Analyzing dynamic properties of intermediate filaments.
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Biomedical subjects
R D Goldman
Publications and source records attributed to R D Goldman.
Keratin incorporation into intermediate filament networks is a rapid process.
The properties of keratin-containing intermediate filament (IF) networks in vivo were studied following the microinjection of biotinylated keratin. Keratin-IFs were biotinylated, disassembled, and separated into type I and type II proteins by ion exchange chromatography. Recombination of these derivatized type I and type II keratins resulted in the formation of 10-nm diameter IF. The type I keratins were microinjected into epithelial cells and observed by immunofluorescence microscopy. Biotin-rich spots were found throughout the cytoplasm at 15-20 min after injection. Short biotinylated fibrous structures were seen at 30-45 min after injection, most of which colocalized with the endogenous bundles of IF (tono-filaments). By 1 1/2 to 2 h after microinjection, extensive biotinylated keratin IF-like networks were evident. These were highly coincident with the endogenous tonofilaments throughout the cell, including those at desmosomal junctions. These results suggest the existence of a relatively rapid subunit incorporation mechanism using numerous sites along the length of the endogenous tonofilament bundles. These observations support the idea that keratin-IFs are dynamic cytoskeletal elements.
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Dynamic aspects of cytoskeletal and karyoskeletal intermediate filament systems during the cell cycle.
IF are major cytoskeletal and karyoskeletal components of eukaryotic cells (Steinert and Roop 1988). In numerous instances, their constituent protein subunits have been shown to be substrates for a variety of kinases such as A-kinase, C-kinase, and Ca++/calmodulin kinase (Geisler and Weber 1988; Inagaki et. 1988; Ando et al. 1991), as well as p34cdc2 (Chou et al. 1990; Peter et al. 1990; Ward and Kirschner 1990; Dessev et al. 1991). To date, all of the phosphorylation sites that have been mapped are in the non-alpha-helical amino- or carboxy-terminal domains (Steinert 1988; Ando et al. 1989, 1991; Geisler et al. 1989; Chou et al. 1991), and these secondary modifications can lead to IF reorganization and/or disassembly in vivo and in vitro (see, e.g., Iganaki et al. 1988; Lamb et al. 1989; Chou et al. 1990; Peter et al. 1990; Heald and McKeon 1990; Dessev et al. 1991). In addition, it is possible that the exchange seen between subunits and polymerized IF in interphase following the microinjection of unpolymerized protein (Vikstrom et al. 1989; Miller et al. 1991) may also be regulated in some fashion by phosphorylation/dephosphorylation reactions. In cultured fibroblasts such as BHK-21, the interphase equilibrium state that favors IF polymerization is shifted dramatically to a disassembled state in mitosis, apparently due to enhanced phosphorylation at specific sites mediated through the activity of p34cdc2. However, in other cells in mitosis, such as HeLa, the mechanisms involved in the regulation of cytoskeletal IF remain unclear. Therefore, no one common mechanism appears to be responsible for IF regulation during cell division. On the basis of the majority of data available, it appears that the regulation of IF phosphorylation plays an important role in the regulation of the supramolecular organization of IF cytoskeletal and karyoskeletal networks, especially in the remodeling events that take place as cells enter and exit mitosis. Although the functional significance of IF phosphorylation during interphase is not as obvious as it is in some mitotic cells, we are tempted to speculate that there may be a connection with mechanisms involved in signal transduction, since IF proteins appear to be targets for kinases known to be activated by second messengers such as Ca++ and cAMP.
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Intermediate filament reorganization during mitosis is mediated by p34cdc2 phosphorylation of vimentin.
As cells enter mitosis, the intermediate filament (IF) networks of interphase BHK-21 cells are depolymerized to form cytoplasmic aggregates of disassembled IFs, and the constituent IF proteins, vimentin and desmin are hyperphosphorylated at several specific sites. We have characterized one of two endogenous vimentin kinases from a particulate fraction of mitotic cell lysates. Through several purification steps, vimentin kinase activity copurifies with histone H1 kinase and both activities bind to p13suc1-Sepharose. The final enriched kinase preparation consists primarily of p34cdc2 and polypeptides of 65 and 110 kd. The purified kinase complex phosphorylates vimentin in vitro at a subset of sites phosphorylated in vivo during mitosis. Furthermore, phosphorylation of in vitro polymerized vimentin IFs by the purified kinase causes their disassembly. Therefore, vimentin is a substrate of p34cdc2 and phosphorylation of vimentin contributes to M phase reorganization of the IF network.
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Lamin dimers. Presence in the nuclear lamina of surf clam oocytes and release during nuclear envelope breakdown.
