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 in the giant muscle cells of the nematode Ascaris lumbricoides; abundance and three-dimensional complexity of arrangements

The body muscle cells of the nematode Ascaris lumbricoides are characterized by massive amounts of intermediate filaments (IF). These occur in all three regions of this giant cell type. They traverse the cytoplasm of the balloon-like belly, which houses the nucleus, and occur as bundles in the arm-like extensions to the nerve. The organization of IF in the third region, the contractile fiber, was analyzed further by serial sections and three-dimensional reconstruction. IF bundles traverse the glycogen-rich lumina of the fiber and reach as baskets around the sarcomeres. Together with numerous dense bodies they form the Z-band-like arrangements. IF bundles reach the plasma membrane at hemidesmosome-like specializations often situated at deep membrane invaginations filled with a fibrillar component of the extracellular matrix. The ultrastructural appearance of IF bundles is connected to the contractional state of the sarcomeres. They appear straight in extended muscle but coil up upon contraction. In the pharynx massive IF bundles are oriented longitudinally. A second type of IF bundles follows the radially oriented sarcomeres. These reveal pronounced Z-band type structures with massive disks. IF surround the sarcomeres and seem to terminate at these disks. We discuss possible functions of the complex IF organization in body muscle and pharynx.     

Intermediate filaments formed de novo from tail-less cytokeratins in the cytoplasm and in the nucleus

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.     

Role of stress fibers in the association of intermediate filaments with microtubules in fibroblast cells.

The association between intermediate filaments (IF) and microtubules (MT) has been demonstrated by several experiments using MT inhibitors and by microinjecting specific antibodies. The actin cytoskeleton has recently been assigned a role in this process of drug induced IF collapse. However, this was not found to be true in large cells with irregular morphology. For instance, in early passage diploid fibroblasts of human origin and in armadillo cell lines, where the cells are large, irregular in shape and exhibit prominent stress fibers ( SF ), depolymerization of MT with nocodazole did not lead to collapse of IF . Instead, the IF formed bundles of coils that seemed to associate with the SF . Disintegration of the SF with cytochalasin B led to the collapse of the IF . It appears that the actin organization in such large cells with extensive SF , is not as contractile as in typical spindle shaped fibroblasts which have relatively less stable actin organization. The stable SF may actually prevent IF collapse.     

Modulation of vimentin containing intermediate filament distribution and phosphorylation in living fibroblasts by the cAMP-dependent protein kinase

Microinjection of the purified catalytic subunit of the cAMP-dependent protein kinase (A-kinase) into living rat embryo fibroblasts leads to dramatic changes in vimentin intermediate filament (IF) organization, involving the collapse of the filaments into tight bundles. In some cell types, this rearrangement of the IF proceeds further, leading to an apparent loss of filament integrity, resulting in a punctate staining pattern throughout the cytoplasm. Both these types of IF rearrangement are fully reversible, and similar to structural changes previously described for IF during mitosis. As shown by electron microscopy, in rat embryo fibroblasts these changes in IF structure do not involve the loss of the 10-nM filament structure but instead correspond to the bundling together of 25 or more individual filaments. Metabolic pulse labeling of injected cells reveals that accompanying these changes in IF organization is a dramatic increase in vimentin phosphorylation which appears maximal when the IF are fully rearranged. However, this increase in IF phosphorylation is not accompanied by any significant increase in soluble vimentin. Analysis of the sites of phosphorylation on vimentin from injected cells by either V8 protease cleavage, or two-dimensional tryptic peptide mapping, revealed increased de novo phosphorylation of two vimentin phosphopeptides after microinjection of A-kinase. These data strongly suggest that the site-specific phosphorylation of vimentin by A-kinase is responsible for the dynamic changes in IF organization observed after injection of the kinase into living cells, and may be involved in similar rearrangement of the IF previously described during mitosis or after heat shock.     

