Absence Of Intermediate Filaments Research Articles

Ultrastructural features of aberrant glial cells isolated from the spinal cord of paralytic rats expressing the amyotrophic lateral sclerosis-linked SOD1G93A mutation.

In the rat model of amyotrophic lateral sclerosis expressing the G93A superoxide dismutase-1 mutation, motor neuron death and rapid paralysis progression are associated with the emergence of a population of aberrant glial cells (AbAs) that proliferate in the degenerating spinal cord. Targeting of AbAs with anti-neoplasic drugs reduced paralysis progression, suggesting a pathogenic potential contribution of these cells accelerating paralysis progression. In the present study, analyze the cellular and ultrastructural features of AbAs following their isolation and establishment in culture during several passages. We found that AbAs exhibit permanent loss of contact inhibition, absence of intermediate filaments and abundance of microtubules, together with an important production of extracellular matrix components. Remarkably, AbAs also exhibited exacerbated ER stress together with a significant abundance of lipid droplets, as well as autophagic and secretory vesicles, all characteristic features of cellular stress and inflammatory activation. Taken together, the present data show AbA cells as a unique aberrant phenotype for a glial cell that might explain their pathogenic and neurotoxic effects.

Cytoskeletal organization in microtentacles

Microtentacles are thin, flexible cell protrusions that have recently been described and whose presence enhances efficient attachment of circulating cells. They are found on circulating tumor cells and can be induced on a wide range of breast cancer cell lines, where they are promoted by factors that either stabilize microtubules or destabilize the actin cytoskeleton. Evidence suggests that they are relevant to the metastatic spread of cancer, so understanding their structure and formation may lead to useful therapies. Microtentacles are formed by microtubules and contain vimentin intermediate filaments, but beyond this, there is little information about their ultrastructure. We have used electron microscopy of high pressure frozen sections and tomography of cryo-prepared intact cells, along with super resolution fluorescence microscopy, to provide the first ultrastructural insights into microtubule and intermediate filament arrangement within microtentacles. By scanning electron microscopy it was seen that microtentacles form within minutes of addition of drugs that stabilize microtubules and destabilize actin filaments. Mature microtentacles were found to be well below one micrometer in diameter, tapering gradually to below 100nm at the distal ends. They also contained frequent branches and bulges suggestive of heterogeneous internal structure. Super resolution fluorescence microscopy and examination of sectioned samples showed that the microtubules and intermediate filaments can occupy different areas within the microtentacles, rather than interacting intimately as had been expected. Cryo-electron tomography of thin regions of microtentacles revealed densely packed microtubules and absence of intermediate filaments. The number of microtubules ranged from several dozen in some areas to just a few in the thinnest regions, with none of the regular arrangement found in axonemes. Improved understanding of the mechanism of microtentacle formation, as well as the resultant structure, will be valuable in developing therapies against metastasis, if the hypothesized role of microtentacles in metastasis is confirmed. This work provides a significant step in this direction.

GFAP and vimentin deficiency alters gene expression in astrocytes and microglia in wild-type mice and changes the transcriptional response of reactive glia in mouse model for Alzheimer's disease.

Reactive astrocytes with an increased expression of intermediate filament (IF) proteins Glial Fibrillary Acidic Protein (GFAP) and Vimentin (VIM) surround amyloid plaques in Alzheimer's disease (AD). The functional consequences of this upregulation are unclear. To identify molecular pathways coupled to IF regulation in reactive astrocytes, and to study the interaction with microglia, we examined WT and APPswe/PS1dE9 (AD) mice lacking either GFAP, or both VIM and GFAP, and determined the transcriptome of cortical astrocytes and microglia from 15- to 18-month-old mice. Genes involved in lysosomal degradation (including several cathepsins) and in inflammatory response (including Cxcl5, Tlr6, Tnf, Il1b) exhibited a higher AD-induced increase when GFAP, or VIM and GFAP, were absent. The expression of Aqp4 and Gja1 displayed the same pattern. The downregulation of neuronal support genes in astrocytes from AD mice was absent in GFAP/VIM null mice. In contrast, the absence of IFs did not affect the transcriptional alterations induced by AD in microglia, nor was the cortical plaque load altered. Visualizing astrocyte morphology in GFAP-eGFP mice showed no clear structural differences in GFAP/VIM null mice, but did show diminished interaction of astrocyte processes with plaques. Microglial proliferation increased similarly in all AD groups. In conclusion, absence of GFAP, or both GFAP and VIM, alters AD-induced changes in gene expression profile of astrocytes, showing a compensation of the decrease of neuronal support genes and a trend for a slightly higher inflammatory expression profile. However, this has no consequences for the development of plaque load, microglial proliferation, or microglial activation.

