Keratin incorporation into intermediate filament networks is a rapid process

The properties of keratin-containing intermediate filament (IF) networks in vivo were studied following the microinjection of biotinylated keratin. Keratin-IFs were biotinylated, disassembled, and separated into type I and type II proteins by ion exchange chromatography. Recombination of these derivatized type I and type II keratins resulted in the formation of 10-nm diameter IF. The type I keratins were microinjected into epithelial cells and observed by immunofluorescence microscopy. Biotin-rich spots were found throughout the cytoplasm at 15-20 min after injection. Short biotinylated fibrous structures were seen at 30-45 min after injection, most of which colocalized with the endogenous bundles of IF (tono-filaments). By 1 1/2 to 2 h after microinjection, extensive biotinylated keratin IF-like networks were evident. These were highly coincident with the endogenous tonofilaments throughout the cell, including those at desmosomal junctions. These results suggest the existence of a relatively rapid subunit incorporation mechanism using numerous sites along the length of the endogenous tonofilament bundles. These observations support the idea that keratin-IFs are dynamic cytoskeletal elements.     

The 'ins' and 'outs' of intermediate filament organization

A major function shared by several types of cytoplasmic intermediate filaments (IFs) is to stabilize cellular architecture against the mechanical forces it is subjected to. As for other fibrous cytoskeletal arrays, a crucial determinant of this function is the spatial organization of IFs in the cytoplasm. However, very few crossbridging proteins are specific for IFs - most IF-associated proteins known to exert a structural role act to tether IFs to other major cytoskeletal elements, such as F-actin, microtubules or adhesion complexes. In addition, IFs are endowed with the ability to participate in their own organization. This intriguing property is probably connected to the unusual degree of sequence diversity and sequence-specific regulation that characterize IF genes and their proteins. This dependence upon a combination of extrinsic and intrinsic determinants contributes to distinguish IFs from other fibrous cytoskeletal polymers and is key to their function.     

Keratin intermediate filament chains in the European common wall lizard (Podarcis muralis) and a potential keratin filament crosslinker

On the basis of sequence homology with mammalian α-keratins, and on the criteria that the coiled-coil segments and central linker in the rod domain of these molecules must have conserved lengths if they are to assemble into viable intermediate filaments, a total of 28 Type I and Type II keratin intermediate filament chains (KIF) have been identified from the genome of the European common wall lizard (Podarcis muralis). Using the same criteria this number may be compared to 33 found here in the green anole lizard (Anole carolinensis) and 25 in the tuatara (Sphenodon punctatus). The Type I and Type II KIF genes in the wall lizard fall in clusters on chromosomes 13 and 2 respectively. Although some differences occur in the terminal domains in the KIF chains of the two lizards and tuatara, the similarities between key indicator residues – cysteine, glycine and proline – are significant. The terminal domains of the KIF chains in the wall lizard also contain sequence repeats commonly based on glycine and large apolar residues and would permit the fine tuning of physical properties when incorporated within the intermediate filaments. The H1 domain in the Type II chain is conserved across the lizards, tuatara and mammals, and has been related to its role in assembly at the 2–4 molecule level. A KIF-like chain (K80) with an extensive tail domain comprised of multiple tandem repeats has been identified as having a potential filament-crosslinking role.     

Cell type heterogeneity of intermediate filament expression in epithelia of the human pituitary gland

In the present study we have localized immunohistochemically the intermediate filament proteins of the human pituitary gland (adenohypophysis, pars intermedia and pars tuberalis) by an indirect immunoperoxidase technique or by double immunofluorescence methods and analysed the individual cytokeratin polypeptides using two-dimensional gel electrophoresis. We found that the expression of cytokeratins in different epithelial cells of the human anterior pituitary gland was heterogeneous. Whereas the endocrine cells only expressed cytokeratins 8 and 18, the folliculo-stellate cells exhibited a reactivity for cytokeratins 7, 8, 18 and 19 as well as for GFAP and vimentin. The squamous epithelial cells of the pars tuberalis and the Ratke's cysts showed a more complex cytokeratin pattern of both squamous and simple type. Whereas in may cystic epithelial cells including the "pseudo-follicles" a triple expression of cytokeratin, vimentin and GFAP could be observed, only some basal cells of squamous epithelial nests coexpressed cytokeratin and vimentin. The differences in the intermediate filament protein distribution are discussed in the light of embryological relationships of the different parts of the human pituitary gland.     

