced in the undisturbed GFAP(-/-)vim(-/-) retinas. After retinal detachment, Müller cells formed subretinal glial scars in the wt mice. In contrast, such scars were not observed in GFAP(-/-)vim(-/-) mice. Müller cells, which normally elongate and thicken in response to detachment, appeared compressed, thin, and "spikey" in the GFAP(-/-)vim(-/-) mice. The end foot region of Müller cells in the GFAP(-/-)vim(-/-) mice often sheared away from the rest of the retina during detachment, corroborating earlier results showing decreased resistance of this region in GFAP(-/-)vim(-/-) retinas to mechanical stress. In regions with end foot shearing, ganglion cells showed intense neurite sprouting, as revealed by anti-neurofilament labeling, a response rarely observed in wt mice.

CONCLUSIONS

Müller cells are subtly different in the GFAP(-/-)vim(-/-) mouse retina before detachment. The end foot region of these cells may be structurally reinforced by the presence of the intermediate filament cytoskeleton, and our data suggest a critical role for these proteins in Müller cell reaction to retinal detachment and participation in subretinal gliosis.

Mechanical responsiveness is essential to all biological systems down to the level of tissues and cells. The intra- and extracellular mechanics of such systems are governed by a series of proteins, such as microtubules, actin, intermediate filaments and collagen. As a general design motif, these proteins self-assemble into helical structures and superstructures that differ in diameter and persistence length to cover the full mechanical spectrum. Gels of cytoskeletal proteins display particular mechanical responses (stress stiffening) that until now have been absent in synthetic polymeric and low-molar-mass gels. Here we present synthetic gels that mimic in nearly all aspects gels prepared from intermediate filaments. They are prepared from polyisocyanopeptides grafted with oligo(ethylene glycol) side chains. These responsive polymers possess a stiff and helical architecture, and show a tunable thermal transition where the chains bundle together to generate transparent gels at extremely low concentrations. Using characterization techniques operating at different length scales (for example, macroscopic rheology, atomic force microscopy and molecular force spectroscopy) combined with an appropriate theoretical network model, we establish the hierarchical relationship between the bulk mechanical properties and the single-molecule parameters. Our results show that to develop artificial cytoskeletal or extracellular matrix mimics, the essential design parameters are not only the molecular stiffness, but also the extent of bundling. In contrast to the peptidic materials, our polyisocyanide polymers are readily modified, giving a starting point for functional biomimetic hydrogels with potentially a wide variety of applications, in particular in the biomedical field.

The displacement loop (D loop) is the product of homology search and DNA strand invasion, constituting a central intermediate in homologous recombination (HR). In eukaryotes, the Rad51 DNA strand exchange protein is assisted in D loop formation by the Rad54 motor protein. Curiously, Rad54 also disrupts D loops. How these opposing activities are coordinated toward productive recombination is unknown. Moreover, a seemingly disparate function of Rad54 is removal of Rad51 from heteroduplex DNA (hDNA) to allow HR-associated DNA synthesis. Here, we uncover features of D loop formation/dissociation dynamics, employing Rad51 filaments formed on ssDNAs that mimic the physiological length and structure of in vivo substrates. The Rad54 motor is activated by Rad51 bound to synapsed DNAs and guided by a ssDNA-binding domain. We present a unified model wherein Rad54 acts as an hDNA pump that drives D loop formation while simultaneously removing Rad51 from hDNA, consolidating both ATP-dependent activities of Rad54 into a single mechanistic step.

The roles of the different molecular domains of intermediate filament (IF) proteins in the assembly and higher order organization of IF structures have recently been studied by various groups but with partially controversial results. To examine the requirement of the aminoterminal (head) and the carboxyterminal (tail) domain of cytokeratins (CKs) for de novo IF formation in the living cell, we have constructed cDNAs coding for intact as well as head- and/or tail-less human CKs 8 and 18 and the naturally tail-less human CK 19, all under the control of the human beta-actin promoter. After transient and stable transfections of mouse 3T3-L1 cells, which are devoid of any CKs, we have studied, with such constructs, the resulting gene products by gel electrophoresis and immunolocalization techniques. By light and electron microscopy we show that extended cytoplasmic IF meshworks are formed from pairs of the type II CK 8 with the type I CKs 18 or 19 as well as from pairs of tail-less CK 8 with tail-less CKs 18 or 19 in the transfected cells, proving that the absence of the tail domain in both types of CKs does not prevent the de novo formation of regular IFs. Most surprisingly, however, we have observed spectacular alterations in the nucleocytoplasmic distribution of the IFs formed from tail-less CKs. In many of the transfected cells, a large part, or all, of the detectable CKs was found to occur in extensive IF bundles in the nucleoplasm. Intranuclear accumulations of CK deposits, however mostly nonfibrillar, were also observed when the cells had been transfected with cDNAs encoding tail-less CKs also lacking their head domains, whereas CKs deleted only in the head domain were found exclusively in the cytoplasm. The specific domain requirements for the assembly of cytoplasmic IF bundles are discussed and possible mechanisms of intranuclear accumulation of IFs are proposed.