Characterization of the intermediate filament apparatus in skin fibroblasts from patients with giant axonal neuropathy: effect of trypsin

Skin fibroblasts from two siblings with giant axonal neuropathy (GAN) were examined by both biochemical and immunocytochemical studies. The presence of intermediate filaments (IF) characteristic of these cells was affected by the growth conditions. Immediately after plating and during the following 24 hours the majority of the cells contained an IF "bundle"; however, after 4-6 days in culture only a minority of the cells retained this structure. We present evidence that trypsinization but not serum concentration is likely to influence the formation of the "bundle." The results indicate that the formation of the "bundle" may result from a defective association or relationship between the cytoskeleton and the plasma membrane.     

Ultrastructural Studies of Carboxyl-Terminal Truncation Mutants of the Neuronal Intermediate Filament Protein Peripherin

Abstract: We have prepared carboxyl-terminal truncation mutants of the neuronal intermediate filament (IF) protein peripherin and examined the assembly characteristics of these mutant proteins in SW13 cells in the presence and absence of vimentin. In the absence of vimentin, tailless peripherin protein (Per-C424) self-assembles into bundles and clumps as observed by immunofluorescence, whereas a peripherin mutant that is missing the tail as well as a small portion of the rod (Per-C356) appears as spherical aggregates. Similar phenotypes are observed when vimentin-positive cells are transfected with Per-C424 or Per-C356. In these cells, the entire IF network is disrupted, and vimentin colocalizes with the mutant peripherin proteins. To examine the morphology of the bundles and clumps formed by Per-C424 at the electron microscopic level, we prepared stable cell lines expressing different levels of this mutant protein. By immunofluorescence, Per-C424 appears as either clumps or bundles of filaments depending on the expression level of the mutant protein. However, under electron microscopy, it is apparent that both clumps and bundles are composed of tightly packed IFs. We were unable to obtain stable cell lines expressing Per-C356, indicating that this mutant may prevent cell proliferation. Using a vector containing an internal ribosomal entry site, we prepared a construct that expresses Per-C356 and green fluorescent protein as a single mRNA, and we were able to isolate cells that expressed Per-C356 by fluorescence-activated cell sorting. Electron microscopic analysis of these cells showed that these aggregates are solid and contain no obvious filamentous structures.     

Kelch Domain of Gigaxonin Interacts with Intermediate Filament Proteins Affected in Giant Axonal Neuropathy

Patients with giant axonal neuropathy (GAN) show progressive loss of motor and sensory function starting in childhood and typically live for less than 30 years. GAN is caused by autosomal recessive mutations leading to low levels of gigaxonin (GIG), a ubiquitously-expressed BTB/Kelch cytoplasmic protein believed to be an E3 ligase substrate adaptor. GAN pathology is characterized by aggregates of intermediate filaments (IFs) in multiple tissues. To delineate the molecular pathway between GIG deficiency and IF pathology, we undertook a proteomic screen to identify the normal binding partners of GIG. Prominent among them were several classes of IFs, including the neurofilament subunits whose accumulation leads to the axonal swellings for which GAN is named. We showed these interactions were dependent on the Kelch domain of GIG. Furthermore, we identified the E3 ligase MYCBP2 and the heat shock proteins HSP90AA1/AB1 as interactors with the BTB domain that may result in the ubiquitination and subsequent degradation of intermediate filaments. Our open-ended proteomic screen provides support to GIG’s role as an adaptor protein, linking IF proteins through its Kelch domain to the ubiquitin pathway proteins via its BTB domain, and points to future approaches for reversing the phenotype in human patients.     