Biological adhesion of the flatworm Macrostomum lignano relies on a duo-gland system and is mediated by a cell type-specific intermediate filament protein

BackgroundFree-living flatworms, in both marine and freshwater environments, are able to adhere to and release from a substrate several times within a second. This reversible adhesion relies on adhesive organs comprised of three cell types: an adhesive gland cell, a releasing gland cell, and an anchor cell, which is a modified epidermal cell responsible for structural support. However, nothing is currently known about the molecules that are involved in this adhesion process.ResultsIn this study we present the detailed morphology of the adhesive organs of the free-living marine flatworm Macrostomum lignano. About 130 adhesive organs are located in a horse-shoe-shaped arc along the ventral side of the tail plate. Each organ consists of exactly three cells, an adhesive gland cell, a releasing gland cell, and an anchor cell. The necks of the two gland cells penetrate the anchor cell through a common pore. Modified microvilli of the anchor cell form a collar surrounding the necks of the adhesive- and releasing glands, jointly forming the papilla, the outer visible part of the adhesive organs. Next, we identified an intermediate filament (IF) gene, macif1, which is expressed in the anchor cells. RNA interference mediated knock-down resulted in the first experimentally induced non-adhesion phenotype in any marine animal. Specifically, the absence of intermediate filaments in the anchor cells led to papillae with open tips, a reduction of the cytoskeleton network, a decline in hemidesmosomal connections, and to shortened microvilli containing less actin.ConclusionOur findings reveal an elaborate biological adhesion system in a free-living flatworm, which permits impressively rapid temporary adhesion-release performance in the marine environment. We demonstrate that the structural integrity of the supportive cell, the anchor cell, is essential for this adhesion process: the knock-down of the anchor cell-specific intermediate filament gene resulted in the inability of the animals to adhere. The RNAi mediated changes of the anchor cell morphology are comparable to situations observed in human gut epithelia. Therefore, our current findings and future investigations using this powerful flatworm model system might contribute to a better understanding of the function of intermediate filaments and their associated human diseases.

Deletion of K1/K10 does not impair epidermal stratification but affects desmosomal structure and nuclear integrity

Keratins K1 and K10 are the most abundant proteins in the upper epidermis where they polymerize to form intermediate filaments (IFs). In addition to their well-established function in providing epidermal stability, K1/K10 (i.e. the dimer between K1 and K10) IFs are supposed to be important for terminal epidermal differentiation and barrier formation. It was previously shown that the imbalanced deletion of one of the partner keratins, K10, disturbed epidermal homoeostasis, although stability was provided by compensatory upregulation of K5/K14, which formed IFs together with the remaining K1. Here, we show that deletion of both partner keratins, K1 and K10, results in lethal postnatal skin fragility in mice. Krt1(-/-);Krt10(-/-) mice revealed that K1/K10 IFs are unexpectedly dispensable for epidermal stratification. Although the stratum corneum was less compact and cornified envelope differentiation was impaired, a dye exclusion assay showed that the development of a functional water barrier was surprisingly independent from the presence of K1/K10 IFs. The deletion of K1/K10 was not compensated by any other keratin pair such as the basal epidermal keratins K5/K14, and electron microscopy revealed total absence of IFs in the suprabasal epidermis. Although plakoglobin was unchanged, the expression of the desmosomal proteins desmoplakin, desmocollin 1 and desmoglein 1 were altered and suprabasal desmosomes were smaller in Krt1(-/-);Krt10(-/-) than in wild-type epidermis suggesting an involvement of K1/K10 IFs in desmosome dynamics. Furthermore, Krt1(-/-);Krt10(-/-) mice showed premature loss of nuclei during epidermal differentiation and lower levels of emerin, lamin A/C and Sun1, revealing a previously unknown function for IFs in maintaining nuclear integrity in the upper epidermis.

