Dynamic organisation of intermediate filaments and associated proteins during the cell cycle

Intermediate filaments, which form the structural framework of both the cytoskeleton and the nuclear lamina in most eukaryotic cells, have been found to be highly dynamic structures. A continuous exchange of subunit proteins at the filament surface and a stabilisation of soluble subunits by chaperone-type proteins may modulate filament structure and plasticity. Recent studies on the cell cycle-dependent interaction of intermediate filaments with associated proteins, and a detailed analysis of intermediate filament phosphorylation in defined subcellular locations at various stages of mitosis, have brought new insights into the molecular mechanisms involved in the mitotic reorganisation of intermediate filaments. Some of these studies have allowed new speculations about the possible cellular functions of cytoplasmic intermediate filaments, and increased our understanding of the specific functions of the lamins and the lamina-associated membrane proteins in the post-mitotic reassembly of the nucleus.     

Changes in the Cytoskeletal and Nuclear Matrix Proteins in Rat Hepatocyte Neoplastic Nodules in Their Relation to the Process of Transformation

In a previous paper (Barboroet al.,1993,Biophys. J.65, 1690–1699) we have shown that cancer development in the resistant hepatocyte model of Solt and Farber is characterized by the progressive unfolding of the higher-order structure of chromatin. A possible functional role of decondensation phenomena in cell transformation cannot be ruled out. Genetic activation involves the relaxation of the superstructure of chromatin, which may be, at least in part, modulated by its interaction with the nuclear matrix. Moreover, recent observations suggest that gene expression can be stimulated by alterations in the organization of the cytoskeleton. Therefore, we have characterized the changes in composition that the nuclear matrix–intermediate filament complex undergoes during the evolution of rat hepatocyte nodules. Dramatic changes in the expression of both the nuclear matrix and intermediate filament proteins occur during transformation; they are, however, related in a different way to the stages of carcinogenesis. Several new nuclear matrix proteins appear in early nodules, isolated 9 weeks after initiation. The subsequent evolution of persistent nodules is also characterized by discrete changes in the composition. Thus, the new synthesis of nuclear matrix proteins reflects the emergence of successive cellular populations, in line with the recent finding that a subset of components of the nuclear matrix is cell type-specific. In contrast, intermediate filament proteins undergo continuing changes. A new keratin with apparent molecular weight of 39 kDa, analogous to human keratin 19, appears in early nodules, and its expression steadily increases up to the 32nd week from initiation; at the same time, the amount of the proteolytic fragments of keratins A and D increases sharply. These findings suggest that the inappropriate expression of keratin 19 may be involved in the epigenetic activation of new cellular programs, through the rearrangement of the cytoskeleton which in turn may perturb nuclear matrix function.     

Association of syncoilin and desmin: linking intermediate filament proteins to the dystrophin-associated protein complex.

We recently identified a novel protein called syncoilin, a putative intermediate filament protein that interacts with alpha-dystrobrevin, a member of the dystrophin-associated protein complex. Syncoilin is found at the neuromuscular junction, sarcolemma, and Z-lines and is thought to be important for muscle fiber integrity. Based on the similar protein structure and cellular localization of syncoilin and desmin, we proposed that these proteins interact in vivo. The data presented confirm an interaction between syncoilin and desmin and demonstrate their co-localization in skeletal muscle. Intriguingly, whereas these proteins interact, COS-7 cell expression studies show that desmin and syncoilin do not assemble into heterofilaments. Furthermore, fractionation assay and immunofluorescence study of H2K myoblasts and myotubes suggest that, unlike typical intermediate filament proteins, syncoilin does not participate in filament formation with any protein. However, it is possible that syncoilin is involved in the anchoring of the desmin intermediate filament network at the sarcolemma and the neuromuscular junction. This interaction is likely to be important for maintaining muscle fiber integrity and may also link the dystrophin-associated protein complex to the cytoskeleton. The dysfunction or absence of syncoilin may result in the disruption of the intermediate filament network leading to muscle necrosis. Syncoilin is therefore an ideal candidate gene for muscular dystrophies and desmin-related myopathies.     

A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells

We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximity-dependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximity-dependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment.     