Differential organization of desmin and vimentin in muscle is due to differences in their head domains

In most myogenic systems, synthesis of the intermediate filament (IF) protein vimentin precedes the synthesis of the muscle-specific IF protein desmin. In the dorsal myotome of the Xenopus embryo, however, there is no preexisting vimentin filament system and desmin's initial organization is quite different from that seen in vimentin-containing myocytes (Cary and Klymkowsky, 1994. Differentiation. In press.). To determine whether the organization of IFs in the Xenopus myotome reflects features unique to Xenopus or is due to specific properties of desmin, we used the injection of plasmid DNA to drive the synthesis of vimentin or desmin in myotomal cells. At low levels of accumulation, exogenous vimentin and desmin both enter into the endogenous desmin system of the myotomal cell. At higher levels exogenous vimentin forms longitudinal IF systems similar to those seen in vimentin-expressing myogenic systems and massive IF bundles. Exogenous desmin, on the other hand, formed a reticular IF meshwork and non-filamentous aggregates. In embryonic epithelial cells, both vimentin and desmin formed extended IF networks. Vimentin and desmin differ most dramatically in their NH2- terminal "head" regions. To determine whether the head region was responsible for the differences in the behavior of these two proteins, we constructed plasmids encoding chimeric proteins in which the head of one was attached to the body of the other. In muscle, the vimentin head- desmin body (VDD) polypeptide formed longitudinal IFs and massive IF bundles like vimentin. The desmin head-vimentin body (DVV) polypeptide, on the other hand, formed IF meshworks and non-filamentous structures like desmin. In embryonic epithelial cells DVV formed a discrete filament network while VDD did not. Based on the behavior of these chimeric proteins, we conclude that the head domains of vimentin and desmin are structurally distinct and not interchangeable, and that the head domain of desmin is largely responsible for desmin's muscle- specific behaviors.     

Cytokeratin filament organization in the ciliated cells of the quail oviduct

With the exception of keratinocytes and some types of cultured cells, ciliated cells appear to be the major cell type which contains the most developed cytokeratin meshwork. We report, here, on the intermediate filament (IF) organization in ciliated cells of the quail oviduct using ultrastructural and immunocytochemical techniques. Special attention was focused on the relationships between IF and other cell organelles. The meshwork of IFs appears as a subapical disk constituted of separate bundles mainly composed of interwoven 10-nm filaments. From this subapical region, a descending bundle connects the array of IFs occupying the basal part of the cell. The nucleus is maintained in a loose network of IFs. In ciliated cells there are no free centrioles, but IFs are related to centriolar appendages (striated rootlets).     

Augmentation of mouse natural killer cell activity by interferon and interferon inducers.

Interferon (IF), in addition to its anti-viral capacity, is increasingly being found to be a regulator of cell division, cell surface antigens, and cell function. To determine whether IF also plays a role in the regulation of natural killer (NK) cell activity in mice, the in vivo and in vitro effects of IF and IF inducers on NK activity were studied. We observed that pyran, lipopolysaccharide, and polyinosinicopolycytidylic acid (poly I:C) as well as crude and purified IF preparations significantly elevated splenic NK levels in normal mice within 3 to 24 hr of i.p. administration. Normal spleen cells treated with poly I:C or IF in vitro also had augmented NK activity. Poly I:C and IF were themselves not cytotoxic and their presence was not required during the lytic process, indicating that IF acts on lymphocytes to activate NK function. The addition of anti-IF in the incubation medium completely blocked the boosting of NK activity by poly I:C or IF. The characteristics of the effector cells activated by IF were consistent with those of NK cells rather than macrophages, since the boosted effector cells were not retained by a rayon column or removed by carbonyl iron. Moreover, they were resistant to treatment with anti-Thy 1.2 serum plus complement, which eliminated mature T cells.     

