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

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

Immunocytochemical studies of intermediate filament aggregates and their relationship to microtubules in cultured skin fibroblasts from patients with giant axonal neuropathy

Giant axonal neuropathy (GAN) is a severe autosomal recessive disease affecting both the peripheral and central nervous systems. It is characterized by segmental axonal ballooning due to large neurofilamentous masses and abnormal aggregation of filaments in other cell types including glial cells. Coomassie blue staining of the detergent-resistant cytoskeleton of cultured skin fibroblasts from three patients with GAN revealed the presence of large cytoplasmic filamentous aggregates in the great majority of cells. The aggregates were birefringent when viewed under polarization microscopy and electron microscopy showed that they were composed of aggregates of 8 to 10 nm intermediate filaments. The aggregates stained with antisera specific for vimentin but did not stain with antibodies to actin, tubulin, or the high molecular weight (HMW) microtubule associated protein. Examination of the fibroblasts containing the vimentin aggregates with antibodies to tubulin and the HMW protein showed that they had a normal distribution of microtubules and that the microtubules present were normally associated with the HMW protein. The results suggest that giant axonal neuropathy is a generalized inborn error of organization of intermediate filaments and that a defect in microtubules or their association with HMW protein is not responsible for the observed aggregation of intermediate filaments in this disease. Further study of GAN may be useful in understanding the function of intermediate filaments.     

Withaferin a alters intermediate filament organization, cell shape and behavior

Withaferin A (WFA) is a steroidal lactone present in Withania somnifera which has been shown in vitro to bind to the intermediate filament protein, vimentin. Based upon its affinity for vimentin, it has been proposed that WFA can be used as an anti-tumor agent to target metastatic cells which up-regulate vimentin expression. We show that WFA treatment of human fibroblasts rapidly reorganizes vimentin intermediate filaments (VIF) into a perinuclear aggregate. This reorganization is dose dependent and is accompanied by a change in cell shape, decreased motility and an increase in vimentin phosphorylation at serine-38. Furthermore, vimentin lacking cysteine-328, the proposed WFA binding site, remains sensitive to WFA demonstrating that this site is not required for its cellular effects. Using analytical ultracentrifugation, viscometry, electron microscopy and sedimentation assays we show that WFA has no effect on VIF assembly in vitro. Furthermore, WFA is not specific for vimentin as it disrupts the cellular organization and induces perinuclear aggregates of several other IF networks comprised of peripherin, neurofilament-triplet protein, and keratin. In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates. The organization of microtubules and actin/microfilaments is also affected by WFA. Microtubules become wavier and sparser and the number of stress fibers appears to increase. Following 24 hrs of exposure to doses of WFA that alter VIF organization and motility, cells undergo apoptosis. Lower doses of the drug do not kill cells but cause them to senesce. In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects.     

The 'ins' and 'outs' of intermediate filament organization

A major function shared by several types of cytoplasmic intermediate filaments (IFs) is to stabilize cellular architecture against the mechanical forces it is subjected to. As for other fibrous cytoskeletal arrays, a crucial determinant of this function is the spatial organization of IFs in the cytoplasm. However, very few crossbridging proteins are specific for IFs - most IF-associated proteins known to exert a structural role act to tether IFs to other major cytoskeletal elements, such as F-actin, microtubules or adhesion complexes. In addition, IFs are endowed with the ability to participate in their own organization. This intriguing property is probably connected to the unusual degree of sequence diversity and sequence-specific regulation that characterize IF genes and their proteins. This dependence upon a combination of extrinsic and intrinsic determinants contributes to distinguish IFs from other fibrous cytoskeletal polymers and is key to their function.     