Tracking melanosomes inside a cell to study molecular motors and their interaction

Cells known as melanophores contain melanosomes, which are membrane organelles filled with melanin, a dark, nonfluorescent pigment. Melanophores aggregate or disperse their melanosomes when the host needs to change its color in response to the environment (e.g., camouflage or social interactions). Melanosome transport in cultured Xenopus melanophores is mediated by myosin V, heterotrimeric kinesin-2, and cytoplasmic dynein. Here, we describe a technique for tracking individual motors of each type, both individually and in their interaction, with high spatial (approximately 2 nm) and temporal (approximately 1 msec) localization accuracy. This method enabled us to observe (i) stepwise movement of kinesin-2 with an average step size of 8 nm; (ii) smoother melanosome transport (with fewer pauses), in the absence of intermediate filaments (IFs); and (iii) motors of actin filaments and microtubules working on the same cargo nearly simultaneously, indicating that a diffusive step is not needed between the two systems of transport. In concert with our previous report, our results also show that dynein-driven retrograde movement occurs in 8-nm steps. Furthermore, previous studies have shown that melanosomes carried by myosin V move 35 nm in a stepwise fashion in which the step rise-times can be as long as 80 msec. We observed 35-nm myosin V steps in melanophores containing no IFs. We find that myosin V steps occur faster in the absence of IFs, indicating that the IF network physically hinders organelle transport.

Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

Ezrin connects the apical F-actin scaffold to membrane proteins in the apical brush border of intestinal epithelial cells. Yet, the mechanisms that recruit ezrin to the apical domain remain obscure. Using stable CACO-2 transfectants expressing keratin 8 (K8) antisense RNA under a tetracycline-responsive element, we showed that the actin-ezrin scaffold cannot assemble in the absence of intermediate filaments (IFs). Overexpression of ezrin partially rescued this phenotype. Overexpression of K8 in mice also disrupted the assembly of the brush border, but ezrin distributed away from the apical membrane in spots along supernumerary IFs. In cytochalasin D-treated cells ezrin localized to a subapical compartment and coimmunoprecipitated with IFs. Overexpression of ezrin in undifferentiated cells showed a Triton-insoluble ezrin compartment negative for phospho-T567 (dormant) ezrin visualized as spots along IFs. Pulse-chase analysis showed that Triton-insoluble, newly synthesized ezrin transiently coimmunoprecipitates with IFs during the first 30 min of the chase. Dormant, but not active (p-T567), ezrin bound in vitro to isolated denatured keratins in Far-Western analysis and to native IFs in pull-down assays. We conclude that a transient association to IFs is an early step in the polarized assembly of apical ezrin in intestinal epithelial cells.

Under stress, the absence of intermediate filaments from Müller cells in the retina has structural and functional consequences.

In epithelial and muscle cells, intermediate filaments (IFs) are important for resistance to mechanical stress. The aim of this study was to elucidate whether IFs are also important for providing resistance to mechanical stress in the Müller cells of the retina and whether this has any pathophysiological consequences. We used mice deficient in IF proteins glial fibrillary acidic protein and/or vimentin (GFAP(-/-), Vim(-/-) and GFAP(-/-) Vim(-/-)), and stress on the retina was applied by excision of the eyes immediately post mortem (compared with in situ fixation) or by inducing a neovascular response to oxygen-induced retinopathy (OIR). The structure of unchallenged retinas was normal, but mechanical stress caused local separation of the inner limiting membrane (ILM) and adjacent tissue from the rest of the retina in GFAP(-/-) Vim(-/-) mice and, to a lesser extent, in Vim(-/-) mice. This detachment occurred within the endfeet of Müller cells, structures normally rich in IFs but IF-free in GFAP(-/-) Vim(-/-) mice. Hypoxia-induced neovascularization was comparable in all groups of mice with respect to the retinal surface area occupied by new vessels. However, the vessels traversed the ILM and penetrated the vitreous body less frequently than in wild-type retinas (31-55% in Vim(-/-), 66-79% in GFAP(-/-) Vim(-/-)). We conclude that IFs are important for maintaining the mechanical integrity of Müller-cell endfeet and the inner retinal layers under a mechanical challenge. Furthermore, the absence of IFs in Müller cells leads to an abnormal response of the vascular system to ischemia, specifically decreased ability of newly formed blood vessels to traverse the ILM.