Redistribution of GFAP and αB-crystallin after thermal stress in C6 glioma cell line

Summary Some intermediate filament (IF) proteins expressed in the development of glia include nestin, vimentin, and glial fibrillary acidic protein (GFAP). However, GFAP is the major intermediate filament protein of mature astrocytes. To determine the organization of GFAP in glial cells, rat GFAP cDNA tagged with enhanced green fluorescent protein (EGFP) was transfected into the rat C6 glioma cell line. After selection, two stable C6-EGFP-GFAP cell lines were established. Stable C6-EGFP-GFAP cell lines with or without heat shock treatment were analyzed by immunocytochemistry, electron microscopy, and Western blot analysis. In the transient transfection study, EGFP-GFAP transiently expressed in C6 cells formed punctate aggregations in the cytoplasm right after transfection, but gradually a filamentous structure of EGFP-GFAP was observed. The protein level of nestin in the C6-EGFP-GFAP stable clone was similar to that in the pEGFP-C1 transfected C6 stable clones and non-transfected C6 cells, whereas the level of vimentin was reduced in Western blotting. Interestingly, the expression level of small heat shock protein αB-crystallin in C6-EGFP-GFAP cells was also enhanced after transfection. Immunostaining patterns of C6-EGFP-GFAP cells showed that GFAP was dispersed as a fine filamentous structure. However, after heat shock treatment, GFAP formed IF bundles in C6-EGFP-GFAP cells. In the meantime, αB-crystallin also colocalized with IF bundles of GFAP in C6-EGFP-GFAP cells. The heat-induced GFAP reorganization we found suggested that small heat shock protein αB-crystallin may play a functional role regulating the cytoarchitecture of GFAP.     

The intermediate-sized filaments in rat kangaroo PtK2 cells. I. Morphology in situ.

The system of the intermediate-sized filaments (IF) of rat kangaroo PtK2 cells which can be specifically demonstrated by immunofluorescence microscopy using certain rabbit autoantibodies and guinea pig antibodies against bovine hoof prekeratin has been studied by electron microscopy. The characteristic ornamental, curved arrays of this system are shown after fixation in situ in both thin sections and whole-cell-preparations to represent bundles of 6 to 11 nm thick filaments extending through the whole cytoplasm, although in some cells they appear to be enriched in the perinuclear region. While many individual IF are recognized in the cytoplasm the tendency of such filaments to aggregate laterally into bundles is one of their prominent features. Among such bundle formations one form that consists of tightly packed IF cemented together in a dense osmiophilic matrix is especially conspicious. The appearance and mode of arrangement of the IF is not significantly altered in cells treated with colcemid and/or cytochalasin B. Spatial relationships of IF with microfilament-containing cables and microtubules as well as with membranous structures are also described. IF are heterogeneous in width and reveal an unstained, apparently hollow core, indicative of a tubular organization. Many IF show small, sometimes periodically arranged lateral projections which seem to be involved in IF cross-linking. Associations with polyribosomes are common. The changes in the IF system during mitosis have also been examined. The structural details of the IF as well as their possible role as cytoskeletal elements involved in the control of cell shape and cytoplasmic architecture are discussed in relation to data on various intermediate-sized filaments from other cell types. The close similarity of the IF of PtK2 cells to aggregates of prekeratin filaments is emphasized. It is suggested that PtK2 cells represent an epithelial cell line growing in a state of balanced semi-keratinization.     

Changes in the organization of the intermediate filament system of human fibroblasts in lysosomal storage diseases and their modeling]

The organization of the system of the vimentin intermediate filaments (IFs) in human fibroblasts in lysosomal storage diseases (Fabry's disease, mannosidosis) and their modelling has been studied in vitro. It was shown that during accumulation of nonhydrolyzable compounds, hypertrophy of the lysosomal compartment is accompanied by formation of ring-shaped bundles IFs, surrounding apparently these increased organelles. The changed organization of IFs is characteristic of polarised pathological cells in monolayer, and after repassage it is retained only at the spreading state; on transition from the discoid to extended cellular form there occurred the centrifugal shift of ring-shaped structures of IFs to active cell border and gradual restoration of radial fibrillar state of IFs. It is suggested that on intralysosomal storage of unsplit compounds reorganization of the vimentin-type IFs in ring-shaped structure is necessary for optimal distribution and stabilization into the cytoplasm of large amounts of increased lysosomes with exo- and endogenous contents. In condition of free spreading (i. e. with diminished cell density) the restoration of normal fibrillar IF organization may be due to the loss of considerable number of hypertrophied lysosomes; the involvement of lysosomal membrane in formation of active cellular border is not to be ruled out.     

