Desmin filaments are stably associated with the outer nuclear surface in chick myoblasts

Eukaryotic cells have highly organized, interconnected intracellular compartments. The nuclear surface and cytoplasmic cytoskeletal filaments represent compartments involved in such an association. Intermediate filaments are the major cytoskeletal elements in this association. Desmin is a muscle-specific structural protein and one of the earliest known muscle-specific genes to be expressed during cardiac and skeletal muscle development. Desmin filaments have been shown to be associated with the nuclear surface in the myogenic cell line C2C12. Previous studies have revealed that mice lacking desmin develop imperfect muscle, exhibiting the loss of nuclear shape and positioning. In the present work, we have analyzed the association between desmin filaments and the outer nuclear surface in nuclei isolated from pectoral skeletal muscle of chick embryos and in primary chick myogenic cell cultures by using immunofluorescence microscopy, negative staining, immunogold, and transmission electron microscopy. We show that desmin filaments remain firmly attached to the outer nuclear surface after the isolation of nuclei. Furthermore, positive localization of desmin persists after gentle washing of the nuclei with high ionic strength solutions. These data suggest that desmin intermediate filaments are stably and firmly connected to the outer nuclear surface in skeletal muscles cells in vivo and in vitro.     

Cytoplasmic intermediate filaments revealed as dynamic and multipurpose scaffolds

Intermediate filaments are cytoskeletal polymers encoded by a large family of differentially expressed genes that provide crucial structural support in the cytoplasm and nucleus of higher eukaryotes. Perturbation of their function accounts for several genetically determined diseases in which fragile cells cannot sustain mechanical and non-mechanical stresses. Recent studies shed light on how this structural support is modulated to meet the changing needs of cells, and reveal a novel role whereby intermediate filaments influence cell growth and death through dynamic interactions with non-structural proteins.     

Connections of intermediate filaments with the nuclear lamina and the cell periphery

We investigated the relationship between intermediate filaments (IFs) and other detergent- and nuclease-resistant filamentous structures of cultured liver epithelial cells (T51B cell line) using whole mount unembedded preparations which were sequentially extracted with Triton X-100 and nucleases. Immunogold labelling and stereoscopic observation facilitated the examination of each filamentous structure and their three-dimensional relationships to each other. After solubilizing phospholipid, nucleic acid and soluble cellular protein, the resulting cytoskeleton preparation consisted of a network of cytokeratin and vimentin IFs linked by 3 nm filaments. The IFs were anchored to and determined the position of the nuclear lamina filaments (NLF) network and the centrioles. The NLF was composed of the nuclear lamina filaments measuring 3-6 nm in diameter which radiated from and anchored to the skeleton of the nuclear pores. The IFs located in the nuclear region appeared to be interwoven with the NLF. At the cell surface, the IFs seemed to be attached to the putative actin filament network. They formed a focally interrupted plexus-like structure at the cell periphery. Fragments of vimentin filaments were found among the filamentous network located at the cell surface, and some filaments terminated blindly there.     

Determinants for intracellular sorting of cytoplasmic and nuclear intermediate filaments

The mechanism by which nuclear and cytoplasmic filaments are sorted in vivo was studied by examining which lamin sequences are required to target an otherwise cytoplasmic IF protein, the small neurofilament subunit (NF-L), to the nuclear lamina. By swapping corresponding domains between NF-L and lamin A, nuclear envelope targeting of NF-L was shown to require the presence of the "head" domain, a 42-amino acid sequence unique to lamin rod domains, a nuclear localization signal and the CAAX motif. Replacement of the entire COOH-terminal tail of lamin A with that of NF-L had no discernible effect on nuclear localization of lamin A, provided the substituted NF-L tail contained a NLS and a CAAX motif. This chimeric protein exhibited characteristics more typical of lamin B than that of the parental lamin A. With regard to cytoplasmic assembly properties, substitution of the head domain of lamin A for that of NF-L did not substantially affect the ability of NF-L to coassemble with vimentin in the cytoplasm. In contrast, insertion of a 42-amino acid sequence unique to lamin rod domains into NF-L profoundly affected NF-L coassembly with vimentin indicating that the 42-amino acid insertion in lamins may be important for sorting lamins from cytoplasmic IF proteins.     

