Dual roles of intermediate filaments in apoptosis

New roles have emerged recently for intermediate filaments (IFs), namely in modulating cell adhesion and growth, and providing resistance to various forms of stress and to apoptosis. In this context, we first summarize findings on the IF association with the cell response to mechanical stress and growth stimulation, in light of growth-related signaling events that are relevant to death-receptor engagement. We then address the molecular mechanisms by which IFs can provide cell resistance to apoptosis initiated by death-receptor stimulation and to necrosis triggered by excessive oxidative stress. In the same way, we examine IF involvement, along with cytolinker participation, in sequential caspase-mediated protein cleavages that are part of the overall cell death execution, particularly those that generate new functional IF protein fragments and uncover neoantigen markers. Finally, we report on the usefulness of these markers as diagnostic tools for disease-related aspects of apoptosis in humans. Clearly, the data accumulated in recent years provide new and significant insights into the multiple functions of IFs, particularly their dual roles in cell response to apoptotic insults.     

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.     

Intermediate filament cytoskeleton of the liver in health and disease

Intermediate filaments (IFs) represent the largest cytoskeletal gene family comprising approximately 70 genes expressed in tissue specific manner. In addition to scaffolding function, they form complex signaling platforms and interact with various kinases, adaptor, and apoptotic proteins. IFs are established cytoprotectants and IF variants are associated with >30 human diseases. Furthermore, IF-containing inclusion bodies are characteristic features of several neurodegenerative, muscular, and other disorders. Acidic (type I) and basic keratins (type II) build obligatory type I and type II heteropolymers and are expressed in epithelial cells. Adult hepatocytes contain K8 and K18 as their only cytoplasmic IF pair, whereas cholangiocytes express K7 and K19 in addition. K8/K18-deficient animals exhibit a marked susceptibility to various toxic agents and Fas-induced apoptosis. In humans, K8/K18 variants predispose to development of end-stage liver disease and acute liver failure (ALF). K8/K18 variants also associate with development of liver fibrosis in patients with chronic hepatitis C. Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated K8/K18, chaperones and sequestosome1/p62 (p62) as their major constituents. MDBs are found in various liver diseases including alcoholic and non-alcoholic steatohepatitis and can be formed in mice by feeding hepatotoxic substances griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDBs also arise in cell culture after transfection with K8/K18, ubiquitin, and p62. Major factors that determine MDB formation in vivo are the type of stress (with oxidative stress as a major player), the extent of stress-induced protein misfolding and resulting chaperone, proteasome and autophagy overload, keratin 8 excess, transglutaminase activation with transamidation of keratin 8 and p62 upregulation.     

Towards a molecular description of intermediate filament structure and assembly

Intermediate filaments (IFs) represent one of the prominent cytoskeletal elements of metazoan cells. Their constituent proteins are coded by a multigene family, whose members are expressed in complex patterns that are controlled by developmental programs of differentiation. Hence, IF proteins found in epidermis differ significantly from those in muscle or neuronal tissues. Due to their fibrous nature, which stems from a fairly conserved central alpha-helical coiled-coil rod domain, IF proteins have long resisted crystallization and thus determination of their atomic structure. Since they represent the primary structural elements that determine the shape of the nucleus and the cell more generally, a major challenge is to arrive at a more rational understanding of how their nanomechanical properties effect the stability and plasticity of cells and tissues. Here, we review recent structural results of the coiled-coil dimer, assembly intermediates and growing filaments that have been obtained by a hybrid methods approach involving a rigorous combination of X-ray crystallography, small angle X-ray scattering, cryo-electron tomography, computational analysis and molecular modeling.     

Recent insight into intermediate filament structure

Intermediate filaments (IFs) are key players in multiple cellular processes throughout human tissues. Their biochemical and structural properties are important for understanding filament assembly mechanisms, for interactions between IFs and binding partners, and for developing pharmacological agents that target IFs. IF proteins share a conserved coiled-coil central-rod domain flanked by variable N-terminal ‘head’ and C-terminal ‘tail’ domains. There have been several recent advances in our understanding of IF structure from the study of keratins, glial fibrillary acidic protein, and lamin. These include discoveries of (i) a knob–pocket tetramer assembly mechanism in coil 1B; (ii) a lamin-specific coil 1B insert providing a one-half superhelix turn; (iii) helical, yet flexible, linkers within the rod domain; and (iv) the identification of coil 2B residues required for mature filament assembly. Furthermore, the head and tail domains of some IFs contain low-complexity aromatic-rich kinked segments, and structures of IFs with binding partners show electrostatic surfaces are a major contributor to complex formation. These new data advance the connection between IF structure, pathologic mutations, and clinical diseases in humans.     