Microinjection of intermediate filament proteins into living cells with and without preexisting intermediate filament network

Human cells grown in monolayer culture were microinjected with intermediate filament subunit proteins. In fibroblasts with a preexisting vimentin network, injected porcine glial fibrillary acidic protein (GFAP) co-localized with the vimentin network within 24 hours. Phosphorylated GFAP variants were found to become dephosphorylated concomitantly with their incorporation into filamentous structures. After microinjection of either porcine GFAP or murine vimentin into human carcinoma cells lacking cytoplasmic intermediate filaments, we observed that different types of filament networks developed. Whereas vimentin was incorporated into short filaments immediately after injection, GFAP was found to aggregate into rodlike structures. This may indicate a differential filament forming ability of these intermediate filament proteins in vivo.     

Expression of intermediate filament proteins in TPA-induced MPC-11 and HL-60 cells.

Under normal culture conditions, the tumor cell lines MPC-11 and HL-60 exhibit high rates of proliferation and show a peculiar expression of intermediate filament proteins as they appear to synthesize only lamin B. A 48-h exposure of murine plasmacytomas MPC-11 to the phorbol ester TPA reduces their growth and induces vimentin synthesis without affecting the composition of their nuclear lamina. When applied to human leukemic promyelocytes HL-60, such treatment promotes their maturation into macrophage-like cells: their proliferative ability is suppressed, a differentiated phenotype is developed, and their content in intermediate filament proteins now includes vimentin and a full complement of lamins A, B, and C. In the present study, a kinetic analysis of vimentin and lamin A/C expression in relation to proliferation and differentiation has been performed in these two cellular systems. Proliferation rates of MPC-11 and HL-60 populations were evaluated by monitoring cell growth and measuring thymidine incorporation. Maturation of HL-60 cells was assessed by Giemsa staining and percentage of adherent cells. Expression of vimentin and lamins A/C was analyzed using immunofluorescence and immunoblotting techniques. Our data show that there is a relationship between the level of vimentin expression and the extent of growth inhibition in both systems. They also suggest that the expression of lamins A/C during the TPA-induced maturation of HL-60 promyelocytes might be part of the processes which lock these cells into the macrophage pathway.     

In vitro assembly properties of mutant and chimeric intermediate filament proteins: insight into the function of sequences in the rod and end domains of IF

The factors and mechanisms regulating assembly of intermediate filament (IF) proteins to produce filaments with their characteristic 10 nm diameter are not fully understood. All IF proteins contain a central rod domain flanked by variable head and tail domains. To elucidate the role that different domains of IF proteins play in filament assembly, we used negative staining and electron microscopy (EM) to study the in vitro assembly properties of purified bacterially expressed IF proteins, in which specific domains of the proteins were either mutated or swapped between a cytoplasmic (mouse neurofilament-light (NF-L) subunit) and nuclear intermediate filament protein (human lamin A). Our results indicate that filament formation is profoundly influenced by the composition of the assembly buffer. Wild type (wt) mouse NF-L formed 10 nm filaments in assembly buffer containing 175 mM NaCl, whereas a mutant deleted of 18 NH2-terminal amino acids failed to assemble under similar conditions. Instead, the mutant assembled efficiently in buffers containing CaCl2 > or = 6 mM forming filaments that were 10 times longer than those formed by wt NF-L, although their diameter was significantly smaller (6-7 nm). These results suggest that the 18 NH2-terminal sequence of NF-L might serve two functions, to inhibit filament elongation and to promote lateral association of NF-L subunits. We also demonstrate that lengthening of the NF-L rod domain, by inserting a 42 aa sequence unique to nuclear IF proteins, does not compromise filament assembly in any noticeable way. Our results suggests that the known inability of nuclear lamin proteins to assemble into 10 nm filaments in vitro cannot derive solely from their longer rod domain. Finally, we demonstrate that the head domain of lamin A can substitute for that of NF-L in filament assembly, whereas substitution of both the head and tail domains of lamins for those of NF-L compromises assembly. Therefore, the effect of lamin A "tail" domain alone, or the synergistic effect of lamin "head" and the "tail" domains together, interferes with assembly into 10-nm filaments.     