Cytoskeletal proteins connecting intermediate filaments to cytoplasmic and nuclear periphery

Intermediate filaments (IFs), together with microtubules and microfilaments build up the cytoskeleton of most eukaryotic cells. Cytoplasmic IFs form a dense filament network radiating from the nucleus and extending to the plasma membrane. The association between the cytoplasmic and nuclear surfaces appears to provide a continuous link important for the organisation of the cytoplasm, for cellular communication, and possibly for the transport into and out of the nucleus. Cytoplasmic IFs approach the nuclear surface, thin fibrils seem to connect the IFs with the nuclear pore complexes and a direct interaction of cytoplasmic IFs with the nuclear lamin B has been observed by in vitro binding studies. However, none of the components that cross-link IFs to the nucleus has been unambiguously identified. Furthermore, if a direct interaction between cytoplasmic IFs and the nuclear lamin B occurs in vivo, the question of how cytoplasmic IFs get access to the nuclear interior remains to be resolved. The association of IFs with the plasma membranes involves different components, some of which are cell type specific. Two specialised complexes in epithelial cells: the desmosome and the hemidesmosome, serve as attachment sites for keratin filaments. Desmoplakin is considered as the cross-linking component of IFs to the desmosomal plaque, whereas BPAG1 (bullous pemphigoid antigen) would cross-link IFs at the hemidesmosomal plaque. In other cell types the modality of how IFs are anchored to the plasma membrane is less well understood. It involves different components such as the spectrin based membrane skeleton, ankyrin, myosin, plectin and certainly many other still unravelled partners. Association between the IFs and cellular membranes plays an important role in determining cell shape and tissue integrity. Thus, the identification and characterisation of the components involved in these interactions will be crucial for understanding the function of intermediate filaments.     

Phosphatase and actin regulator 4 is associated with intermediate filaments in adult neural stem cells and their progenitor astrocytes

Phosphatase and actin regulator 4 (Phactr4) is a newly discovered protein that inhibits protein phosphatase 1 and shows actin-binding activity. We previously found that Phactr4 is expressed in the neurogenic niche in adult mice, although its precise subcellular localization and possible function in neural stem cells (NSCs) is not yet understood. Here, we show that Phactr4 formed punctiform clusters in the cytosol of subventricular zone-derived adult NSCs and their progeny in vitro. These Phactr4 signals were not associated with F-actin fibers but were closely associated with intermediate filaments such as nestin and glial fibrillary acidic protein (GFAP) fibers. Direct binding of Phactr4 with nestin and GFAP filaments was demonstrated using Duolink protein interaction analyses and immunoprecipitation assays. Interestingly, when nestin fibers were de-polymerized during the mitosis or by the phosphatase inhibitor, Phactr4 appeared to be dissociated from nestin, suggesting that their protein interaction is regulated by the protein phosphorylation. These results suggest that Phactr4 forms functional associations with intermediate filament networks in adult NSCs.     

Interferon gamma polymorphisms and their interaction with smoking are associated with lung function