COOH-terminal truncated human cardiac MyBP-C alters myosin filament organization

Myosin-binding protein C (MyBP-C) is thought to play structural and/or regulatory role in striated muscles. The cardiac isoform of MyBP-C is one of the disease genes associated with familial hypertrophic cardiomyopathy and most of the mutations produce COOH truncated proteins. In order to determine the consequences of these mutations on myosin filament organization, we have characterized the effect of a 52-kDa NH2-terminal peptide of human cardiac MyBP-C on the alpha-myosin heavy chain (alpha-MyHC) filament organization. This peptide lacks the COOH-terminal MyHC-binding site and retains the two MyHC-binding domains located in the N-terminal part of MyBP-C. For this characterization, cDNA constructs (rat alpha-MyHC, full-length and truncated human cardiac MyBP-C) were transiently expressed singly or in pairwise combination in COS cells. In conformity with previous works performed on the skeletal isoform of MyBP-C, we observed that full-length cardiac MyBP-C organizes the MyHC into dense structures of uniform width. While the truncated protein is stable and can interact with MyHC in COS cells, it does not result in the same organization of sarcomeric MyHC that is seen with the full-length MyBP-C. These results suggest that the presence of truncated cardiac MyBP-C could, at least partly, disorganize the sarcomeric structure in patients with familial hypertrophic cardiomyopathy.     

Keratin 20 helps maintain intermediate filament organization in intestinal epithelia

Of the >20 epithelial keratins, keratin 20 (K20) has an unusual distribution and is poorly studied. We began to address K20 function, by expressing human wild-type and Arg80-->His (R80H) genomic (18 kb) and cDNA K20 in cells and mice. Arg80 of K20 is conserved in most keratins, and its mutation in epidermal keratins causes several skin diseases. R80H but not wild-type K20 generates disrupted keratin filaments in transfected cells. Transgenic mice that overexpress K20 R80H have collapsed filaments in small intestinal villus regions, when expressed at moderate levels, whereas wild-type K20-overexpressing mice have normal keratin networks. Overexpressed K20 maintains its normal distribution in several tissues, but not in the pancreas and stomach, without causing any tissue abnormalities. Hence, K20 pancreatic and gastric expression is regulated outside the 18-kb region. Cross-breeding of wild-type or R80H K20 mice with mice that overexpress wild-type K18 or K18 that is mutated at the conserved K20 Arg80-equivalent residue show that K20 plays an additive and compensatory role with K18 in maintaining keratin filament organization in the intestine. Our data suggest the presence of unique regulatory domains for pancreatic and gastric K20 expression and support a significant role for K20 in maintaining keratin filaments in intestinal epithelia.     

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

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

Giant axonal neuropathy: a conditional mutation affecting cytoskeletal organization

Giant axonal neuropathy (GAN) results from autosomal recessive mutations (gan-) that affect cytoskeletal organization; specifically, intermediate filaments (IFs) are found collapsed into massive bundles in a variety of different cell types. We studied the gan- fibroblast lines WG321 and WG139 derived from different GAN patients. Although previous studies implied that the gan- IF phenotype was constitutive, we find that it is conditional. That is, when cells were grown under the permissive condition of medium containing over 2% fetal calf serum, most cells had normal IF organization. IF bundles formed when gan- cells were transferred to the nonpermissive condition of low (0.1%) serum. Microtubule organization appeared normal in the presence or absence of serum. The effect of serum starvation was largely blocked or reversed by the addition of BSA to the culture media. We found no evidence that the gan- phenotype depends upon progress through the cell cycle. We discuss the possible role of serum effects in the etiology of GAN and speculate as to the molecular nature of the gan- defect.     

Sarcoplasmic reticulum and intermediate filament organization in cultured neonatal cardiac muscle cells

Cardiac muscle cells from 3-day-old rat neonates were cultured for periods of 2 to 56 days. In order to facilitate ultrastructural studies on the organization of the sarcoplasmic reticulum, the cells were prepared for transmission electron microscopy according to a regimen including postfixation in reduced osmium ferrocyanide. The nonjunctional sarcoplasmic reticulum (NJSR) was organized as a loose, fenestrated sleeve around the exterior of bundles of myofilaments and was particularly prominent at the level of the Z line. The only recognizable junctional elements of the sarcoplasmic reticulum were in a peripheral location. Reduced osmium ferrocyanide was also useful in distinguishing intermediate (10 nm) filaments, since it understained Z substance, which often obscured these structures. Intermediate filaments were arranged both at the Z line and the intercalated disc, in parallel strands, approximately at right angles to the myofilaments.     

Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro

To investigate the sequences important for assembly of keratins into 10- nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.     

BBLN-1 is essential for intermediate filament organization and apical membrane morphology

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A requirement for cytoplasmic dynein and dynactin in intermediate filament network assembly and organization

We present evidence that vimentin intermediate filament (IF) motility in vivo is associated with cytoplasmic dynein. Immunofluorescence reveals that subunits of dynein and dynactin are associated with all structural forms of vimentin in baby hamster kidney-21 cells. This relationship is also supported by the presence of numerous components of dynein and dynactin in IF-enriched cytoskeletal preparations. Overexpression of dynamitin biases IF motility toward the cell surface, leading to a perinuclear clearance of IFs and their redistribution to the cell surface. IF-enriched cytoskeletal preparations from dynamitin-overexpressing cells contain decreased amounts of dynein, actin-related protein-1, and p150Glued relative to controls. In contrast, the amount of dynamitin is unaltered in these preparations, indicating that it is involved in linking vimentin cargo to dynactin. The results demonstrate that dynein and dynactin are required for the normal organization of vimentin IF networks in vivo. These results together with those of previous studies also suggest that a balance among the microtubule (MT) minus and plus end-directed motors, cytoplasmic dynein, and kinesin are required for the assembly and maintenance of type III IF networks in interphase cells. Furthermore, these motors are to a large extent responsible for the long recognized relationships between vimentin IFs and MTs.     

Stability and association with the cytomatrix of mitochondrial DNA in spontaneously immortalized mouse embryo fibroblasts containing or lacking the intermediate filament protein vimentin

To extend previous observations demonstrating differences in number, morphology, and activity of mitochondria in spontaneously immortalized vim(+) and vim(-) fibroblasts derived from wild-type and vimentin knockout mice, some structural and functional aspects of mitochondrial genome performance and integrity in both types of cells were investigated. Primary Vim(+/+) and Vim(-/-) fibroblasts, which escaped terminal differentiation by immortalization were characterized by an almost twofold lower mtDNA content in comparison to that of their primary precursor cells, whereby the average mtDNA copy number in two clones of vim(+) cells was lower by a factor of 0.6 than that in four clones of vim(-) cells. However, during serial subcultivation up to high passage numbers, the vim(+) and vim() fibroblasts increased their mtDNA copy number 1.5- and 2.5-fold, respectively. While early-passage cells of the vim(+) and vim(-) fibroblast clones differed only slightly in the ratio between mtDNA content and mitochondrial mass represented by mtHSP70 protein, after ca. 300 population doublings the average mtDNA/mtmass ratio in the vim(+) and vim() cells was increased by a factor of 2 and 4.5, respectively. During subcultivation, both types of cells acquired the fully transformed phenotype. These findings suggest that cytoskeletal vimentin filaments exert a strong influence on the mechanisms controlling mtDNA copy number during serial subcultivation of immortalized mouse embryo fibroblasts, and that vimentin deficiency causes a disproportionately enhanced mtDNA content in high-passage vim(-) fibroblasts. Such a role of vimentin filaments was supported by the stronger retention potential for mtDNA and mtDNA polymerase (gamma) detected in vim(+) fibroblasts by Triton X-100 extraction of mitochondria and agaroseembedded cells. Moreover, although the vim(+) and vim(-) fibroblasts were equally active in generating free radicals, the vim(-) cells exhibited higher levels of immunologically detectable 8-oxoG and mismatch repair proteins MSH2 and MLH1 in their mitochondria. Because in vim(-) fibroblasts only one point mutation was detected in the mtDNA D-loop control region, these cells are apparently able to efficiently remove oxidatively damaged nucleobases. On the other hand, a number of large-scale mtDNA deletions were found in high-passage vim(-) fibroblasts, but not in low-passage vim(-) cells and vim(+) cells of both low and high passage. Large mtDNA deletions were also induced in young vim(-) fibroblasts by treatment with the DNA intercalator ethidium bromide, whereas no such deletions were found after treatment of vim(+) cells. These results indicate that in immortalized vim(-) fibroblasts the mitochondrial genome is prone to large-scale rearrangements, probably due to insufficient control of mtDNA repair and recombination processes in the absence of vimentin.     

