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PubMed1998

Intermediate filament disassembly in cultured dorsal root ganglion neurons is associated with amino-terminal head domain phosphorylation of specific subunits.

We previously reported that activation of protein kinase A in cultured rat dorsal root ganglion neurons, treated concomitantly with low concentrations of okadaic acid that selectively inhibit protein phosphatase-2A, enhanced the Triton X-100 solubility of neurofilament triplet proteins. We now show that peripherin and alpha-internexin follow the same fragmentation profile as the neurofilament subunits, consistent with the notion that all five cytoplasmic intermediate filament proteins in these neurons form an integrated filamentous network whose assembly can be modulated by protein kinase A. Similar to the situation previously observed for the light neurofilament subunit, there was a strong correlation between phosphorylation of the amino-terminal head domain of peripherin and filament fragmentation. In contrast, insignificant levels of 32P were incorporated into alpha-internexin under conditions promoting disassembly, indicating that phosphorylation of this protein is not involved directly in filament fragmentation. The situation for the mid-sized neurofilament subunit (NFM) was not as clear-cut. Phosphopeptide mapping of NFM revealed many head and tail domain phosphorylation sites. However, changes in NFM head domain phosphorylation under conditions promoting filament disassembly were not as pronounced as for peripherin.

Animals↗
PubMed1990

Stratum corneum antibodies detected by hemagglutination are not directed against keratin intermediate filaments.

Autoantibodies to stratum corneum (SC) occur in virtually all normal adult human sera. These antibodies may be directed against various antigens of the SC. They have been detected by indirect immunofluorescence, passive hemagglutination (HA), immune adherence, and most recently by enzyme immunoassay and immunoblot methods. The purpose of our study was to examine whether antibodies to SC antigens as detected by passive HA are similar to the keratin intermediate filament (KIF) reactive antibodies. SC antigen preparation was prepared from psoriatic scales by the trypsin-phenol-water (TPW) extraction method. KIFs were prepared by 8 M urea extraction of normal callus or psoriatic scales. The anti-SC antibody titers of normal human sera were determined by passive HA before and after absorption with TPW-SC antigen preparation and upon absorption with KIFs. Similarly, titers of anti-KIF antibodies were determined on absorbed and unabsorbed sera by immunoblot assay. The results of this study indicate that the absorption of the sera with KIFs did not affect the titer of antibodies to TPW-extractable SC antigens whereas the titer of KIF antibodies dropped. KIF-reactive antibodies, on the other hand, were not affected by absorption with TPW-SC antigen, whereas the latter absorbed out the corresponding reactive antibodies. These results indicate that antibodies directed against SC antigen are different from the KIF-reactive antibodies.

Amino Acids↗
PubMed1992

[The role of intermediate filaments in forming cellular processes induced in fibroblasts by the tumor promoter TPA].

A tumor promoter phorbol 12-myristate 13-acetate (PMA) induces characteristic reversible changes in the cell shape in certain fibroblastic lines. This reaction to PMA may be regarded as a prototype of reorganizations involving formation of stable cytoplasmic processes. Two specific drugs, Taxol and Colcemid, were used to study the role of microtubules and vimentin-containing intermediate filaments (IF) in the development of PMA-induced reorganizations. A short (I h) exposure to PMA induced formation of processes in the control cells rather than in the Colcemid treated cells having depolymerized microtubules and the IF that collapsed around the nucleus. A longer (3-4 h) exposure to PMA of the colcemid-treated cells induced a partial reversal of the IF collapse; those parts of peripheral lamellae that contained IF were transformed into narrow noncontractile processes. It is suggested that the local interaction of the IF with the actin system is an essential step in the formation of processes from lamellae.

Actins↗
PubMed2001

Spatiotemporal analysis of flow-induced intermediate filament displacement in living endothelial cells.

The distribution of hemodynamic shear stress throughout the arterial tree is transduced by the endothelium into local cellular responses that regulate vasoactivity, vessel wall remodeling, and atherogenesis. Although the exact mechanisms of mechanotransduction remain unknown, the endothelial cytoskeleton has been implicated in transmitting extracellular force to cytoplasmic sites of signal generation via connections to the lumenal, intercellular, and basal surfaces. Direct observation of intermediate filament (IF) displacement in cells expressing green fluorescent protein-vimentin has suggested that cytoskeletal mechanics are rapidly altered by the onset of fluid shear stress. Here, restored images from time-lapse optical sectioning fluorescence microscopy were analyzed as a four-dimensional intensity distribution function that represented IF positions. A displacement index, related to the product moment correlation coefficient as a function of time and subcellular spatial location, demonstrated patterns of IF displacement within endothelial cells in a confluent monolayer. Flow onset induced a significant increase in IF displacement above the nucleus compared with that measured near the coverslip surface, and displacement downstream from the nucleus was larger than in upstream areas. Furthermore, coordinated displacement of IF near the edges of adjacent cells suggested the existence of mechanical continuity between cells. Thus, quantitative analysis of the spatiotemporal patterns of flow-induced IF displacement suggests redistribution of intracellular force in response to alterations in hemodynamic shear stress acting at the lumenal surface.