The nuclear lamina of surf clam oocytes contains dimers of 67-kDa lamin which are stabilized by both noncovalent interactions and disulfide bonds. The latter can be reduced but re-form when the reducing agent is removed. The cysteine residues involved in these disulfide bonds are inaccessible to alkylating agents unless the protein is unfolded in urea. During nuclear envelope breakdown the lamin is released as a mixture of oligomers in which disulfide-stabilized dimers are associated noncovalently with lamin monomers. Concurrent with solubilization, both dimers and monomers are phosphorylated to a similar extent, indicating that the interactions which maintain these complexes are not destabilized by lamin phosphorylation. Our results suggest the existence of two types of interactions between the lamin molecules in the polymer, which react differently to phosphorylation during nuclear envelope breakdown.
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Dynamic properties of intermediate filaments: disassembly and reassembly during mitosis in baby hamster kidney cells.
A morphological analysis of the organizational changes in the type III intermediate filament (IF) system in dividing baby hamster kidney (BHK-21) cells was carried out by immunofluorescence and immunoelectron microscopy. The most dramatic change occurred during prometaphase, when the typical network of long 10-nm-diameter IF characteristic of interphase cells disassembled into aggregates containing short 4-6 nm filaments. During anaphase-telophase, arrays of short IF reappeared throughout the cytoplasm, and, in cytokinesis, the majority of IF were longer and concentrated in a juxtanuclear cap. These results demonstrate that the relatively stable IF cytoskeletal system of interphase cells is partitioned into daughter cells during mitosis by a process of disassembly and reassembly. This latter process occurs in a series of morphologically distinct steps at different stages of the mitotic process.
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Dynamic aspects of intermediate filament networks in BHK-21 cells.
A procedure was developed for the conjugation of vimentin with biotin. Biotinylated vimentin was then microinjected into BHK-21 cells and the fate of the labeled protein was determined at various times postinjection by indirect immunofluorescence. Microinjected vimentin could be traced through a specific sequence of morphological changes ultimately resulting in the formation of a filamentous network. The injected protein was first detected in spots dispersed throughout the cytoplasm. Subsequently, these spots appeared to cluster near the nucleus where they merged into a diffuse "cap." This cap coincided with a concentration of endogenous intermediate filaments and eventually gave rise to a filamentous network that was coincident with the endogenous intermediate filament network as determined by double-label immunofluorescence. The results indicate that the incorporation of exogenous vimentin into a filamentous network is initiated in a perinuclear region and progresses in a polarized fashion toward the cell surface.
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Phosphorylation and disassembly of intermediate filaments in mitotic cells.
As baby hamster kidney (BHK-21) cells enter mitosis, networks of intermediate filaments (IFs) are transformed into cytoplasmic aggregates of protofilaments. Coincident with this morphological change, the phosphate content of vimentin increases from 0.3 mol of Pi per mol of protein in interphase to 1.9 mol of Pi per mol of protein in mitosis. A similar increase in phosphate content is observed with desmin, from 0.5 mol of Pi per mol of protein to 1.5 mol of Pi per mol of protein. Fractionation of mitotic cell lysates by hydroxylapatite column chromatography reveals the presence of two IF protein kinase activities, designated as IF protein kinase I and IF protein kinase II. Comparison of two-dimensional 32P-labeled phosphopeptide maps of vimentin and desmin phosphorylated in vivo in mitosis, and in vitro using partially purified kinase fractions, reveals extensive similarity in the two sets of phosphorylation sites. Phosphorylation of in vitro polymerized IFs by IF protein kinase II induces complete disassembly as determined by negative-stain electron microscopy. The results support the idea that the disassembly of IFs in mitosis is regulated by the phosphorylation of its subunit proteins.
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Fractionation of desmosomes and comparison of the polypeptide composition of desmosomes prepared from two bovine epithelial tissues.