Cell type heterogeneity of intermediate filament expression in epithelia of the human pituitary gland

Summary In the present study we have localized immunohistochemically the intermediate filament proteins of the human pituitary gland (adenohypophysis, pars intermedia and pars tuberalis) by an indirect immunoperoxidase technique or by double immunofluorescence methods and analysed the individual cytokeratin polypeptides using two-dimensional gel electrophoresis. We found that the expression of cytokeratins in different epithelial cells of the human anterior pituitary gland was heterogeneous. Whereas the endocrine cells only expressed cytokeratins 8 and 18, the folliculo-stellate cells exhibited a reactivity for cytokeratins 7, 8, 18 and 19 as well as for GFAP and vimentin. The squamous epithelial cells of the pars tuberalis and the Ratke's cysts showed a more complex cytokeratin pattern of both squamous and simple type. Whereas in many cystic epithelial cells including the pseudo-follicles a triple expression of cytokeratin, vimentin and GFAP could be observed, only some basal cells of squamous epithelial nests coexpressed cytokeratin and vimentin. The differences in the intermediate filament protein distribution are discussed in the light of embryological relationships of the different parts of the human pituitary gland.     

Abnormal intermediate filament organization alters mitochondrial motility in giant axonal neuropathy fibroblasts

Giant axonal neuropathy (GAN) is a rare disease caused by mutations in the GAN gene, which encodes gigaxonin, an E3 ligase adapter that targets intermediate filament (IF) proteins for degradation in numerous cell types, including neurons and fibroblasts. The cellular hallmark of GAN pathology is the formation of large aggregates and bundles of IFs. In this study, we show that both the distribution and motility of mitochondria are altered in GAN fibroblasts and this is attributable to their association with vimentin IF aggregates and bundles. Transient expression of wild-type gigaxonin in GAN fibroblasts reduces the number of IF aggregates and bundles, restoring mitochondrial motility. Conversely, silencing the expression of gigaxonin in control fibroblasts leads to changes in IF organization similar to that of GAN patient fibroblasts and a coincident loss of mitochondrial motility. The inhibition of mitochondrial motility in GAN fibroblasts is not due to a global inhibition of organelle translocation, as lysosome motility is normal. Our findings demonstrate that it is the pathological changes in IF organization that cause the loss of mitochondrial motility.     

Sarcoplasmic reticulum and intermediate filament organization in cultured neonatal cardiac muscle cells

Cardiac muscle cells from 3-day-old rat neonates were cultured for periods of 2 to 56 days. In order to facilitate ultrastructural studies on the organization of the sarcoplasmic reticulum, the cells were prepared for transmission electron microscopy according to a regimen including postfixation in reduced osmium ferrocyanide. The nonjunctional sarcoplasmic reticulum (NJSR) was organized as a loose, fenestrated sleeve around the exterior of bundles of myofilaments and was particularly prominent at the level of the Z line. The only recognizable junctional elements of the sarcoplasmic reticulum were in a peripheral location. Reduced osmium ferrocyanide was also useful in distinguishing intermediate (10 nm) filaments, since it understained Z substance, which often obscured these structures. Intermediate filaments were arranged both at the Z line and the intercalated disc, in parallel strands, approximately at right angles to the myofilaments.     

Withaferin a alters intermediate filament organization, cell shape and behavior

Withaferin A (WFA) is a steroidal lactone present in Withania somnifera which has been shown in vitro to bind to the intermediate filament protein, vimentin. Based upon its affinity for vimentin, it has been proposed that WFA can be used as an anti-tumor agent to target metastatic cells which up-regulate vimentin expression. We show that WFA treatment of human fibroblasts rapidly reorganizes vimentin intermediate filaments (VIF) into a perinuclear aggregate. This reorganization is dose dependent and is accompanied by a change in cell shape, decreased motility and an increase in vimentin phosphorylation at serine-38. Furthermore, vimentin lacking cysteine-328, the proposed WFA binding site, remains sensitive to WFA demonstrating that this site is not required for its cellular effects. Using analytical ultracentrifugation, viscometry, electron microscopy and sedimentation assays we show that WFA has no effect on VIF assembly in vitro. Furthermore, WFA is not specific for vimentin as it disrupts the cellular organization and induces perinuclear aggregates of several other IF networks comprised of peripherin, neurofilament-triplet protein, and keratin. In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates. The organization of microtubules and actin/microfilaments is also affected by WFA. Microtubules become wavier and sparser and the number of stress fibers appears to increase. Following 24 hrs of exposure to doses of WFA that alter VIF organization and motility, cells undergo apoptosis. Lower doses of the drug do not kill cells but cause them to senesce. In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects.     

Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro

To investigate the sequences important for assembly of keratins into 10- nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.     

BBLN-1 is essential for intermediate filament organization and apical membrane morphology

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A requirement for cytoplasmic dynein and dynactin in intermediate filament network assembly and organization

We present evidence that vimentin intermediate filament (IF) motility in vivo is associated with cytoplasmic dynein. Immunofluorescence reveals that subunits of dynein and dynactin are associated with all structural forms of vimentin in baby hamster kidney-21 cells. This relationship is also supported by the presence of numerous components of dynein and dynactin in IF-enriched cytoskeletal preparations. Overexpression of dynamitin biases IF motility toward the cell surface, leading to a perinuclear clearance of IFs and their redistribution to the cell surface. IF-enriched cytoskeletal preparations from dynamitin-overexpressing cells contain decreased amounts of dynein, actin-related protein-1, and p150Glued relative to controls. In contrast, the amount of dynamitin is unaltered in these preparations, indicating that it is involved in linking vimentin cargo to dynactin. The results demonstrate that dynein and dynactin are required for the normal organization of vimentin IF networks in vivo. These results together with those of previous studies also suggest that a balance among the microtubule (MT) minus and plus end-directed motors, cytoplasmic dynein, and kinesin are required for the assembly and maintenance of type III IF networks in interphase cells. Furthermore, these motors are to a large extent responsible for the long recognized relationships between vimentin IFs and MTs.     

A role for disulfide bonding in keratin intermediate filament organization and dynamics in skin keratinocytes

We recently reported that a trans-dimer, homotypic disulfide bond involving Cys367 in keratin 14 (K14) occurs in an atomic-resolution structure of the interacting K5/K14 2B domains and in keratinocyte cell lines. Here we show that a sizable fraction of the K14 and K5 protein pools participates in interkeratin disulfide bonding in primary cultures of mouse skin keratinocytes. By comparing the properties of wild-type K14 with a completely cysteine-free variant thereof, we found that K14-dependent disulfide bonding limited filament elongation during polymerization in vitro but was necessary for the genesis of a perinuclear-concentrated network of keratin filaments, normal keratin cycling, and the sessile behavior of the nucleus and whole cell in keratinocytes studied by live imaging. Many of these phenotypes were rescued when analyzing a K14 variant harboring a single Cys residue at position 367. These findings establish disulfide bonding as a novel and important mechanism regulating the assembly, intracellular organization, and dynamics of K14-containing intermediate filaments in skin keratinocytes.     

Keratin 20 serine 13 phosphorylation is a stress and intestinal goblet cell marker

Keratin polypeptide 20 (K20) is an intermediate filament protein with preferential expression in epithelia of the stomach, intestine, uterus, and bladder and in Merkel cells of the skin. K20 expression is used as a marker to distinguish metastatic tumor origin, but nothing is known regarding its regulation and function. We studied K20 phosphorylation as a first step toward understanding its physiologic role. K20 phosphorylation occurs preferentially on serine, with a high stoichiometry as compared with keratin polypeptides 18 and 19. Mass spectrometry analysis predicted that either K20 Ser(13) or Ser(14) was a likely phosphorylation site, and Ser(13) was confirmed as the phospho-moiety using mutation and transfection analysis and generation of an anti-K20-phospho-Ser(13) antibody. K20 Ser(13) phosphorylation increases after protein kinase C activation, and Ser(13)-to-Ala mutation interferes with keratin filament reorganization in transfected cells. In physiological contexts, K20 degradation and associated Ser(13) hyperphosphorylation occur during apoptosis, and chemically induced mouse colitis also promotes Ser(13) phosphorylation. Among mouse small intestinal enterocytes, K20 Ser(13) is preferentially phosphorylated in goblet cells and undergoes dramatic hyperphosphorylation after starvation and mucin secretion. Therefore, K20 Ser(13) is a highly dynamic protein kinase C-related phosphorylation site that is induced during apoptosis and tissue injury. K20 Ser(13) phosphorylation also serves as a unique marker of small intestinal goblet cells.     

Immunoelectron microscopic studies of desmin (skeletin) localization and intermediate filament organization in chicken cardiac muscle

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.     