Animals↗
PubMed2005

Intermediate filaments and vesicular membrane traffic: the odd couple's first dance?

During the last two decades, much attention has been focused on the regulation of membrane traffic by the actin and microtubule cytoskeletal networks. Their dynamic and polarized behavior and associated motors provide a logical framework from which architectural and movement cues can be communicated to organelles. The study of these cytoskeletal systems has been greatly aided by pharmacological agents. In contrast, intermediate filaments (IFs) have largely been neglected as a potential player in membrane traffic, both because a comprehensive pharmacology to perturb them does not exist and because they lack the intrinsic polarity and specific motors that make the other cytoskeletal systems attractive. In this review, we will discuss evidence suggesting that IFs may play roles in controlling organelle positioning and in membrane protein targeting. Furthermore, we will discuss potential mechanisms by which IFs may regulate the localization and function of organelles.

Adaptor Protein Complex 3↗
PubMed1990

Heterogeneity in spinal radial glia demonstrated by intermediate filament expression and HRP labelling.

Considerable evidence indicates that radial glial cells play an active role in guiding growing neurites during development of the vertebrate CNS. In this paper we describe subpopulations of radial glial in the spinal cord of the axolotl. Amphibians maintain radial glia throughout life, and subpopulations are described using anatomical criteria following filling of individual cells with horseradish peroxidase and immunocytochemical staining with a range of intermediate filament antibodies. Radial glial cells in specific regions of the spinal cord stain with a range of antibodies specific to human keratins 8 and 18, and to glial fibrillary acid protein (GFAP). Some of these antibodies show selective localized to specific regions of individual glial cell processes. Immunoblotting analysis indicates that two keratins are present in the axolotl CNS corresponding to the two earliest embryonic keratins of vertebrates, keratin 8 and 18. Comparisons of molecular weight indicate that these may correspond to keratins identified in Xenopus laevis, the genes of which have been cloned. Axolotl GFAP is also identified in Western blots and may be present in two forms of differing molecular weight. These results are discussed in terms of the likely role of radial glial cells, and comparisons are drawn between the keratin and GFAP types seen in the axolotl spinal cord and of those in other vertebrate groups.

Ambystoma↗
PubMed1986

Effects of pemphigus antibody on the organization of microtubules and keratin-intermediate filaments in cultured human keratinocytes.

It has been suggested that pemphigus antibodies (PA) react with the surface molecules on keratinocytes, and induce the production and release of proteases resulting in acantholysis. If this is the case, the immunoreactions on the cell surface may send signals to the interior of the cell across the membrane. The present study was carried out to determine whether or not cytoskeletons [microtubules (MT) and keratin-intermediate filaments (KIF)] respond to PA-immunoreactions in cultured human keratinocytes, by indirect immunofluorescence microscopy using anti-keratin and anti-alpha-tubulin antibodies. During incubation for 30 min to 72 h in a PA-containing medium with a normal concentration of Ca2+ (1.2 mM), no changes in MT or KIF organization were detected. Alterations in the organization of these filaments were observed 96 h after addition of PA. When cells grown in a normal medium for 5-7 days were transferred to a medium containing PA and a low level of Ca2+ (0.07-0.14 mM) the reorganization of KIFs and MTs occurred after 1 h incubation. However, no reorganization of the cytoskeletons was detected in the absence of cell detachment. These observations suggest that the pemphigus antibody-induced reorganization of MTs and KIFs does not precede acantholysis and is probably secondary to it, but is not a direct transmembrane response. The present study also showed that immunofluorescence microscopy of KIFs may be one of the most sensitive methods for detecting early cell-to-cell dissociation in cultured keratinocytes.

Autoantibodies↗
PubMed1992

The desmoplakin carboxyl terminus coaligns with and specifically disrupts intermediate filament networks when expressed in cultured cells.