Keeping the LINC: the importance of nucleocytoskeletal coupling in intracellular force transmission and cellular function

Providing a stable physical connection between the nucleus and the cytoskeleton is essential for a wide range of cellular functions and it could also participate in mechanosensing by transmitting intra- and extra-cellular mechanical stimuli via the cytoskeleton to the nucleus. Nesprins and SUN proteins, located at the nuclear envelope, form the LINC (linker of nucleoskeleton and cytoskeleton) complex that connects the nucleus to the cytoskeleton; underlying nuclear lamins contribute to anchoring LINC complex components at the nuclear envelope. Disruption of the LINC complex or loss of lamins can result in disturbed perinuclear actin and intermediate filament networks and causes severe functional defects, including impaired nuclear positioning, cell polarization and cell motility. Recent studies have identified the LINC complex as the major force-transmitting element at the nuclear envelope and suggest that many of the aforementioned defects can be attributed to disturbed force transmission between the nucleus and the cytoskeleton. Thus mutations in nesprins, SUN proteins or lamins, which have been linked to muscular dystrophies and cardiomyopathies, may weaken or completely eliminate LINC complex function at the nuclear envelope and result in impaired intracellular force transmission, thereby disrupting critical cellular functions.     

Mouse models of laminopathies

The A‐ and B‐type lamins are nuclear intermediate filament proteins in eukaryotic cells with a broad range of functions, including the organization of nuclear architecture and interaction with proteins in many cellular functions. Over 180 disease‐causing mutations, termed ‘laminopathies,’ have been mapped throughout LMNA, the gene for A‐type lamins in humans. Laminopathies can range from muscular dystrophies, cardiomyopathy, to Hutchinson–Gilford progeria syndrome. A number of mouse lines carrying some of the same mutations as those resulting in human diseases have been established. These LMNA‐related mouse models have provided valuable insights into the functions of lamin A biogenesis and the roles of individual A‐type lamins during tissue development. This review groups these LMNA‐related mouse models into three categories: null mutants, point mutants, and progeroid mutants. We compare their phenotypes and discuss their potential implications in laminopathies and aging.     

The biology of desmin filaments: how do mutations affect their structure, assembly, and organisation?

Desmin, the major intermediate filament (IF) protein of muscle, is evolutionarily highly conserved from shark to man. Recently, an increasing number of mutations of the desmin gene has been described to be associated with human diseases such as certain skeletal and cardiac myopathies. These diseases are histologically characterised by intracellular aggregates containing desmin and various associated proteins. Although there is progress regarding our knowledge on the cellular function of desmin within the cytoskeleton, the impact of each distinct mutation is currently not understood at all. In order to get insight into how such mutations affect filament assembly and their integration into the cytoskeleton we need to establish IF structure at atomic detail. Recent progress in determining the dimer structure of the desmin-related IF-protein vimentin allows us to assess how such mutations may affect desmin filament architecture.     

Intermediate filaments exchange subunits along their length and elongate by end-to-end annealing

Actin filaments and microtubules lengthen and shorten by addition and loss of subunits at their ends, but it is not known whether this is also true for intermediate filaments. In fact, several studies suggest that in vivo, intermediate filaments may lengthen by end-to-end annealing and that addition and loss of subunits is not confined to the filament ends. To test these hypotheses, we investigated the assembly dynamics of neurofilament and vimentin intermediate filament proteins in cultured cells using cell fusion, photobleaching, and photoactivation strategies in combination with conventional and photoactivatable fluorescent fusion proteins. We show that neurofilaments and vimentin filaments lengthen by end-to-end annealing of assembled filaments. We also show that neurofilaments and vimentin filaments incorporate subunits along their length by intercalation into the filament wall with no preferential addition of subunits to the filament ends, a process which we term intercalary subunit exchange.     

Intracellular assembly and sorting of intermediate filament proteins: role of the 42 amino acid lamin insert