Response to Quinlan and Nilsson: Astroglia sitting at the controls?

We agree with Roy Quinlan and Michael Nilsson that selective disruption of intermediate filaments using a ‘magic bullet’, such as RNA interference (RNAi) technologies or peptide inhibition, might improve regeneration in the retina [1]. Quinlan and Nilsson also raised the issue of the wider implications of our data for neuroregeneration in humans. Our work suggests that astroglia are pivotal in determining the permissiveness of the adult mammalian CNS for at least some aspects of neuroregeneration, such as the migration or integration of immature neurons and neurite outgrowth. Consistent with this possibility, Fred Gage and colleagues suggested that adult neurogenesis occurs only at specific regions in the CNS (i.e. the subventricular zone and the dentate gyrus of the hippocampus) because astrocytes allow it there and not elsewhere [2]. Understanding on a molecular level how astrocytes induce or restrict regeneration might provide the key to controlling regeneration in the clinic. Comparison of gene expression in the regeneration-supporting environment of GFAPVim mice with that in the standard regeneration-restrictive adult CNS might lead to the identification of molecular pathways that control CNS regeneration. Naturally, such knowledge might provide targets that are more amenable to therapy than intermediate filaments themselves. Why is the regenerative capacity of the CNS limited, and is it even wise to contemplate playing with it? We would like to think that the increasing complexity of multicellular organisms – in particular, the development of the ability to acquire, process, store and use information – required tight control over the plasticity of the neuronal network. Hence, evolution might have opted for less regeneration. This might mean that a system of brakes on regeneration evolved in higher organisms. If so, where should we look for them? One such brake might be neuron-specific. The expression profiles of many genes in mammalian CNS neurons change during ontogenesis. In several cases, this change coincides with the loss of neuronal regenerative capacity around birth [3,4]. For example, in rodents Bcl-2 expression is high during an early developmental stage and declines at birth [5]. In mice, overexpression of Bcl-2 prolonged the regeneration-permissive window after birth [6]. Conceivably, the first brake on neuroregeneration is an intrinsic program that makes it very difficult for neurons to regenerate after a certain point during ontogenesis. We propose that mature astrocytes constitute a second brake. Considered since Rudolf Virchow’s time to be merely glue between the active and multifunctional neurons, astroglia increasingly appear to be at the controls. They modulate synaptogenesis [7], set the threshold for neuronal signaling [8] and have multiple roles in CNS diseases. In addition, immature glialfibrillary acidic protein (GFAP)-positive glial cells seem to be the predominant stem cells in the adult CNS [9]. Astrocytes become activated in essentially all known CNS pathologies [10]. This ‘reactive gliosis’ remains enigmatic but its complexity certainly extends beyond the questionable century-old concept of a ‘glial scar’ as a mechanical obstacle to axonal regeneration. Previously, we showed that absence of intermediate filaments in astrocytes affects the quality of post-traumatic glial scars [11,12]. In the highlighted paper [13], we show that it renders the CNS environment more permissive to the integration of transplanted cells and the outgrowth of their neurites. Thus, mature astrocytes with their characteristic GFAP expression might constitute the second brake on CNS regeneration, a brake that becomes progressively stronger as the CNS matures and ages. Is there a third brake on CNS regeneration? Many have been proposed, including the presence of myelin-associated growth-inhibiting molecules or the lack of neurotrophic and growth-supporting factors [14]. We suggest looking also in the immune system. A growing body of experimental evidence shows that the immune response can promote tissue repair in CNS injury and disease [15,16]. In the optic nerve crush model, simultaneous injury of the eye lens enhances the regeneration of axons of the ganglion cells [17]. Lens injury leads to macrophage accumulation in the vitreous body, and intravitreal injections of a monocyte-activating agent exert a comparable regeneration-promoting effect [17]. Macrophage-conditioned medium also induced axonal regeneration in vitro [18]. However, activated microglia inhibit both baseline and injury-triggered hippocampal neurogenesis in the adult [19]. Interestingly, the complement system, an evolutionarily ancient arm of humoral immunity, has recently been linked to regeneration outside the CNS [20,21]. Although the picture is still very incomplete, modulation of the immune response might prove to be important in controlling neuroregeneration in the adult. Corresponding authors: Milos Pekny (milos.pekny@medkem.gu.se), Dong Feng Chen (dfchen@vision.eri.harvard.edu). Update TRENDS in Neurosciences Vol.27 No.5 May 2004 243