Specific interactions between desmoplakins I and II (DP I and II) and other desmosomal or cytoskeletal molecules have been difficult to determine in part because of the complexity and insolubility of the desmosome and its constituents. We have used a molecular genetic approach to investigate the role that DP I and II may play in the association of the desmosomal plaque with cytoplasmic intermediate filaments (IF). A series of mammalian expression vectors encoding specific predicted domains of DP I were transiently expressed in cultured cells that form (COS-7) and do not form (NIH-3T3) desmosomes. Sequence encoding a small antigenic peptide was added to the 3' end of each mutant DP cDNA to facilitate immunolocalization of mutant DP protein. Light and electron microscopical observations revealed that DP polypeptides including the 90-kD carboxy-terminal globular domain of DP I specifically colocalized with and ultimately resulted in the complete disruption of IF in both cell lines. This effect was specific for IF as microtubule and microfilament networks were unaltered. This effect was also specific for the carboxyl terminus of DP, as the expression of the 95-kD rod domain of DP I did not visibly alter IF networks. Immunogold localization of COS-7 cells transfected with constructs including the carboxyl terminus of DP demonstrated an accumulation of mutant protein in perinuclear aggregates within which IF subunits were sequestered. These results suggest a role for the DP carboxyl terminus in the attachment of IF to the desmosome in either a direct or indirect manner.

3T3 Cells↗
PubMed1991

Dual parameter flow cytometry for deoxyribonucleic acid and intermediate filament proteins of residual mature teratoma. All tumor cells are aneuploid.

Most testicular germ cell tumors of adults are presumably derived from polyploid carcinoma in situ. Thus, one would expect that even highly differentiated teratoma components are aneuploid and that it is unlikely to find diploid tumor cell (sub)populations. We studied 10 residual mature teratomas (RMTs) using a dual parameter flow cytometry procedure. Nuclear DNA was stained with propidium iodide and cytoplasmic intermediate filament proteins, in particular, cytokeratins, with fluorescein isothiocyanate-labeled specific monoclonal antibodies. Cells in RMTs, immunoreactive with antibodies to cytokeratins were considered to be tumor cells. These were always found to be aneuploid, in agreement with the available cytogenetic data on these tumors. The diploid cells present in RMTs were devoid of cytokeratins; therefore, these cells represent the nonmalignant normal host stromal and inflammatory cells. These results, in accordance with our earlier finding, indicate that diploid testicular germ cell tumors are extremely rare in adults, and that even the histologically benign somatic tissues in RMT after polychemotherapy are aneuploid.

Adult↗
PubMed2005

Intermediate filaments interact with dormant ezrin in intestinal epithelial cells.

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

Animals↗
PubMed1983

Use of antibodies to intermediate filaments in the diagnosis of metastatic amelanotic malignant melanoma.

Immunofluorescent staining of tissue from a lung tumor detected 12 years after excision of a primary malignant melanoma of the skin was negative for prekeratin and positive for vimentin, indicating that the tumor was not epithelial in origin and excluding carcinoma from the differential diagnosis. Complementary conventional staining with hematoxylin-eosin confirmed the melanocytic origin of the tumor, indicating that it was probably an amelanotic metastasis of the original malignant melanoma. The findings in this case demonstrate the potential usefulness of immunohistochemical microscopic characterization of specific intermediate filament proteins in the diagnosis of otherwise ambiguous cases of amelanotic melanoma.

Antibodies↗
PubMed1990

Cytoskeletal reorganizations responsible for the phorbol ester-induced formation of cytoplasmic processes: possible involvement of intermediate filaments.

The tumor promoter phorbol 12-myristate 13-acetate (PMA) induces characteristic reversible changes of cell shape in certain fibroblastic lines: motile lamellas are transformed into noncontractile narrow processes; simultaneously, the actin microfilament network of lamellas is locally disorganized. This reaction to PMA may be regarded as a prototype of reorganizations involving formation of stable cytoplasmic processes. Specific drugs, Taxol and Colcemid, were used to study the role of microtubules and vimentin-containing intermediate filaments (IF) in the development of PMA-induced reorganizations. PMA readily induced formation of noncontractile processes in Taxol-treated fibroblasts; these cells had a profoundly altered microtubular system but noncollapsed IF. A short (1 hr) exposure to PMA induced formation of processes in control cells but not in the Colcemid-treated cells, which had depolymerized microtubules and IF that collapsed around the nucleus. Longer (3-4 hr) exposure of the Colcemid-treated cells to PMA induced partial reversal of the IF collapse; those parts of the peripheral lamellas that contained IF were transformed into narrow noncontractile processes. It is suggested that the local interaction of IF with the actin system is an essential step in the formation of processes from lamellas. The microtubular system controls distribution of IF in the cytoplasm and thus plays an indirect role in the reorganization of the actin cortex.