Interactions between genetic and environmental determinants are likely to be important in the pathogenesis of chronic obstructive pulmonary disease. We hypothesized that interferon gamma (IFNG) single nucleotide polymorphisms (SNPs) and their interaction with smoking are associated with the rate of decline or level of lung function in smokers. We studied four SNPs in IFNG in 585 non-Hispanic whites (NHW) who had the fastest (n =280) or the slowest (n=305) decline of FEV₁% predicted selected from among continuous smokers followed for 5 years in the NHLBI Lung Health Study. We also studied 1061 NHW with the lowest (n=530) or the highest (n=531) baseline lung function at the beginning of the LHS. Two SNPs were associated with baseline levels of lung function and the p values were 0.008 for +2197T/C in a dominant model and 0.002 for +5171A/G in a recessive model. However, after adjusting for confounding factors, only +5171A/G was still significant (p=0.001 for the recessive model). In addition, there was a significant genotype and smoking interaction with p=0.006 for the +5171A/G (GG vs.GA + AA) for the baseline lung function. When comparing individuals with GG versus individuals with AG + AA for low lung function, the adjusted odds ratios decreased significantly as pack-years increased. No association was found in the rate of decline study. There was an association between IFNG genotype and baseline of lung function and this association was modified by cigarette smoking.This work was supported by grants from the Canadian Institutes of Health Research and National Institutes of Heath Grant 5R01HL064068-04. The Lung Health Study was supported by contract N01-HR-46002 from the Division of Lung Diseases of the National Heart, Lung, and Blood Institute.     

Synemin and vimentin are components of intermediate filaments in avian erythrocytes

Synemin, a high-molecular-weight protein associated with intermediate filaments in muscle, and vimentin, an intermediate-filament subunit found in many different cell types, have been identified by immunologic and electrophoretic criteria as components of intermediate filaments in mature avian erythrocytes. Desmin, the predominant subunit of intermediate filaments in muscle, has not been detected in these cells. Two dimensional immunoautoradiography of proteolytic fragments of synemin and vimentin demonstates that the erythrocyte proteins are highly homologous, if not identical, to their muscle counterparts. Double immunoflurorescence reaveals that erythrocyte synemin and vimentin co-localize in a cytoplasmic network of sinuous filaments that extends from the nucleus to the plasma membrane and resists aggregation by colcemid. Erythrocytes that are attached to glass cover slips can be sonicated to remove nuclei and nonadherent regions of the plasma membrane; this leaves elliptical patches of adherent membrane that retain mats of vimentin- and synemin-containing intermediate filaments, as seen by immunofluorescence and rotary shadowing. Similarly, mechanical enucleation of erythrocyte ghosts in suspension allows isolation of plasma membranes that retain a significant fraction of the synemin and vimentin, as assayed by electrophoresis, and intermediate filaments, as seen in thin sections. Both synemin and vimentin remain insoluble along with spectrin and actin, in solutions containing nonionic detergent and high salt. However, brief exposure of isolated membrane to distilled water releases the synemin and vimentin together in nearly pure form, before the release of significant amounts of spectrin and actin. These data suggest that avian erythrocyte intermeditate filaments are somehow anchored to the plasma membrane; erythrocytes may thus provide a simple system for the study of intermediate filaments and their mode of interaction with membranes. In addition, these data, in conjunction with previous data from muscle, indicate that synemin is capable of associating with either desmin or vimentin and may thus perform a special role in the structure or function of intermediate filaments in erythrocytes as well as muscle.     

Caveolae in fibroblast-like synoviocytes: static structures associated with vimentin-based intermediate filaments

The fibroblast-like synoviocyte is a CD13-positive cell-type containing numerous caveolae, both single and interconnected clusters. In unstimulated cells, all single caveolae at the cell surface and the majority of those localized deeper into the cytoplasm were freely accessible from the medium, as judged from electron microscopy of synoviocytes exposed to the membrane impermeable marker Ruthenium Red. Caveolar internalization could be induced by a CD13 antibody or by cholera toxin B subunit (CTB). Thus, in experiments using sequential labeling with Alexa 488- and 594-conjugated CTB, about 50% of CTB-positive caveolae were internalized by 5 min of chase, and these remained inaccessible from the cell surface for periods up to 24 h. No colocalization with an endosomal marker, EEA1, or Lysotracker was observed, indicating that internalized caveolae clusters represent a static compartment. Vimentin was identified as the most abundant protein in detergent resistant membranes (DRM’s), and by immunogold electron microscopy caveolae were seen in intimate contact with intermediate-size filaments. These observations indicate that vimentin-based filaments are responsible for the spatio-temporal fixation of caveolae clusters. RECK, a glycosylphosphatidylinositol-anchored protein acting as a negative regulator of cell surface metalloproteinases, was also localized to the caveolae clusters. We propose that these clusters function as static reservoirs of specialized lipid raft domains where proteins involved in cell–cell interactions, such as CD13, can be sequestered by binding to RECK in a regulatory manner.     