Time and cell cycle dependent formation of heterogeneous tubulin arrays induced by colchicine in Triticum aestivum root meristem

We have investigated the appearance and reorganization of tubulin-containing arrays induced by colchicine in the root meristem of wheat Triticum aestivum, using immunostaining and electron microscopy. Colchicine caused depolymerization of microtubules and formation of tubulin cortical strands composed of filamentous material only in C-mitotic cells. After prolonged exposure to the drug, both interphase and C-mitotic cells acquired needle-type bundles, arranged as different crystalloids and/or macrotubules. The unmodified tyrosinated form of alpha-tubulin was detected within microtubules in control cells, but was not found within cortical strands. It was identified, however, within needle-type bundles. The modified acetylated form of alpha-tubulin, which was absent in control cells, was detected within needle-type bundles. Thus, cortical strands were transitory arrays, transformed into needle-type bundles during prolonged exposure to colchicine. Cortical strands appeared in a cell cycle-dependent manner, whereas needle-type bundles were cell cycle stable arrays. The diverse morphological organization, intracellular distribution and stability of tubulin-containing arrays may be associated with heterogeneity of alpha-tubulin isoforms. We assume that non-microtubular arrays substitute for microtubules in conditions where normal tubulin polymerization is inhibited.     

Quantitative changes in microtubule distribution correlate with guard cell function in Arabidopsis

Radially arranged cortical microtubules are a prominent feature of guard cells. We observed guard cells expressing GFP-tubulin (GFP-TUA6) with confocal microscopy and found recognizable changes in the appearance of microtubules when stomata open or close (Eisinger et al., 2012). In the present study, analysis of fluorescence distribution showed a dramatic increase in peak intensities of microtubule bundles within guard cells as stomata open. This increase was correlated with an increase in the total fluorescence that could be attributed to polymerized tubulin. Adjacent pavement cells did not show similar changes in peak intensities or integrated fluorescence when stomatal apertures changed. Imaging of RFP-tagged end binding protein 1 (EB1) and YFP-tagged α-tubulin expressed in the same cell revealed that the number of microtubules with growing ends remained constant, although the total amount of polymerized tubulin was higher in open than in closed guard cells. Taken together, these results indicate that the changes in microtubule array organization that are correlated with and required for normal guard cell function are characterized by changes in microtubule clustering or bundling.     

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.     

Co-expression of cytokeratins and neurofilament proteins in a permanent cell line: cultured rat PC12 cells combine neuronal and epithelial features

The cytoskeleton of the rat cultured cell line PC12, which is widely used in cell biology as a model system for neuron-like differentiation, displays an unusual combination of intermediate-sized filaments (IFs). As determined by electron microscopy, immunolocalization, and biochemical analyses, these cells contain, in addition to neurofilaments, an extended meshwork of bundles of cytokeratin IFs comprising cytokeratins A and D, equivalent to human cytokeratin polypeptides Nos. 8 and 18, irrespective of whether they are grown in the presence or absence of nerve growth factor. The two IF systems differ in their fibrillar arrays, the neurofilaments being concentrated in perinuclear aggregates similar to those found in certain neuroendocrine tumors of epithelial origin. We conclude that PC12 cells permanently co-express IFs of both the epithelial and the neuronal type and thus present an IF combination different from those of adrenal medulla cells and pheochromocytomas, i.e., the putative cells of origin of the line PC12. The IF cytoskeleton of PC12 cells resembles that of various neuroendocrine tumors derived from epithelial cells. The results show that the development of a number of typical neuronal differentiation features is compatible with the existence of an epithelial type IF cytoskeleton, i.e., cytokeratins. The implications of these findings concerning the validity of the PC12 cell line as a model for neuronal differentiation and possible explanations of the origin of cells with this type of IF co-expression are discussed.