Alkaloids↗
PubMed1996

Intermediate filament proteins in TPA-treated skeletal muscle cells in culture.

The cocarcinogenic phorbol ester 13-tetradecanoyl-O-phorbol acetate selectively and reversibly inhibits the ongoing differentiation programme of chick muscle cells in culture. 13-tetradecanoyl-O-phorbol acetate promptly blocks spontaneous contractions in mature myotubes and induces them to retract, forming giant myosacs and concurrently stress fibre-like structures are assembled. Using indirect immunofluorescence to localise desmin, the muscle specific intermediate filament protein, it was shown that its distribution is longitudinally oriented in mature myotubes. In myosacs, desmin has a reticular pattern although not as linearly oriented as in control myotubes. Using gel electrophoresis of control and 13-tetradecanoyl-O-phorbol acetate treated cell extracts, three major protein bands were observed with molecular weight of 43, 50 and 55 kDa. They migrate as actin, desmin and vimentin, respectively. The 50 kDa and 55 kDa proteins were expressed more in 13-tetradecanoyl-O-phorbol acetate-treated cells. The 50 kDa band was confirmed as desmin by immunoblotting using anti-chicken desmin antibody. Two-dimensional gel electrophoresis analysis showed the appearance of more acidic isoforms of the 50 and 55 kDa proteins 13-tetradecanoyl-O- phorbol in acetate-treated cells. The 43 kDa protein was seen as three distinct isoforms in control cells and as only two isoforms in 13-tetradecanoyl-O-phorbol acetatetreated cells.

Actinin↗
PubMed1991

Intermediate filaments in normal thyrocytes: modulation of vimentin expression in primary cultures.

In dog thyrocyte primary cultures, the antagonistic effects of thyrotropin (TSH) and epidermal growth factor (EGF) on differentiation expression were accompagnied by distinct long-term morphological changes: TSH-treated cells showed an epitheloid morphology; EGF reversibly induced a fusiform shape. Using indirect immunofluorescence microscopy and two-dimensional gel electrophoresis, we studied the modifications in the distribution and synthesis of the intermediate filament proteins of the cytoskeleton in response to TSH and EGF. These factors had little effect on the expression of cytokeratins 8 and 18, which were expressed in 98% of cells. However, TSH induced a profound redistribution of cytokeratins (and actin) with the appearance of a marked staining of cell junctions. Vimentin was coexpressed with cytokeratins in about 40% of cells from normal thyroid follicles freshly isolated by collagenase. During culture, immunostained vimentin network progressively developed in 90% of control and EGF-treated cells simultaneously with vimentin synthesis. In contrast, only 20% of TSH-treated cells reacted with vimentin antibody and we observed a marked decrease in vimentin synthesis in response to TSH. Therefore, vimentin synthesis, which should occur in at least some normal thyroid follicles in vivo, was inhibited in vitro by TSH which promotes differentiation expression. However, EGF-treated cells thereafter cultured with TSH regained an epitheloid morphology and differentiation in spite of the persistency of a complete network of vimentin.

Actins↗
PubMed1991

Basaloid carcinoma of salivary glands, a variety of undifferentiated adenocarcinoma. Immunohistochemical study of intermediate filament proteins in 24 cases.

Among adenoid cystic carcinomas of salivary glands (ACCs), the solid basaloid type has a poor prognosis similar to that of undifferentiated adenocarcinomas. We studied 24 cases in immunohistochemistry using antibodies reactive with keratins of various molecular weights, vimentin, S-100 protein, and its A and B subunits. Our findings were correlated with the histological pattern and with the variable degree of differentiation of these carcinomas. In comparison with other types of ACC, intermediate filament proteins in this group were weakly expressed. The co-expression of cytokeratin and vimentin was noted in some cases. Additional features noted were the presence of cribriform cavities associated with solid lobules and areas of necrosis giving a comedocarcinomatous pattern. In these two variants, cells characterized by the dual expression of cytokeratin and S-100 protein were seen. In the highly malignant anaplastic variety, only a few cells were weakly positive with antisera to cytokeratin and vimentin. This group shows similarities to undifferentiated adenocarcinomas of salivary glands. Such similarities could be explained by the common origin of these tumours from intercalated ducts.

Carcinoma, Transitional Cell↗