Vimentin intermediate filaments undergo spatial reorganization in cultured smooth muscle cells in response to contractile activation; however, the role of vimentin in the physiological properties of smooth muscle has not been well elucidated. Tracheal smooth muscle strips were loaded with antisense oligonucleotides (ODNs) against vimentin and then cultured for 2 days to allow for protein degradation. Treatment with vimentin antisense, but not sense, ODNs suppressed vimentin protein expression; neither vimentin antisense nor sense ODNs affected protein levels of desmin and actin. Force development in response to ACh stimulation or KCl depolarization was lower in vimentin-deficient tissues than in vimentin sense ODN- or non-ODN-treated muscle strips. Passive tension was also depressed in vimentin-depleted muscle tissues. Vimentin downregulation did not attenuate increases in myosin light chain (MLC) phosphorylation in response to contractile stimulation or basal MLC phosphorylation. In vimentin sense ODN-treated or non-ODN-treated smooth muscle strips, the desmosomal protein plakoglobin was primarily localized in the cell periphery. The membrane-associated localization of plakoglobin was reduced in vimentin-depleted muscle tissues. These studies suggest that vimentin filaments play an important role in mediating active force development and passive tension, which are not regulated by MLC phosphorylation. Vimentin downregulation impairs the structural organization of desmosomes, which may be associated with the decrease in force development.

Intermediate filament proteins form an essential part of the cytoskeleton and provide topological order to cells and tissues. These features result from their intrinsic property of self-organization and their response to extrinsic cues. Keratins represent the largest subgroup among all intermediate filament proteins and are differentially expressed as pairs of type I and type II intermediate filament proteins in epithelia. Their primary function is to impart mechanical strength to cells. This function is illustrated by patients with keratin mutations and by gene-deficient mice. Additional functions include their participation in the response to stress, cell signalling and apoptosis, and thus the keratin cytoskeleton appears far more dynamic than previously anticipated. This may result from hyperphosphorylation and possibly from interaction with associated proteins. How signalling networks affect keratin organization, turnover and function and vice versa will be a major challenge for future investigations.

We studied the localization of desmin (skeletin), the major protein subunit of muscle-type intermediate filaments, in adult chicken cardiac muscle by high resolution immunoelectron microscopic labeling of ultrathin frozen sections of the intact fixed tissues. We carried out single labeling for desmin and double labeling for both desmin and either vinculin or alpha-actinin. In areas removed from the intercalated disk membranes, we observed desmin labeling between adjacent Z-bands in every interfibrillar space. Where these spaces were wide and contained mitochondria, convoluted strands of desmin labeling bridged between the periphery of neighboring Z-bands and the mitochondria. The intermediate filaments appeared to be organized in a more three-dimensional manner within the interfibrillar spaces of cardiac as compared to skeletal muscle. Near the intercalated disks, desmin labeling was intense within the interfibrillar spaces, but was completely segregated from the microfilament attachment sites (fascia adherens) where vinculin and alpha-actinin were localized. Desmin therefore appears to play no role in the attachment of microfilaments to the intercalated disk membrane. We discuss the role of intermediate filaments in the organization of cardiac and skeletal striated muscle in the light of these and other results.

Considerable sequence data have been collected from the intermediate filament proteins and other alpha-fibrous proteins including myosin, tropomyosin, paramyosin, desmoplakin and M-protein. The data show that there is a clear preference for some amino acids to occur in specific positions within the heptad substructure that characterizes the sequences which form the coiled-coil rod domain in this class of proteins. The results also indicate that although there are major similarities between the various proteins there are also key differences. In all cases, however, significant regularities in the linear disposition of the acidic and the basic residues in the coiled-coil segments can be related to modes of chain and molecular aggregation. In particular a clear trend has been observed which relates the mode of molecular aggregation to the number of interchain ionic interactions per heptad pair.