Immunoelectron microscopic studies of desmin (skeletin) localization and intermediate filament organization in chicken skeletal muscle

We studied the localization of desmin (skeletin), the major subunit of muscle-type intermediate filaments, by high resolution immunoelectron microscopy in adult chicken skeletal muscle. Immunoferritin labeling of ultrathin frozen sections of intact fixed sartorius muscle showed the presence of desmin between adjacent Z-bands and as strands peripheral to Z-bands, forming apparent connections between the Z-bands with adjacent sarcolemma, mitochondria, and nuclei. We observed no desmin labeling, however, in the vicinity of the T-tubules. In addition, intermediate filaments were morphologically discernible at the level of the Z-bands in plastic sections of glycerol-extracted muscle that had been infused with unlabeled antidesmin antibodies. Our results indicate that the desmin present in adult skeletal muscle, that had previously been detected by immunofluorescence light microscopy, is largely if not entirely in the form of intermediate filaments. The results provide evidence that these filaments serve to interconnect myofibrils at the level of their Z-bands, and to connect Z-bands with other specific structures and organelles in the myotube, but not with the T-tubule system.     

Keratin function in skin epithelia: a broadening palette with surprising shades

Keratins make up the largest subgroup of intermediate filament (IF) proteins and form a dynamic network of 10-12 nm filaments, built from type I/type II heterodimers, in the cytoplasm of epithelial cells. A major function of keratin IFs is to protect epithelial cells from mechanical and non-mechanical stresses that cause cell rupture and death. Interference with this role is the root cause of a large number of inherited epithelial fragility conditions. Additional functions, non-mechanical in nature, are manifested in a way that depends on the specific keratin and on the epithelial context. The recent discovery of unusual mutations affecting keratin proteins has uncovered a novel dimension of their mechanical support function, and has synergized with mouse genetics to reveal a role in skin pigmentation. Other studies extended the role of keratin proteins in regulating the response to pro-apoptotic signals, and revealed their ability to modulate protein synthesis and cell size in epithelial cells challenged to grow.     

Evidence for an intermediate in tau filament formation

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule-associated protein tau. In vitro, fibrillization of full-length, unphosphorylated recombinant tau can be induced under near-physiological conditions by treatment with various agents, including anionic surfactants. Here we examine the pathway through which anionic surfactants promote tau fibrillization using a combination of electron microscopy and fluorescence spectroscopy. Protein and surfactant first interacted in solution to form micelles, which then provided negatively charged surfaces that accumulated tau aggregates. Surface aggregation of tau protein was followed by the time-dependent appearance of a thioflavin S reactive intermediate that accumulated over a period of hours. The intermediate was unstable in the absence of anionic surfaces, suggesting it was not filamentous. Fibrillization proceeded after intermediate formation with classic nucleation-dependent kinetics, consisting of lag phase followed by the exponential increase in filament lengths, followed by an equilibrium phase reached in approximately 24 h. The pathway did not require protein insertion into the micelle hydrophobic core or conformational change arising from mixed micelle formation, because anionic microspheres constructed from impermeable polystyrene were capable of qualitatively reproducing all aspects of the fibrillization reaction. It is proposed that the progression from amorphous aggregation through intermediate formation and fibrillization may underlie the activity of other inducers such as hyperphosphorylation and may be operative in vivo.     

Maintenance of the intestinal tube in Caenorhabditis elegans: the role of the intermediate filament protein IFC-2

The Caenorhabditis elegans intestinal lumen is surrounded by a dense cytoplasmic network that is laterally attached to the junctional complex and is referred to as the endotube. It localizes to the terminal web region which anchors the microvillar actin filament bundles and is particularly rich in intermediate filaments. To examine their role in intestinal morphogenesis and function, C. elegans reporter strains were generated expressing intestine-specific CFP-tagged intermediate filament polypeptide IFB-2. When these animals were treated with dsRNA against intestinal intermediate filament polypeptide IFC-2, the endotube developed multiple bubble-shaped invaginations that protruded into the enterocytic cytoplasm. The irregularly widened lumen remained surrounded by a continuous IFB-2::CFP-labeled layer. Comparable but somewhat mitigated phenotypic changes were also noted in wild-type N2 worms treated with ifc-2 (RNAi). Junctional complexes were ultrastructurally and functionally normal and the apical domain of intestinal cells was also not altered. These observations demonstrate that IFC-2 is important for structural maintenance of the intestinal tube but is not needed for establishment of the endotube and epithelial cell polarity.