Nuclear and cytoplasmic intermediate filament (IF) proteins segregate into two independent cellular networks by mechanisms that are poorly understood. We examined the role of a 42 amino acid (aa) insert unique to vertebrate lamin rod domains in the coassembly of nuclear and cytoplasmic IF proteins by overexpressing chimeric IF proteins in human SW13+ and SW13- cells, which contain and lack endogenous cytoplasmic IF proteins, respectively. The chimeric IF proteins consisted of the rod domain of human nuclear lamin A/C protein fused to the amino and carboxyl-terminal domains of the mouse neurofilament light subunit (NF-L), which contained or lacked the 42 aa insert. Immunofluorescence microscopy was used to follow assembly and targeting of the proteins in cells. Chimeric proteins that lacked the 42 aa insert colocalized with vimentin, whereas those that contained the 42 aa insert did not. When overexpressed in SW13- cells, chimeric proteins containing the 42 aa formed very short or broken cytoplasmic filaments, whereas chimeric proteins that lacked the insert assembled efficiently into long, stable cytoplasmic filaments. To examine the roles of other structural motifs in intracellular targeting, we added two additional sequences to the chimera, a nuclear localization signal (NLS) and a CAAX motif, which are found in nuclear IF proteins. Addition of an NLS alone or an NLS in combination with the CAAX motif to the chimera with the 42 aa insert resulted in cagelike filament that assembled close to the nuclear envelope and nuclear lamina-like targeting, respectively. Our results suggest that the rod domains of eukaryotic nuclear and cytoplasmic IF proteins, which are related to each other, are still compatible upon deletion of the 42 aa insert of coassembly. In addition, NF-L end domains can substitute for the corresponding lamin domains in nuclear lamina targeting.     

Discrimination between the nuclear lamin and intermediate filament (cytokeratin/vimentin) proteins of rat hepatic tumor cells by differential solubility and electrophoretic criteria

1. The major proteins which comprise the high salt/detergent-insoluble cytoskeletal matrix of rat hepatic tumor cells containing abnormal (Mallory body-like) aggregates of intermediate filaments were distinguished on the basis of electrophoretic mobility and differential solubility. 2. Gel electrophoresis of the intermediate filament-enriched cytoskeletal fraction of Mallory body hepatic tumor cells revealed the presence of: (a) intermediate filament proteins typical of cultured liver epithelial cells (cytokeratins A and D, vimentin), (b) some residual actin and, (c) two peptides of Mr = 68,000-72,000. 3. Analysis of the products of filament disassembly/reassembly mixtures indicated that the two Mr = 68,000-72,000 peptide species had the solubility characteristics of nuclear lamins. 4. The presence of nuclear lamin proteins in the high salt/detergent-resistant fraction of cultured liver cells was consistent with the resolution of residual nuclear-like structures in extracted cell monolayers. 5. Thus, while cytokeratin/vimentin-class intermediate filament proteins and nuclear lamins co-isolate from rat liver cells under conditions of high salt/detergent extraction, these two types of cytoskeletal proteins could be distinguished on the basis of their differential solubility and molecular weight.     

Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells.

The intermediate filament proteins desmin and vimentin and the muscle tropomyosins were the major protein phosphate acceptors in 8-day-old myotubes incubated for 4 h in medium containing radiolabeled phosphate. The addition of isoproterenol or 8-bromo-cyclic AMP (BrcAMP) resulted in a two- to threefold increase in incorporation of 32PO4 into both desmin and vimentin, whereas no changes in the incorporation of 32PO4 into tropomyosin or other cellular proteins were observed. The BrcAMP- or hormonally induced increase in 32PO4 incorporation into desmin and vimentin was independent of protein synthesis and was not caused by stimulation of protein phosphate turnover. In addition, BrcAMP did not induce significant changes in the specific activity of the cellular ATP pool. These data suggest that the observed increase in 32PO4 incorporation represented an actual increase in phosphorylation of the intermediate filament proteins desmin and vimentin. Two-dimensional tryptic analysis of desmin from 8-day-old myotubes revealed five phosphopeptides of which two showed a 7- to 10-fold increase in 32PO4 incorporation in BrcAMP-treated myotubes. Four of the phosphopeptides identified in desmin labeled in vivo were also observed in desmin phosphorylated in vitro by bovine heart cAMP-dependent protein kinase. Although phosphorylation of desmin and vimentin was apparent in myogenic cells at all stages of differentiation, BrcAMP- and isoproterenol-induced increases in phosphorylation of these proteins were restricted to mature myotubes. These data strongly suggest that in vivo phosphorylation of the intermediate filament proteins desmin and vimentin is catalyzed by the cAMP-dependent protein kinases and that such phosphorylation may be regulated during muscle differentiation.     