Desmosomes isolated from bovine tongue mucosa or muzzle epidermis appeared identical by ultrastructural analyses but had some differences in their polypeptide compositions as determined by SDS-PAGE. These preparations were extracted in 9 M urea, 10 mM Tris-HCl (pH 9), and 25 mM B-mercaptoethanol and then centrifuged at 240,000g for 30 min. The urea-soluble and insoluble fractions were analyzed by SDS-PAGE. The urea soluble fractions of both tongue and muzzle desmosomes were enriched in polypeptides of 240, 210, 81, and 75 kDa and also polypeptides (40 to 70 kDa) that were keratin-like, as determined by immunoblotting analyses with keratin antisera. The urea insoluble fraction of tongue desmosomes contained glycoproteins of 165, 160, 140, 110, and 100 kDa, while this fraction from muzzle contained glycoproteins of 165, 115, and 105 kDa. Ultrastructural examinations of insoluble pellets obtained from urea extracted tongue and muzzle desmosomes showed that most of the components at the cytoplasmic faces of the desmosomes were removed, while the membrane regions of the desmosomes resisted the treatment. The urea soluble proteins were dialyzed against 10 mM Tris-HCl (pH 7.6), and the resulting preparation was pelleted by centrifugation and examined by electron microscopy. Ultrastructural examination of this material revealed that it had assembled into a fibrillar meshwork, similar to the fibrillar region adjacent to the submembranous plaque of isolated desmosomes. Thus, treatment of isolated desmosomes with 9 M urea allowed the fractionation of membrane-associated desmosomal proteins from cytoplasmic desmosomal proteins. A comparison of these fractions from tongue and muzzle indicated that the polypeptide compositions of the desmosomes varied between tissues, especially with respect to the fractions enriched in either glycoproteins or keratin.
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A type III intermediate filament gene is expressed in mature neurons.
A cDNA (199E) specific for the 57 kd neural IF protein has been isolated from a PC12 cell lambda gt11 library. Antibody eluted from the fusion protein produced by 199E recognizes the 57 kd protein on immunoblots and, in PC12 cells, labels a pattern of fibrillar structures identical to that seen with 57 kd antiserum. In situ hybridization using antisense RNA transcripts labels areas of the nervous system known to contain the 57 kd protein. 199E hybridizes with a single mRNA species of approximately 2.0 kb from PC12 cells. A 199E-reactive message can be detected as early as E10 in rat embryos. Southern analyses suggest that there is only one gene for this protein. Amino acid sequence predicted from 199E indicates that the 57 kd protein is a type III IF protein like vimentin and desmin. Thus, expression of IF structural genes in neurons is not limited to the type IV neuronal IF triplet proteins.
Adrenal Gland Neoplasms↗
Isolation of cDNAs encoding desmosomal plaque proteins: evidence that bovine desmoplakins I and II are derived from two mRNAs and a single gene.
Desmoplakins (DPs) I and II (approximately equal to 240 and approximately equal to 210 kDa) are major components of the internal portion of the desmosomal cytoplasmic plaque. Desmosomes play a crucial role in cell-cell adhesion and serve as specific attachment sites for cytoplasmic intermediate filaments. Although DP-I and -II are closely related molecules, their structure (i.e., amino acid or DNA sequence) has not been determined. In addition, it is not known whether these proteins are derived from one or more genes or whether they result from posttranscriptional or posttranslational events. This paper describes the isolation and characterization of eight DP cDNA clones from a bovine lambda gt11 expression library. Fusion proteins from six of these clones selected antibodies that reacted with DP-I and -II and two selected antibodies that reacted with DP-I alone. Antibodies made against fusion protein produced by the DP1A clone reacted specifically with DP-I and -II on immunoblots. When used for indirect immunofluorescence on bovine tongue cryostat sections and cultured mouse keratinocytes, these antibodies produced a typical desmosomal staining pattern. RNA blot analysis demonstrated hybridization of three DP-I/II cDNA probes with two messages of approximately equal to 7.5 and approximately equal to 9.5 kilobases in bovine tongue RNA. In contrast, a cDNA clone that affinity-purified antibodies reacting with DP-I only hybridized exclusively with the 9.5-kilobase band. Southern blots of genomic DNA digested with a panel of restriction enzymes were hybridized with one probe derived from a DP-I/II clone and with one from a DP-I clone. Both probes hybridized with single bands of the same size in each digested sample of DNA. Together, these data suggest that DP-I and DP-II are translated from two separate messages in bovine tongue and that these messages may be derived from a single gene.
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Distribution of a novel 57 kDa intermediate filament (IF) protein in the nervous system.