A role for disulfide bonding in keratin intermediate filament organization and dynamics in skin keratinocytes

We recently reported that a trans-dimer, homotypic disulfide bond involving Cys367 in keratin 14 (K14) occurs in an atomic-resolution structure of the interacting K5/K14 2B domains and in keratinocyte cell lines. Here we show that a sizable fraction of the K14 and K5 protein pools participates in interkeratin disulfide bonding in primary cultures of mouse skin keratinocytes. By comparing the properties of wild-type K14 with a completely cysteine-free variant thereof, we found that K14-dependent disulfide bonding limited filament elongation during polymerization in vitro but was necessary for the genesis of a perinuclear-concentrated network of keratin filaments, normal keratin cycling, and the sessile behavior of the nucleus and whole cell in keratinocytes studied by live imaging. Many of these phenotypes were rescued when analyzing a K14 variant harboring a single Cys residue at position 367. These findings establish disulfide bonding as a novel and important mechanism regulating the assembly, intracellular organization, and dynamics of K14-containing intermediate filaments in skin keratinocytes.     

Cordycepin rapidly collapses the intermediate filament networks into juxtanuclear caps in fibroblasts and epidermal cells

The nucleoside analog 3'-deoxyadenosine (cordycepin) rapidly collapses the intermediate filaments into juxtanuclear caps in interphase fibroblasts and keratinocytes. A minimum of 80 micrograms/ml cordycepin or 20 micrograms/ml cordycepin in combination with 2 micrograms/ml of the deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenosine (EHNA) to inhibit its degradation is required to see these effects. This is the same concentration required for cordycepin to arrest cells at the onset of mitosis and depolymerize the microtubules to small asters. Cordycepin enters the cells rapidly and is phosphorylated to 3'-dATP with a concomitant drop in ATP levels. However, the direct reduction of ATP levels does not mimic the same rapid effects of cordycepin on either the intermediate filaments or microtubules. In addition, similar effects are not produced by a variety of other adenosine analogs with alterations in the 2'- and 3'-ribose positions. Although other pharmacological reagents result in alterations of the fibroblastic intermediate filaments, cordycepin is unusual because of the rapidity with which the fibroblastic intermediate filaments collapse into the juxtanuclear caps. The juxtanuclear caps have a morphology different from that of the perinuclear bundles of intermediate filaments that arise after long-term depolymerization of the microtubules. The keratin fibers in the epidermal cells retract to a perinuclear ring when treated with cordycepin.     

Curcumin inhibits cell motility and alters microfilament organization and function in prostate cancer cells

Curcumin is a dietary phytochemical associated with anti-tumorigenic effects, but the mechanisms by which it inhibits cancer cell growth and metastasis are not completely understood. For example, little information is available regarding the effects of curcumin on cytoskeletal organization and function. In this study, time-lapse video and immunofluorescence labeling methods were used to demonstrate that curcumin significantly alters microfilament organization and cell motility in PC-3 and LNCaP human prostate cancer cells in vitro. Curcumin rapidly arrests cell movements and subsequently alters cell shape in the highly motile PC-3 cell line, but has a less noticeable effect on the relatively immobile LNCaP cell line. Stress fibers are augmented, and the overall quantity of f-actin appears to increase in both types of cells following curcumin treatment. Cytochalasin B (CB) disrupts microfilament organization in both cell lines, and causes vigorous membrane blebbing in PC-3 cells, but not LNCaP cells. Pre-treatment of cells with curcumin suppresses changes in microfilament organization caused by CB, and blocks PC-3 membrane blebbing. At least some of the effects of curcumin appear to be mediated by protein kinase C (PKC), as treatment with the PKC inhibitor bisindolylmaleimide inhibits the ability of curcumin to block CB-induced membrane blebbing. These findings demonstrate that curcumin exerts significant effects on the actin cytoskeleton in prostate cancer cells, including altering microfilament organization and function. This is a novel observation that may represent an important mechanism by which curcumin functions as a chemopreventative agent, and as an inhibitor of angiogenesis and metastasis.