Topographic regulation of neuronal intermediate filaments by phosphorylation, role of peptidyl-prolyl isomerase 1: significance in neurodegeneration

The neuronal cytoskeleton is tightly regulated by phosphorylation and dephosphorylation reactions mediated by numerous associated kinases, phosphatases and their regulators. Defects in the relative kinase and phosphatase activities and/or deregulation of compartment-specific phosphorylation result in neurodegenerative disorders. The largest family of cytoskeletal proteins in mammalian cells is the superfamily of intermediate filaments (IFs). The neurofilament (NF) proteins are the major IFs. Aggregated forms of hyperphosphorylated tau and phosphorylated NFs are found in pathological cell body accumulations in the central nervous system of patients suffering from Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis. The precise mechanisms for this compartment-specific phosphorylation of cytoskeletal proteins are not completely understood. In this review, we focus on the mechanisms of neurofilament phosphorylation in normal physiology and neurodegenerative diseases. We also address the recent breakthroughs in our understanding the role of different kinases and phosphatases involved in regulating the phosphorylation status of the NFs. In addition, special emphasis has been given to describe the role of phosphatases and Pin1 in phosphorylation of NFs.     

Intermediate filaments: from cell architecture to nanomechanics

Intermediate filaments (IFs) constitute a major structural element of animal cells. They build two distinct systems, one in the nucleus and one in the cytoplasm. In both cases, their major function is assumed to be that of a mechanical stress absorber and an integrating device for the entire cytoskeleton. In line with this, recent disease mutations in human IF proteins indicate that the nanomechanical properties of cell-type-specific IFs are central to the pathogenesis of diseases as diverse as muscular dystrophy and premature ageing. However, the analysis of these various diseases suggests that IFs also have an important role in cell-type-specific physiological functions.     

Intermediate filaments in cardiomyopathy

Intermediate filament (IF) proteins are critical regulators in health and disease. The discovery of hundreds of mutations in IF genes and posttranslational modifications has been linked to a plethora of human diseases, including, among others, cardiomyopathies, muscular dystrophies, progeria, blistering diseases of the epidermis, and neurodegenerative diseases. The major IF proteins that have been linked to cardiomyopathies and heart failure are the muscle-specific cytoskeletal IF protein desmin and the nuclear IF protein lamin, as a subgroup of the known desminopathies and laminopathies, respectively. The studies so far, both with healthy and diseased heart, have demonstrated the importance of these IF protein networks in intracellular and intercellular integration of structure and function, mechanotransduction and gene activation, cardiomyocyte differentiation and survival, mitochondrial homeostasis, and regulation of metabolism. The high coordination of all these processes is obviously of great importance for the maintenance of proper, life-lasting, and continuous contraction of this highly organized cardiac striated muscle and consequently a healthy heart. In this review, we will cover most known information on the role of IFs in the above processes and how their deficiency or disruption leads to cardiomyopathy and heart failure.     

Suppression of Extensive Neurofilament Phosphorylation Rescues α-Internexin/Peripherin-Overexpressing PC12 Cells from Neuronal Cell Death

Intermediate filament (IF) overproduction induces abnormal accumulation of neuronal IF, which is a pathological indicator of some neurodegenerative disorders. In our study, α-Internexin- and peripherin-overexpressing PC12 cells (pINT-EGFP and pEGFP-peripherin) were used as models to study neuropathological pathways responsible for neurodegenerative diseases. Microarray data revealed that Cdk5-related genes were downregulated and Cdk5 regulatory subunit-associated protein 3 (GSK-3α and GSK-3β) were upregulated in pINT-EGFP cells. Increased expression of phosphorylated neurofilament and aberrant activation of Cdk5 and GSK-3β were detected in both pEGFP-peripherin and pINT-EGFP cells by Western blotting. In addition, pharmacological approaches to retaining viability of pINT-EGFP and pEGFP-peripherin cells were examined. Treatment with Cdk5 inhibitor and GSK-3β inhibitor significantly suppressed neuronal death. Dynamic changes of disaggregation of EGFP-peripherin and decrease in green fluorescence intensity were observed in pEGFP-peripherin and pINT-EGFP cells by confocal microscopy after GSK-3β inhibitor treatment. We conclude that inhibition of Cdk5 and GSK-3β suppresses neurofilament phosphorylation, slows down the accumulation of neuronal IF in the cytoplasm, and subsequently avoids damages to cell organelles. The results suggest that suppression of extensive neurofilament phosphorylation may be a potential strategy for ameliorating neuron death. The suppression of hyperphosphorylation of neuronal cytoskeletons with kinase inhibitors could be one of potential therapeutic treatments for neurodegenerative diseases.     