Muscle intermediate filaments and their links to membranes and membranous organelles

Intermediate filaments (IFs) play a key role in the integration of structure and function of striated muscle, primarily by mediating mechanochemical links between the contractile apparatus and mitochondria, myonuclei, the sarcolemma and potentially the vesicle trafficking apparatus. Linkage of all these membranous structures to the contractile apparatus, mainly through the Z-disks, supports the integration and coordination of growth and energy demands of the working myocyte, not only with force transmission, but also with de novo gene expression, energy production and efficient protein and lipid trafficking and targeting. Desmin, the most abundant and intensively studied muscle intermediate filament protein, is linked to proper costamere organization, myoblast and stem cell fusion and differentiation, nuclear shape and positioning, as well as mitochondrial shape, structure, positioning and function. Similar links have been established for lysosomes and lysosome-related organelles, consistent with the presence of widespread links between IFs and membranous structures and the regulation of their fusion, morphology and stabilization necessary for cell survival.     

Actin filaments are associated with the septate junctions of invertebrates

Septate junctions are almost ubiquitous in the tissues of invertebrates but are never found in those of vertebrates. In spite of their widespread occurrence and hence obvious importance to the invertebrates, their precise function has remained elusive although they have been variously considered to be regions of cell-cell coupling, permeability barriers or adhesion sites. This report demonstrates that elements of the cytoskeletal system insert into the cytoplasmic face of septate junctions. Actin filaments, identified by virtue of their capacity to bind the S1 subfragment of rabbit myosin, are associated with the membranes of septate junctions. Cytochalasin D, an actin depolymerizer, leads to disorganization of the intramembrane components of these junctions. These data suggest that a primary role of septate junctions could be to maintain intercellular cohesion and hence tissue integrity. The assembly and localization of these junctions may be mediated, directly or indirectly, by the cytoplasmic actin filaments associated with their lateral membranes.     

The beaded intermediate filaments and their potential functions in eye lens

The elongated fiber cells of the eye lens contain a unique cytoskeletal system, the beaded chain filaments (BFs). The BFs had been morphologically identified more than two decades ago, but the precise identity of their subunit molecules remained unknown. Recently, use of recombinant DNA approaches, refined morphological and immunochemical studies and experiments with mutant mice have allowed the molecular dissection of these structures and provided clues about their potential functins. The BFs represent a highly specialized network of intermediate filaments (IFs) juxtaposed to the plasma membrane. They are obligate heteropolymers composed of two lens-specific polypeptides, filensin and phakinin. In this review we discuss the properties, molecular interactions and in situ arrangement of these two proteins, and comment on their potential roles during lens development.     

Specific types of prosomes are associated to subnetworks of the intermediate filaments in PtK1 cells.

Prosomes are small ribonucleoprotein (RNP) particles of unique morphology in the electron microscope but of variable protein and RNA composition, depending on the differentiation state of the cells studied. They were initially observed as subcomplexes of untranslated mRNP. In previous studies, we found that prosomes are associated to the intermediate filaments (IF) of cytokeratin type in HeLa and PtK1 cells. Here we have studied in detail the association of prosomal antigens with the IF networks in PtK1 cells. Contrary to our earlier conclusions, in these cells the vimentin fibers also carry prosomes which, thus, distribute in between the two types of networks. During the selective collapse of the IF induced by acrylamide, and upon recovery after the withdrawal of the drug, no dissociation of the prosome and IF networks of cytokeratin- and vimentin-type could be observed. These data show that even in a dynamic situation, prosome and IF antigens do not dissociate, indicating strongly that they are located on one and the same structure. Furthermore, the differential distribution of specific prosomal antigens between both types of intermediate filament networks indicates that prosomes do not ubiquitously populate the intermediate filaments but occupy subnetworks of either vimentin or cytokeratin type.     