A 57 kDa protein, that is not vimentin, is the major component of intermediate filaments (IF) obtained after 2 cycles of in vitro assembly from PC12 cells (Parysek and Goldman, 1987). By use of an antiserum to the 57 kDa protein, a cross-reacting antigen (of identical molecular weight) was detected on immunoblots of IF preparations and by immunofluorescence of various rat tissues. Immunolocalization studies on 3-4 micron frozen sections of tongue, small intestine, and adrenal gland showed bright labeling of nerve bundles and fine-caliber nerve fibers. The chromaffin cells and ganglion cells of the adrenal medulla also were labeled. In the nervous system, intense labeling was seen in small-caliber nerve fibers in sciatic nerve and spinal cord dorsal roots, as well as in the dorsal white columns and dorsal root ganglia. Of the ganglion cells, preferential labeling was seen in small-sized ganglion cells, whereas a monoclonal antibody to the 150 and 200 kDa neurofilament triplet (NFT) components labeled the large-sized ganglion cells. In the areas of the brain thus far examined with 57 kDa antiserum, there was labeling of components of cranial nerves and labeling of thin fibers in several areas, including the granular layer of the cerebellum and the corticospinal tract in the brain stem. For each tissue, adjacent sections treated with vimentin or glial fibrillary acidic protein antibody revealed labeling patterns distinct from that seen with either the 57 kDa or NFT antibodies. These results indicate that the 57 kDa IF protein is a neural IF component. Furthermore, this protein is distributed in only certain neuronal elements; these elements may be unified by an as yet unrecognized pattern of function in the nervous system.
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The relationship between intermediate filaments and microfilaments before and during the formation of desmosomes and adherens-type junctions in mouse epidermal keratinocytes.
Actin, keratin, vinculin and desmoplakin organization were studied in primary mouse keratinocytes before and during Ca2+-induced cell contact formation. Double-label fluorescence shows that in cells cultured in low Ca2+ medium, keratin-containing intermediate filament bundles (IFB) and desmoplakin-containing spots are both concentrated towards the cell center in a region bounded by a series of concentric microfilament bundles (MFB). Within 5-30 min after raising Ca2+ levels, a discontinuous actin/vinculin-rich, submembranous zone of fluorescence appears at cell-cell interfaces. This zone is usually associated with short, perpendicular MFB, which become wider and longer with time. Later, IFB and the desmoplakin spots are seen aligned along the perpendicular MFB as they become redistributed to cell-cell interfaces where desmosomes form. Ultrastructural analysis confirms that before the Ca2+ switch, IFB and desmosomal components are found predominantly within the perimeter defined by the outermost of the concentric MFB. Individual IF often splay out, becoming interwoven into these MFB in the region of cell-substrate contact. In the first 30 min after the Ca2+ switch, areas of submembranous dense material (identified as adherens junctions), which are associated with the perpendicular MFB, can be seen at newly formed cell-cell contact sites. By 1-2 h, IFB-desmosomal component complexes are aligned with the perpendicular MFB as the complexes become redistributed to cell-cell interfaces. Cytochalasin D treatment causes the redistribution of actin into numerous patches; keratin-containing IFB undergo a concomitant redistribution, forming foci that coincide with the actin-containing aggregates. These results are consistent with an IF-MF association before and during desmosome formation in the primary mouse epidermal keratinocyte culture system, and with the temporal and spatial coordination of desmosome and adherens junction formation.
Actin Cytoskeleton↗
Evidence for heterogeneity in the 160/165 x 10(3) Mr glycoprotein components of desmosomes.
We have prepared both monoclonal and polyclonal antibody preparations directed against the 160/165 x 10(3) Mr glycoproteins (desmogleins) of bovine tongue epithelial desmosomes. The polyclonal antibody preparation recognizes desmosomes in a number of mouse tissues, e.g. mouse skin, heart, bladder and trachea, as determined by immunofluorescence microscopy. Furthermore, the polyclonal antibodies recognize polypeptide(s), present in the high salt, Triton-insoluble residues ('cytoskeleton preparations') of mouse skin, heart, bladder and trachea, which comigrate with the 160/165 x 10(3) Mr glycoproteins of bovine tongue epithelial desmosomes as determined by 'Western' immunoblotting. Conversely, the monoclonal 160/165 x 10(3) Mr antibody preparation recognizes desmosomes of stratified squamous epithelial tissues but not desmosomes in other tissue types. Moreover, whereas the monoclonal antibodies recognize 160/165 x 10(3) Mr polypeptides in mouse skin cell cytoskeletons they show no immunoreactivity with the cytoskeleton preparations of mouse bladder, trachea and heart following immunoblotting. These results suggest therefore that although there are conserved epitopes of the 160/165 x 10(3) Mr glycoproteins there are also epitopes of these molecules which vary from tissue to tissue. Double label immunofluorescence observations of cryostat sections of mouse skin using the monoclonal antibodies and antibodies directed against desmoplakin, a plaque component of desmosomes, reveal that the monoclonal antibodies do not recognize certain desmosomes in basal cells which are recognized by desmoplakin antibodies. Indeed, double label observations of cryostat sections of mouse skin using the monoclonal antibodies and human autoantibodies which react with hemidesmosomal components suggest that the monoclonal antibodies stain desmosomes located along the apical surfaces of basal cells but fail to recognize desmosomes along the lateral surfaces of these same cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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Characterization of intermediate filaments in PC12 cells.