Nestin promotes the phosphorylation-dependent disassembly of vimentin intermediate filaments during mitosis

The expression of the intermediate filament (IF) protein nestin is closely associated with rapidly proliferating progenitor cells during neurogenesis and myogenesis, but little is known about its function. In this study, we examine the effects of nestin expression on the assembly state of vimentin IFs in nestin-free cells. Nestin is introduced by transient transfection and is positively correlated with the disassembly of vimentin IFs into nonfilamentous aggregates or particles in mitotic but not interphase cells. This nestin-mediated disassembly of IFs is dependent on the phosphorylation of vimentin by the maturation/M-phase-promoting factor at ser-55 in the amino-terminal head domain. In addition, the disassembly of vimentin IFs during mitosis appears to be a unique feature of nestin-expressing cell types. Furthermore, when the expression of nestin is downregulated by the nestin-specific small interfering RNA in nestin-expressing cells, vimentin IFs remain assembled throughout all stages of mitosis. Previous studies suggest that nonfilamentous vimentin particles are IF precursors and can be transported rapidly between different cytoplasmic compartments along microtubule tracks. On the basis of these observations, we speculate that nestin may play a role in the trafficking and distribution of IF proteins and potentially other cellular factors to daughter cells during progenitor cell division.     

Kinin-mediated inflammation in neurodegenerative disorders

The mediatory role of kinins in both acute and chronic inflammation within nervous tissues has been widely described. Bradykinin, the major representative of these bioactive peptides, is one of a few mediators of inflammation that directly stimulates afferent nerves due to the broad expression of specific kinin receptors in cell types in these tissues. Moreover, kinins may be delivered to a site of injury not only after their production at the endothelium surface but also following their local production through the enzymatic degradation of kininogens at the surface of nerve cells. A strong correlation between inflammatory processes and neurodegeneration has been established. The activation of nerve cells, particularly microglia, in response to injury, trauma or infection initiates a number of reactions in the neuronal neighborhood that can lead to cell death after the prolonged action of inflammatory substances. In recent years, there has been a growing interest in the effects of kinins on neuronal destruction. In these studies, the overexpression of proteins involved in kinin generation or of kinin receptors has been observed in several neurologic disorders including neurodegenerative diseases such Alzheimer's disease and multiple sclerosis as well as disorders associated with a deficiency in cell communication such as epilepsy. This review is focused on recent findings that provide reliable evidence of the mediatory role of kinins in the inflammatory responses associated with different neurological disorders. A deeper understanding of the role of kinins in neurodegenerative diseases is likely to promote the future development of new therapeutic strategies for the control of these disorders. An example of this could be the prospective use of kinin receptor antagonists.     

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.     

"Heads and tails" of intermediate filament phosphorylation: multiple sites and functional insights

Intermediate filaments (IFs) are major components of the mammalian cytoskeleton. They are among the most abundant cellular phosphoproteins; their phosphorylation typically involves multiple sites at repeat or unique motifs, preferentially within the "head" or "tail" domains. Phosphorylation and dephosphorylation are essential for the regulation of IF dynamics by modulating the intrinsic properties of IFs: solubility, conformation and filament organization, and, in addition, for the regulation of other IF post-translational modifications. These phosphorylation-regulated properties dictate generalized and context-dependent IF functions that reflect their tissue-specific expression. Most important among IF phosphorylation-mediated functions are the regulation of IF cellular or subcellular compartmentalization, levels and turnover, binding with associated proteins, susceptibility to cell stresses (including apoptosis), tissue-specific functions and IF-associated disease pathogenesis (where IF hyperphosphorylation also serves as a tissue-injury marker).     

Karyoptosis: A novel type of cell death caused by chronic autophagy inhibition

Macroautophagy/autophagy influences onset and progression of several human neurodegenerative diseases, because of its critical role as a regulator of neuronal proteostasis and organelle quality control. In many neurodegenerative diseases, impairment in autophagy is thought to play a fundamental part in the terminal phases of cellular degeneration and death. However, the ultimate mechanism of neuronal cell death remains elusive. In a recent study we have identified a new form of regulated cell death, which arises upon autophagy inhibition.     

Keratin-containing inclusions affect cell morphology and distribution of cytosolic cellular components

Many neurodegenerative diseases are characterized by the presence of protein aggregates bundled with intermediate filaments (IFs) and similar structures, known as Mallory bodies (MBs), are observed in various liver diseases. IFs are anchored at desmosomes and hemidesmosomes, however, interactions with other intercellular junctions have not been determined. We investigated the effect of IF inclusions on junction-associated and cytosolic proteins in various cultured cells. We performed gene transfection of the green fluorescent protein (GFP)-tagged cytokeratin (CK) 18 mutant arg89cys (GFP-CK18 R89C) in cultured cells and observed CK aggregations as well as loss of IF networks. Among various junction-associated proteins, zonula occludens-1 and beta-catenin were colocalized with CK aggregates on immunofluorescent analyses. Similar results were obtained on immunostaining for cytosolic proteins, 14-3-3 zeta protein, glucose-6-phosphate dehydrogenase and DsRed. E-cadherin, a basolateral membrane protein in polarized epithelia, was present on both the apical and basolateral domains in GFP-CK18 R89C-transfected cells. Furthermore, cells containing CK aggregates were significantly larger than GFP-tagged wild type CK18 (GFP-WT CK18)-transfected or non-transfected cells (P < 0.01) and sometimes their morphology was significantly altered. Our data indicate that CK aggregates affect not only cell morphology but also the localization of various cytosolic components, which may affect the cellular function.     