Intermediate filaments in the testis of the teleost mosquito fish Gambusia affinis holbrooki: a light and electron microscope immunocytochemical study and Western blotting analysis

A light and electron microscope immunocytochemical study and Western blotting analysis has been performed on intermediate filaments (vimentin, desmin and cytokeratins) in the testis of the teleost fish Gambusia affinis holbrooki. An immunoreaction to vimentin was observed in the epithelium of the efferent ducts, testicular canal and their surrounding peritubular cells. Positive vimentin immunostaining was also observed in the cells located around seminiferous tubules (boundary cells), Leydig cells, interstitial fibroblasts, chromatophores, and blood vessel endothelial cells. In contrast to mammals, no vimentin immunoreactivity was found in the Sertoli cells. Immunoreactivity to desmin was weak in the epithelial cells of the efferent ducts and testicular canal and intense in the peritubular cells that surrounded these ducts. Desmin immunoreactivity was also observed in the seminiferous tubule boundary cells. The immunoreactivity was weak in the boundary cells that surrounded germ cell cysts containing spermatogonia or spermatocytes and intense in the boundary cells around cysts with elongated or mature spermatids. Immunoreactivity towards cytokeratins was observed only in testicular blood vessels. Cytokeratin immunolabelling was intense in the endothelium and weak in the vascular smooth muscle cells. No cytokeratin immunoreactivity was found in the Sertoli cells, germ cells, interstitial cells or in the efferent duct epithelium. The absence of intermediate filaments in the Sertoli cells, the absence of cytokeratins in the epithelium of the sperm excretory ducts, and the presence of desmin filaments in these epithelial cells are the most important differences with regards to the intermediate filament phenotype in mammalian testes.

The presence or absence of a vimentin-type intermediate filament network affects the shape of the nucleus in human SW-13 cells.

Human SW-13 cells express the intermediate filament protein vimentin in a mosaic pattern (Hedberg, K. K. and Chen, L. B. (1986). Exp. Cell Res. 163, 509-517). We have isolated SW-13 clones that do (vim+) or do not (vim-) synthesize vimentin as analyzed using anti-intermediate filament immunofluorescence, electron microscopy and two-dimensional gel analysis of detergent-extracted preparations. Vimentin is the only cytoplasmic intermediate filament protein present in the vim+ cells, and the vim- cells do not contain any detectable cytoplasmic intermediate filament system. The presence or absence of intermediate filaments did not observably affect the distribution of mitochondria, endoplasmic reticulum, microtubules or actin stress fibers when these structures were visualized by fluorescence microscopy. However, electron microscopy and anti-lamin A/C immunofluorescence studies showed that nuclear morphology in vim- cells was frequently characterized by large folds or invaginations, while vim+ cells had a more regular or smooth nuclear shape. When vim- cells were transfected with a mouse vimentin expression plasmid, the synthesis of a mouse vimentin filament network restored the smooth nuclear morphology characteristic of vim+ cells. Conversely, when vim+ cells were transfected with a carboxy-terminally truncated mutant vimentin, expression of the mutant protein disrupted the organization of the endogenous vimentin filaments and resulted in nuclei with a prominently invaginated morphology. These results indicated that in SW-13 cells the vimentin filament system affects the shape of the nucleus.