A型核纤层蛋白在氧化应激和DNA损伤反应中的作用

核纤层蛋白是一种存在于真核细胞核膜下的中间丝纤维蛋白,是细胞核中重要的骨架蛋白,对维持细胞核的结构和功能具有重要作用。其基因突变会引起一系列的遗传性疾病,称为核纤层蛋白病。这些疾病在细胞水平表现出氧化应激和DNA损伤的特征,提示核纤层蛋白在氧化应激和DNA损伤反应中具有重要作用。本文主要就A型核纤层蛋白在氧化应激、DNA损伤反应中的作用机制进行综述。     

Heat shock causes the collapse of the intermediate filament cytoskeleton in Drosophila embryos

Heat shock has a dramatic effect on the organization of the cytoplasm, causing the intermediate filament cytoskeleton to aggregate at the nucleus. This has previously been shown in cultured Drosophila and mammalian cells. In this paper we analyze the heat lability of the intermediate filament cytoskeleton in early Drosophila embryos by indirect immunofluorescence. At all stages of embryogenesis tested, the intermediate filament cytoskeleton, which is maternally provided, is severely disturbed by 30 min heat shock at 37 degrees C. After the nuclei have migrated to the subcortical cytoplasm, it collapses around them. Nuclei in all heat-shocked embryos are considerably enlarged and become displaced. Embryos before cellular blastoderm stage, in which heat shock protein synthesis is not inducible, are irreversibly arrested in development by heat shock. Embryos at or after cellular blastoderm, which do synthesize heat shock proteins in response to stress, are also immediately arrested in development but continue development when returned to 25 degrees C. We discuss the possibility that cytoplasmic events such as the intermediate filament cytoskeleton rearrangement may be involved in heat shock-mediated phenocopy induction.     

Evolution of homologous domains of cytoplasmic intermediate filament proteins and lamins

The earliest gene duplications in the evolution of the intermediate filament proteins created the ancestors of acidic keratins, basic keratins, nonepithelial intermediate filament proteins, and lamins. Biochemistry and function of cytoplasmic intermediate filaments differ greatly from those of lamins. Cytoplasmic intermediate filament proteins have a different cellular location than lamins, form different types of supramolecular structures, and are missing a protein segment found in lamins; but the data presented here indicate that the cytoplasmic intermediate filaments do not have a common ancestor separate from the ancestor of lamins. In the non-epithelial intermediate filament branch, the ancestor of neurofilament proteins and the common ancestor of desmin, vimentin, and glial fibrillary acidic protein (GFAP) diverged first. By evolutionary criteria, the intermediate filament protein recently discovered in neuronal cells does not belong to the neurofilament family but is more closely related to desmin, vimentin, and GFAP. Sequences of different sub-domains yield different evolutionary trees, possibly indicating existence of sub- domain-specific functions.      

PtK2细胞的核骨架—核纤层——中间纤维体系

本文用选择性系列抽提的方法结合整装细胞电镜技术和DGD包埋-去包埋超薄切片技术,在电镜下清晰地显示了PtK 2细胞的核骨架-核纤层-中间纤维体系的精细结构。处于分裂中期的细胞经抽提后可以看到,染色体残余与中间纤维仍然保持一定的联系。用免疫荧光技术对抽提后的PtK 2细胞进行分析结果表明:其中间纤维能同时与AE1和AE3反应;能与Lamin B反应的单抗可以特异地定位于其核周,而Lamin A(C)的单抗除了与其核纤层蛋白有很强的反应外还与中间纤维有交叉反应。此外,在分裂期细胞中可以看到Lamin A(C)可能与染色体能特异结合;与HeLa细胞不一样。PtK 2细胞的核骨架成份不能与280kD的核骨架蛋白单抗反应。双向电泳结果显示出PtK 2细胞的核骨架-核纤层-中间纤维体系的组成成份与HeLa细胞相比有较大的差异,而且这种差异主要反映在核骨架组份上,TdR的处理也能导致其组份发生变化。     

免疫胶体金标记显示波形纤维与核孔复合体的结合

中间纤维与细胞核的关系是一个亟待解决解决的重要问题。本文采用火鸡红细胞作为研究材料,首先用细胞分级抽提结合免疫印迹反应显示火鸡红细胞中间纤维蛋白为波形纤维蛋白。然后,我们采用细胞分级抽提结合包埋前免疫胶体金标记的方法显示胞质中间纤维被抗波形纤维蛋白抗体－蛋白Ａ－胶体金特异标记。同时,我们显示结合于核孔复合体上的胞质纤维被抗波形纤维蛋白抗体－蛋白Ａ－胶体金所特异标记。本文结果表明,结合于核孔复合体上