The small heat shock protein alphaB-crystallin interacts with intermediate filament proteins. Using a co-sedimentation assay, we showed that in vitro binding of alphaB-crystallin to peripherin and vimentin was temperature-dependent. Specifically, a synthetic peptide representing the first ten residues of alphaB-crystallin was involved in this interaction. When cells were submitted to different stress conditions such as serum starvation, hypertonic stress, or heat shock, we observed a dynamic reorganisation of the intermediate filament network, and concomitant recruitment of alphaB-crystallins on intermediate filament proteins. Under normal conditions alphaB-crystallin was extracted from cells by detergent. In stressed cells, alphaB-crystallin colocalised with intermediate filament proteins, and became resistant to detergent extraction. The intracellular state of alphaB-crystallin seemed to correlate directly with the remodelling of the intermediate filament network in response to stress. This suggested that alphaB-crystallin functions as a molecular chaperone for intermediate filament proteins.

The spatial and temporal immunoexpression of the intermediate filament (IF) protein nestin and its relationship to glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and its receptor flt-1 (VEGF-R1) in reactive astroglia was examined following stab wounds or transplants of fetal CNS tissue into the adult brain. Since developmentally regulated proteins and gene transcripts can be reexpressed in reactive astroglia following certain brain injuries, we analyzed the nestin profile in these experimental paradigms in order to more fully understand the nature of the gliotic "scar." Nestin expression was transiently up-regulated in some but not all astrocytes which often had a different morphology than the typical stout, stellate GFAP (+) cells; the processes of the nestin (+) cells tended to be slender and elongated. In reactive astroglia from the mature brain, nestin expression was robust but generally localized to the wound or graft site, peaked at 7-10 days postoperative, and was absent by 28 days, whereas GFAP (+) astrocytes were far more widespread and persisted for many months. Only nestin was strongly expressed immediately adjacent to early stab wounds, whereas GFAP (+) cells were located further from the wound sites. In contrast, there was marked nestin/GFAP colocalization at the graft/host interface. Semiquantitative analysis combined with confocal microscopy revealed a unique compartmentalization of protein expression; processes from single astrocytes could be entirely nestin (+), GFAP (+), or could show coexpression. At 4, 7, and 14 days postoperative, 41, 58, and 32% of the immunoexpression, respectively, was accounted for by nestin at the graft/host interface, and it was essentially undetectable at 28 days postoperative. In situ hybridization studies showed nestin transcripts within GFAP (+) cells primarily between 4 and 10 days postoperative and absent by 28 days. Many nestin (+) astrocytes, as shown by electron microscopy, were closely related to the vasculature. Therefore we further examined the expression of vascular endothelial growth factor (VEGF), an endothelial cell mitogen associated with angiogenesis. Nestin colocalized with VEGF in some astrocytes (7%) but far more prominently with the VEGF flt-1 receptor (25%). Early astroglial activation may involve several different IF components and possibly a distinct astrocytic population that shows a rapid, transient nestin expression adjacent to injury sites. Expression of the nestin IF phenotype within affected astrocytes in the surgical vicinity may be indicative of a reversion to an immature phenotype that might be less susceptible to attendant hypoxia after injury. Since injured astrocytes are well known to express many bioactive compounds, such transient reexpression of early, developmentally regulated proteins may be a hallmark for the elaboration of growth factors such as VEGF.

Shear stress plays a significant role in endothelial cell biology and atherosclerosis development. Previous work by our group has shown that fluid flow stimulates important functional changes in cells through protein expression regulation. Peroxiredoxins (PRX) are a family of antioxidant enzymes but have yet to be investigated in response to shear stress. Studies have shown that oscillatory shear stress (OS) increases reactive oxygen species (ROS) levels in endothelial cells, whereas laminar shear stress (LS) blocks this response. We hypothesized that PRX are responsible for the anti-oxidative effect of LS. To test this hypothesis, bovine aortic endothelial cells (BAEC) were subjected to LS (15 dyn/cm(2)), OS (+/-5 dyn/cm(2), 1 Hz), or static conditions for 24 h. Using Western blot and immunofluorescence staining, all six isoforms of PRX were identified in BAEC. When compared with OS and static, exposure to chronic LS up-regulated PRX 1 levels intracellularly. LS also increased expression of PRX 5 relative to static controls, but not OS. PRX exhibited broad subcellular localization, with distribution in the cytoplasm, Golgi, mitochondria, and intermediate filaments. In addition, PRX 1 knock down, using specific small interference RNA, attenuated LS-dependent reactive oxygen species reduction in BAEC. However, PRX 5 depletion did not. Together, these results suggest that PRX 1 is a novel mechanosensitive antioxidant, playing an important role in shear-dependent regulation of endothelial biology and atherosclerosis.

