Giant axonal neuropathy alters the structure of keratin intermediate filaments in human hair.

Giant axonal neuropathy (GAN) follows an autosomal recessive genetic inheritance and impedes the peripheral and central nervous system due to axonal swellings that are packed with neurofilaments. The patients display a number of phenotypes, including hypotonia, muscle weakness, decreased reflexes, ataxia, seizures, intellectual disability, pale skin and often curled hair. We used X-ray diffraction and tensile testing to determine potential changes to the structure of keratin intermediate filaments (IFs) in the hair of patients with GAN. A statistically significant decrease in the 47 and the 27 Å diffraction signals were observed. Tensile tests determined that the hair was slightly stiffer, stronger and more extensible in GAN patients. These results suggest that the structure of keratin IFs in hair is altered in GAN, and the findings are compatible with an increased positional disorder of the keratin tetramers within the hair fibres.

Methods for Determining the Cellular Functions of Vimentin Intermediate Filaments.

The type III intermediate filament protein vimentin was once thought to function mainly as a static structural protein in the cytoskeleton of cells of mesenchymal origin. Now, however, vimentin is known to form a dynamic, flexible network that plays an important role in a number of signaling pathways. Here, we describe various methods that have been developed to investigate the cellular functions of the vimentin protein and intermediate filament network, including chemical disruption, photoactivation and photoconversion, biolayer interferometry, soluble bead binding assay, three-dimensional substrate experiments, collagen gel contraction, optical-tweezer active microrheology, and force spectrum microscopy. Using these techniques, the contributions of vimentin to essential cellular processes can be probed in ever further detail.

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.

Intermediate Filaments Play a Pivotal Role in Regulating Cell Architecture and Function.

Intermediate filaments (IFs) are composed of one or more members of a large family of cytoskeletal proteins, whose expression is cell- and tissue type-specific. Their importance in regulating the physiological properties of cells is becoming widely recognized in functions ranging from cell motility to signal transduction. IF proteins assemble into nanoscale biopolymers with unique strain-hardening properties that are related to their roles in regulating the mechanical integrity of cells. Furthermore, mutations in the genes encoding IF proteins cause a wide range of human diseases. Due to the number of different types of IF proteins, we have limited this short review to cover structure and function topics mainly related to the simpler homopolymeric IF networks composed of vimentin, and specifically for diseases, the related muscle-specific desmin IF networks.

Giant axonal neuropathy-associated gigaxonin mutations impair intermediate filament protein degradation.

Giant axonal neuropathy (GAN) is an early-onset neurological disorder caused by mutations in the GAN gene (encoding for gigaxonin), which is predicted to be an E3 ligase adaptor. In GAN, aggregates of intermediate filaments (IFs) represent the main pathological feature detected in neurons and other cell types, including patients' dermal fibroblasts. The molecular mechanism by which these mutations cause IFs to aggregate is unknown. Using fibroblasts from patients and normal individuals, as well as Gan-/- mice, we demonstrated that gigaxonin was responsible for the degradation of vimentin IFs. Gigaxonin was similarly involved in the degradation of peripherin and neurofilament IF proteins in neurons. Furthermore, proteasome inhibition by MG-132 reversed the clearance of IF proteins in cells overexpressing gigaxonin, demonstrating the involvement of the proteasomal degradation pathway. Together, these findings identify gigaxonin as a major factor in the degradation of cytoskeletal IFs and provide an explanation for IF aggregate accumulation, the subcellular hallmark of this devastating human disease.

Inroads into the structure and function of intermediate filament networks.

Although intermediate filaments are one of three major cytoskeletal systems of vertebrate cells, they remain the least understood with respect to their structure and function. This is due in part to the fact that they are encoded by a large gene family which is developmentally regulated in a cell and tissue type specific fashion. This article is in honor of Ueli Aebi. It highlights the studies on IF that have been carried out by our laboratory for more than 40 years. Many of our advances in understanding IF are based on conversations with Ueli which have taken place during adventurous and sometimes dangerous hiking and biking trips throughout the world.

Vimentin intermediate filaments modulate the motility of mitochondria.

Interactions with vimentin intermediate filaments (VimIFs) affect the motility, distribution, and anchorage of mitochondria. In cells lacking VimIFs or in which VimIF organization is disrupted, the motility of mitochondria is increased relative to control cells that express normal VimIF networks. Expression of wild-type VimIF in vimentin-null cells causes mitochondrial motility to return to normal (slower) rates. In contrast, expressing vimentin with mutations in the mid-region of the N-terminal non-α-helical domain (deletions of residues 41-96 or 45-70, or substitution of Pro-57 with Arg) did not inhibit mitochondrial motility even though these mutants retain their ability to assemble into VimIFs in vivo. It was also found that a vimentin peptide consisting of residues 41-94 localizes to mitochondria. Taken together, these data suggest that VimIFs bind directly or indirectly to mitochondria and anchor them within the cytoplasm.

Insights into the mechanical properties of epithelial cells: the effects of shear stress on the assembly and remodeling of keratin intermediate filaments.