Parietal and visceral endoderm differ in their expression of intermediate filaments

Two layers of extra-embryonic endoderm, viz. the parietal endoderm (PE) and the visceral endoderm (VE), arise in the mouse embryo shortly after implantation. Both cell populations apparently originate from the primitive endoderm of the blastocyst. While the endoderm differentiation has been studied both in the embryo and in the embryonal carcinoma model system, the investigation has been hampered by the paucity of unequivocal markers of differentiation, especially in the case of the PE. Here we show that the PE and VE of mouse conceptuses differ in their expression of intermediate filaments: while both cell types contain cytokeratin, expression of vimentin was only revealed in the cells of the PE. The association between the differentiation of PE and the appearance of vimentin filaments is discussed.     

Organization of intermediate filaments and their association with collagen-containing phagosomes in mouse decidual cells

We have analyzed the distribution of intermediate filaments (IF) in the cytoplasm of mature decidual cells of mice. IF were scattered throughout the cytoplasm of these cells although there was a preferential accumulation around the nuclei. In many cells a large area of the cytoplasm was occupied by a rich network of IF that extended from the perinuclear region toward the cell surface. Thin bundles of IF crossed the cytoplasm without a preferential orientation. IF were also seen in close association with nuclear pore complexes, gap junctions, mitochondria, and lysosomes. A very developed network of IF surrounded phagosomes that contained collagen fibrils. Longitudinal and cross sections of these phagosomes showed a very close association of IF with the phagosome membrane.     

The function of intermediate filaments in cell shape and cytoskeletal integrity

This study describes the development and use of a specific method for disassembling intermediate filament (IF) networks in living cells. It takes advantage of the disruptive effects of mimetic peptides derived from the amino acid sequence of the helix initiation 1A domain of IF protein chains. The results demonstrate that at 1:1 molar ratios, these peptides disassemble vimentin IF into small oligomeric complexes and monomers within 30 min at room temperature in vitro. Upon microinjection into cultured fibroblasts, these same peptides induce the rapid disassembly of IF networks. The disassembly process is accompanied by a dramatic alteration in cell shape and the destabilization of microtubule and actin-stress fiber networks. These changes in cell shape and IF assembly states are reversible. The results are discussed with respect to the roles of IF in cell shape and the maintenance of the integrity and mechanical properties of the cytoplasm, as well as the stability of the other major cytoskeletal systems.     

Cytoplasmic intermediate filament proteins of invertebrates are closer to nuclear lamins than are vertebrate intermediate filament proteins; sequence characterization of two muscle proteins of a nematode.

The giant body muscle cells of the nematode Ascaris lumbricoides show a complex three dimensional array of intermediate filaments (IFs). They contain two proteins, A (71 kd) and B (63 kd), which we now show are able to form homopolymeric filaments in vitro. The complete amino acid sequence of B and 80% of A have been determined. A and B are two homologous proteins with a 55% sequence identity over the rod and tail domains. Sequence comparisons with the only other invertebrate IF protein currently known (Helix pomatia) and with vertebrate IF proteins show that along the coiled-coil rod domain, sequence principles rather than actual sequences are conserved in evolution. Noticeable exceptions are the consensus sequences at the ends of the rod, which probably play a direct role in IF assembly. Like the Helix IF protein the nematode proteins have six extra heptads in the coil 1b segment. These are characteristic of nuclear lamins from vertebrates and invertebrates and are not found in vertebrate IF proteins. Unexpectedly the enhanced homology between lamins and invertebrate IF proteins continues in the tail domains, which in vertebrate IF proteins totally diverge. The sequence alignment necessitates the introduction of a 15 residue deletion in the tail domain of all three invertebrate IF proteins. Its location coincides with the position of the karyophilic signal sequence, which dictates nuclear entry of the lamins. The results provide the first molecular support for the speculation that nuclear lamins and cytoplasmic IF proteins arose in eukaryotic evolution from a common lamin-like predecessor.