Brain CHIP: removing the culprits in neurodegenerative disease

A factor that is common to the most-frequent neurodegenerative diseases is the accumulation of abnormal proteins that are associated with cellular dysfunction. Contrary to years of speculation, recent evidence suggests that soluble intermediates--not the visible pathological aggregates associated with disease--are the cause of neurotoxicity. These findings suggest that aggregate formation might be an adaptive stress response that is facilitated by neuronal protein triage molecules. In particular, the molecular co-chaperone CHIP (C terminus of HSC70-interacting protein) has been linked to several of these disorders, serving as a crucial catalyst for the ubiquitination of several heat shock protein (HSP)70 client proteins that are involved in neurodegenerative disease. Therefore, understanding the mechanisms that are involved in CHIP-mediated protein trafficking might provide invaluable clues to neuronal function, both in normal and diseased conditions.     

Molecular mechanism of intermediate filament recognition by plakin proteins

The plakin family of cytolinkers interacts with intermediate filaments (IFs) through plakin repeat domain (PRD) and linker modules. Recent structure/function studies have established the molecular basis of envoplakin-PRD and periplakin-linker interactions with vimentin. Both plakin modules share a broad basic groove which recognizes acidic rod elements on IFs, a mechanism that is applicable to other plakin family members. This review postulates a universal IF engagement mechanism that illuminates the specific effects of pathogenic mutations associated with diseases including arrhythmogenic right ventricular cardiomyopathy, and reveals how diverse plakin proteins offer tailored IF tethering to ensure stable, dynamic and regulated cellular structures.     

Cracks in the foundation: keratin filaments and genetic disease

In the past three years, defects in the genes that encode intermediate filament (IF) proteins have been found to be responsible for some inherited skin diseases, and others have been implicated in certain motor neuron diseases and cardiomyopathies. This article reviews how knowledge of IF structure led to the discovery of genetic disorders of IFs, and how the clinical manifestations of these diseases have confirmed the notion that IFs provide the mechanical strength of cells.     

Rearrangement of the vimentin cytoskeleton during adipose conversion: formation of an intermediate filament cage around lipid globules

During adipose conversion of murine 3T3-L1 cells, the arrangement of vimentin intermediate filaments (IFs) changes from an extended fibrillar state to a complex cage formation tightly associated with the forming lipid globules. The fully developed cage complex surrounding the lipid globule consists of a monolayer of groups of regularly spaced vimentin IFs that in turn is closely ensheathed by a special endoplasmic reticulum cisterna. The same IF cage is also seen in other adipocytes in culture and in tissues. The specificity of the association of lipid globules with vimentin IFs during adipose conversion is discussed as a special form of compartmentalization supporting adipogenesis and is taken as an example of a possible IF function in relation to a cell differentiation process.     

Patterns of expression of cytoskeletal proteins in human thyroid gland and thyroid carcinomas

By two-dimensional gel electrophoresis of proteins insoluble in detergents and high-salt buffer and immunofluorescence microscopy with a panel of polypeptide-specific antibodies to proteins of intermediate filaments (IF) and desmosomes, we have characterized the cytoskeletons of normal human thyroid gland, several kinds of benign lesion (goiter, Hashimoto's and Graves' diseases, adenomas), and the major thyroid carcinomas (follicular, papillary, medullary, and anaplastic). In all these tissues, desmoplakins and cytokeratins 7, 8, 18, and 19 were identified. While cytokeratins 8 and 18 occurred in all epithelial cells and cytokeratin 7 was also rather widespread, cytokeratin 19 occurred in amounts variable between the different types of tissues and in normal thyroid gland was restricted to certain clusters of follicular epithelial cells. Of all samples studied, in none did we detect cytokeratins commonly associated with stratified epithelia such as cytokeratins 4-6, 10, and 13-17, indicating that these are infrequent, if at all present, in such tissues. Coexpression of cytokeratins with vimentin appears to occur constitutively in follicular epithelial cells of normal thyroid gland and is also frequent in the diverse carcinomas, though to various degrees. Medullary carcinomas are exceptional, not only because they express neuroendocrine markers, but also because they coexpress combinations of cytokeratin IFs with neurofilaments and/or vimentin IFs in some cases, but not all. The results are discussed in relation to states of cell differentiation in normal and diseased thyroid gland and with respect to their value in tumor diagnosis.