The effects of shear stress on the keratin intermediate filament (KIF) cytoskeleton of cultured human alveolar epithelial (A549) cells have been investigated. Under normal culture conditions, immunofluorescence revealed a delicate network of fine tonofibrils containing KIFs, together with many nonfilamentous, keratin-containing "particles," mostly containing either keratin 8 (K8) or 18 (K18), but not both. Triton X-100 extracted approximately 10% of the cellular keratin, and this was accompanied by a loss of the particles but not the KIFs. Shear stress dramatically reduced the soluble keratin component and transformed the fine bundles of KIFs into thicker, "wavy" tonofibrils. Both effects were accompanied by the disappearance of most keratin particles and by increased phosphorylation of K8 and K18 on serine residues 73 and 33, respectively. The particles that remained after shearing were phosphorylated and were closely associated with KIFs. We suggest that keratin particles constitute a reservoir of protein that can be recruited into KIFs under flow, creating a more robust cytoskeleton able to withstand shear forces more effectively.

Intermediate filaments: versatile building blocks of cell structure.

Cytoskeletal intermediate filaments (IF) are organized into a dynamic nanofibrillar complex that extends throughout mammalian cells. This organization is ideally suited to their roles as response elements in the subcellular transduction of mechanical perturbations initiated at cell surfaces. IF also provide a scaffold for other types of signal transduction that together with molecular motors ferries signaling molecules from the cell periphery to the nucleus. Recent insights into their assembly highlight the importance of co-translation of their precursors, the hierarchical organization of their subunits in the formation of unit-length filaments (ULF) and the linkage of ULF into mature apolar IF. Analyses by atomic force microscopy reveal that mature IF are flexible and can be stretched to over 300% of their length without breaking, suggesting that intrafilament subunits can slide past one another when exposed to mechanical stress and strain. IF also play a role in the organization of organelles by modulating their motility and providing anchorage sites within the cytoplasm.

Purification of bovine lens and bacterially expressed human vimentin.

INTRODUCTIONIntermediate filaments (IF) are major cytoskeletal systems of vertebrate and many nonvertebrate cells whose expression is cell-type specific and developmentally regulated. This protocol describes a method for purifying one type of IF, vimentin, from bovine lens tissue. Purification of human vimentin expressed in Escherichia coli is also described. These methods are useful in the preparation of other IF protein subunits for microinjection studies as well.

Preparation and Microinjection of x-Rhodamine-Labeled Vimentin.

INTRODUCTIONIntermediate filaments (IF) are major cytoskeletal systems of vertebrate and many nonvertebrate cells whose expression is cell-type specific and developmentally regulated. This protocol describes the x-rhodamine labeling of one type of IF, vimentin, and a method for microinjection of the labeled vimentin into cultured cells. IF dynamics can then be examined with fluorescence microscopy.

Keratin 8 phosphorylation by protein kinase C delta regulates shear stress-mediated disassembly of keratin intermediate filaments in alveolar epithelial cells.

Phosphorylation of keratin intermediate filaments (IF) is known to affect their assembly state and organization; however, little is known about the mechanisms regulating keratin phosphorylation. In this study, we demonstrate that shear stress, but not stretch, causes disassembly of keratin IF in lung alveolar epithelial cells (AEC) and that this disassembly is regulated by protein kinase C delta-mediated phosphorylation of keratin 8 (K8) Ser-73. Specifically, in AEC subjected to shear stress, keratin IF are disassembled, as reflected by their increased solubility. In contrast, AEC subjected to stretch showed no changes in the state of assembly of IF. Pretreatment with the protein kinase C (PKC) inhibitor, bisindolymaleimide, prevents the increase in solubility of either K8 or its assembly partner K18 in shear-stressed AEC. Phosphoserine-specific antibodies demonstrate that K8 Ser-73 is phosphorylated in a time-dependent manner in shear-stressed AEC. Furthermore, we showed that shear stress activates PKC delta and that the PKC delta peptide antagonist, delta V1-1, significantly attenuates the shear stress-induced increase in keratin phosphorylation and solubility. These data suggested that shear stress mediates the phosphorylation of serine residues in K8, leading to the disassembly of IF in alveolar epithelial cells. Importantly, these data provided clues regarding a molecular link between mechanically induced signal transduction and alterations in cytoskeletal IF.

The nucleoskeleton: lamins and actin are major players in essential nuclear functions.

The nucleoskeleton is composed of many interacting structural proteins that provide the framework for DNA replication, transcription and a variety of other nuclear functions. For example, the type-V intermediate filament proteins, the lamins, and their associated proteins (e.g. Lap2alpha) play important roles in DNA replication and transcription. Furthermore, actin, actin-related proteins and other actin-associated proteins likewise appear to be important in nuclear functions because they are components of chromatin-remodeling complexes and are involved in mRNA synthesis, processing and transport. Newly described nuclear proteins that contain both actin- and lamin-binding domains could be involved in regulating molecular crosstalk between these two types of nucleoskeletal proteins. This range of activities might help to explain why genetic defects in some of the nucleoskeletal proteins contribute to an ever-expanding list of human diseases.