Keratins 8 (K8) and 18 are the primary intermediate filaments of simple epithelia. Phosphorylation of keratins at specific sites affects their organization, assembly dynamics, and their interaction with signaling molecules. A number of keratin in vitro and in vivo phosphorylation sites have been identified. One example is K8 Ser-73, which has been implicated as an important phosphorylation site during mitosis, cell stress, and apoptosis. We show that K8 is strongly phosphorylated on Ser-73 upon stimulation of the pro-apoptotic cytokine receptor Fas/CD95/Apo-1 in HT-29 cells. Kinase assays showed that c-Jun N-terminal kinase (JNK) was also activated with activation kinetics corresponding to that of K8 phosphorylation. Furthermore, K8 was also phosphorylated on Ser-73 by JNK in vitro, yielding similar phosphopeptide maps as the in vivo phosphorylated material. In addition, co-immunoprecipitation studies revealed that part of JNK is associated with K8 in vivo, correlating with decreased ability of JNK to phosphorylate the endogenous c-Jun. Taken together, K8 is a new cytoplasmic target for JNK in Fas receptor-mediated signaling. The functional significance of this phosphorylation could relate to regulation of JNK signaling and/or regulation of keratin dynamics.

The distribution of plectin in the cytoplasm of Rat1 and glioma C6 cells was examined using a combination of double and triple immunofluorescence microscopy and interference reflection microscopy. In cells examined shortly after subcultivation (less than 48 h), filamentous networks of plectin structures, resembling and partially colocalizing with vimentin filaments, were observed as reported in previous studies. In cells kept attached to the substrate without growth for periods of 72 h to 8 days (stationary cultures), thick fibrillary plectin structures were observed. These structures were located at the end of actin filament bundles and showed co-distribution with adhesion plaques (focal contacts), vinculin, and vimentin. Only relatively large adhesion plaques (dash-like contacts) were decorated by antibodies to plectin, smaller dot-like contacts at the cell edges remained undecorated. Moreover, in stationary Rat1 cells plectin structures were found to be predominantly colocalized with actin stress fibers. However, after treatment of such cells with colcemid, plectin's distribution changed dramatically. The protein was no longer associated with actin structures, but was distributed diffusely throughout the cytoplasm. After a similar treatment with cytochalasin B, plectin's association with stress fibers again was completely abolished, although stress fibers were still present. The association of plectin with focal contact-associated intermediate filaments was demonstrated also by immunogold electron microscopy of quick-frozen, deep-etched replicas of rat embryo fibroblasts. These data confirm previous reports suggesting a relationship between intermediate filaments on the one hand, and actin stress fibers and their associated plasma membrane junctional complexes, on the other. Furthermore, the data establish plectin as a novel component of focal contact complexes and suggest that plectin plays a role as mediator between intermediate filaments and actin filaments.

Salmonella species translocate effector proteins into the host cell cytoplasm using a type III secretion system (TTSS). The translocation machinery probably contacts the eukaryotic cell plasma membrane to effect protein transfer. Data presented here demonstrate that both SspB and SspC, components of the translocation apparatus, are inserted into the epithelial cell plasma membrane 15 min after Salmonella typhimurium infection. In addition, a yeast two-hybrid interaction between SspC and an eukaryotic intermediate filament protein was identified. Three individual carboxyl-terminal point mutations within SspC that disrupt the yeast two-hybrid interaction were isolated. Strains expressing the mutant SspC alleles were defective for invasion, translocation of effector molecules and membrane localization of SspC. These data indicate that insertion of SspC into the plasma membrane of target cells is required for invasion and effector molecule translocation and that the carboxyl terminus of SspC is essential for these functions.

The chymotryptically excised middle domain of desmin slightly exceeds in length the structurally conserved alpha-helical middle region documented in all intermediate filament proteins by amino acid sequence data. This rod domain is a protofilament derivative with a tetrameric organization, thus indicating the presence of two double-stranded coiled-coil units. We now show by immunoelectron microscopy that Fab fragments of a desmin-specific monoclonal antibody mixed with the rod lead to dumb-bell-shaped structures. The tagging of both ends together with the length of the rod (48 nm) argues for an antiparallel orientation of the two coiled-coils without a major stagger. This information combined with the lateral 21 nm periodicity of the intermediate filament observed by us and others leads to a structural hypothesis similar to those entertained from X-ray data on wool alpha-keratins, although here an antiparallel tetrameric unit of some 60 to 66 nm is invoked, which has never been isolated. The structure that we discuss allows for the existence of both the particles, and the antibody experiment strongly supports the antiparallel orientation postulated in both approaches. The tube-like filament structure proposed for the intermediate filament agrees with recent mass per unit length measurements and allows for two minor classes of intermediate filaments with different values in this property as also found experimentally.