Cytoskeletal organization and tissue patterns of epithelia in the sponge Ephydatia mülleri.

Comparative morphological studies on cytoskeletal patterns of sponge basal epithelium at the tissue level have been performed by diverse methods, including immunofluorescence microscopy, and scanning and transmission electron microscopy of stained whole mounts, thin sections or replicas. These methods give results consistent with each other and show the importance of actin assemblies, which function in addition to the microtubular system and in the absence of intermediate filaments. Actin microfilaments indeed are involved in the formation of cables and networks closely associated with the plasma membrane. Both the cables and the networks result from arrangements of microfilaments into bundles of variable size, and the two types of assembly are probably interconvertible. Microfilaments appear to be implicated in the establishment of spot desmosomes and as devices for cell-to-substratum attachment. Due to the desmosomal articulations from cell to cell, the actin cytoskeleton is framed throughout the complete epithelium. It supports the unitary nature of the entire tissue, which is constructed and functions as a whole. It therefore establishes the "histoskeleton" of basic epithelial tissues. The histoskeleton is involved in all epithelial activities but is not uniformly organized into identical cell patterns at the tissue level because activities are sequential and not synchronized in all cells. Similar cytoskeletal patterns exist only in groups of cells, and this suggests that, at a given time, the multiple functions of the epithelium may be mediated by the occurrence of several multicellular functional units within a single epithelial tissue.

Absence of intermediate filaments in a human adrenal cortex carcinoma-derived cell line

Subclones of a human adrenal cortex carcinoma-derived cell line (SW13) are described which by immunofluorescence lack detectable expression of any of the five known classes of intermediate filament (IF) proteins. Further investigation for vimentin and keratins in these subclones by two-dimensional gel analysis and by immunoblotting gave results consistent with the immunofluorescence results. Despite the apparent absence of IFs, SW13 subclones have organized actin and microtubule cytoskeletal networks, maintain an epithelial shape and colony pattern, and grow well in culture. Although a rat hepatoma cell line which similarly appears to have ceased IF expression has been reported [1], this is the first such report of a human cell line. Although rare, these cases provide evidence that IFs in general are not essential to growth in culture, nor are the keratin-containing IFs in particular necessarily responsible for the ‘cobblestone’ morphology or colony-type growth pattern characteristic of cultured epithelial cells.

Absence of Intermediate Filaments in a Human Adrenal Cortex Carcinoma‐Derived Cell Linea

Absence of Intermediate Filaments in a Human Adrenal Cortex Carcinoma‐Derived Cell Line^a

Effect of retinoic acid on growth and morphological differentiation of mouse NB2a neuroblastoma cells in culture

We have characterized the effects of retinoic acid (RA) on the growth, morphology and biosynthesis of cytoskeletal proteins in NB2a mouse neuroblastoma cells. In addition, the morphological and biochemical changes were compared to those induced by dibutyryl cyclic AMP (db cAMP). Growth inhibition by RA was concentration-dependent and was first detected 24 h after addition of RA. The proliferation of RA-treated NB2a was more dependent on serum than was the proliferation of untreated cultures and RA decreased the saturation density of NB2a cells grown in serum. Morphological changes induced by RA include the formation of an elaborate network of branching neurites in NB2a cells. In contrast, neurites induced by db cAMP or serum deprivation were bipolar and unbranching. Ultrastructural observations of neurites induced by RA revealed dendritic characteristics such as polysomes, spines and absence of intermediate filaments, while neurites induced by db cAMP had axonal characteristics such as filament bundles, absence of ribosomes, and the formation of membrane densities when neurite endings contacted another cell body. These morphological differences were also reflected in a number of changes in the biosynthesis of cytoskeletal proteins. These results suggest that NB2a cells treated with RA and db cAMP are a model system for the study of distinct stages of differentiation.