A 57 kDa protein is the major polypeptide in intermediate filament (IF)-enriched cytoskeletal preparations obtained from the neuronal cell line PC12 (rat pheochromocytoma). Under the conditions used to assemble IF in vitro from other cultured cell lines, 10 nm filaments are formed after 2 cycles of disassembly-assembly from PC12 IF-enriched cytoskeletal preparations; the 57 kDa protein is the major component of the final IF pellet. The 57 kDa protein is immunologically related to the BHK-21 fibroblast 55 kDa protein (vimentin), but a comparison of the peptide maps of PC12 57 kDa and BHK 55 kDa indicates that they are different proteins. With the use of a polyclonal antiserum to the PC12 57 kDa protein, immunofluorescence observations of PC12 cells not treated with NGF reveal a juxtanuclear "knot"-like structure. After NGF treatment, the "knots" are less prominent and many IF arrays are seen coursing through the cytoplasm and extending into the neurites. These immunofluorescence observations of the distribution of IF are corroborated by fine-structural analyses. SDS-PAGE analyses indicate that IF-enriched cytoskeletons isolated from NGF-treated cells have a polypeptide composition similar to that of untreated cells, that is, the 57 kDa protein remains the major polypeptide. SDS-PAGE and immunoblotting analyses show that untreated and NGF-treated PC12 cells also contain relatively minor amounts of the 68, 150, and 200 kDa neurofilament triplet (NFT) proteins. Under immunofluorescence, only 5% of untreated PC12 cells are found to contain a juxtanuclear "knot" labeled with NFT antibodies, but with time following NGF treatment, the number of fluorescent cells increases. After about 2 weeks of NGF treatment, all of the PC12 cells appear to contain NFT antibody-positive filamentous structures. As assessed by immunofluorescence, the NFT polypeptides appear to codistribute with the 57 kDa protein in both untreated and NGF-treated PC12 cells. These data indicate that PC12 cells contain IF composed of a complex set of polypeptides, including a previously unidentified 57 kDa IF protein. While NGF may induce production of NFT polypeptides, there does not appear to be a "switch" from known mesenchymal IF polypeptide expression to NFT polypeptide expression upon stimulation of PC12 cells with NGF.
Adrenal Gland Neoplasms↗
Evidence for an interaction between the cell surface and intermediate filaments in cultured fibroblasts.
Intermediate filaments (IF) were found in close proximity to the plasma membrane in substrate attached baby hamster kidney cells (BHK-21) and chick embryo fibroblasts (CEF) as well as cells removed from their substrate in the absence of trypsin. However, in cells removed with trypsin, it appeared that IF had retracted away from the membrane. In cells with abundant extracellular matrix (ECM), colchicine induced massive cables of IF, which appeared to interact with specialized areas of the inner plasma membrane. In cells lysed to extract most microfilaments and cytoplasmic constituents, the intact IF network which remained was closely associated with the ECM. From these ultrastructural observations it was concluded that IF interact in some way with a "cell membrane complex" defined as comprising the plasma membrane and molecules attached to its inner and outer surfaces. In order to investigate the possibility that components of the membrane complex may co-isolate with IF, native intermediate filaments (NIF) were prepared. In addition to the structural subunits and other associated polypeptides, a approximately 220 kd species which reacted specifically with antibodies directed against the ECM protein fibronectin (FN) was observed; 220 kd was still present after NIF were isolated under pH conditions where FN is more soluble, suggesting that its presence was not simply due to the coprecipitation of two insoluble proteins. Immunofluorescence and immunogold localization confirmed that FN is a component of the cell membrane complex with which IF appeared to interact.
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Relationship between intermediate filaments and microfilaments in cultured fibroblasts: evidence for common foci during cell spreading.
Spreading and fully spread chick embryo fibroblasts (CEF) were examined by double-label fluorescence microscopy using the actin-specific probe rhodamine-phalloidin and an antibody directed against CEF intermediate filaments (IF). During midspreading, a striking relationship became discernible: statistical analysis showed that approximately half of the cell population exhibited one or more phase-dense, phalloidin-binding nodules that appeared to act as foci from which IF diverged. Coincidence between actin-containing structures and IF was not limited to these centers; IF could also frequently be seen running in close parallel arrays with stress fibers. Ultrastructural analysis confirmed the presence of non-membrane-bound out-pocketings along the length of stress fibers from which 10-nm IF diverged. These structures varied in size and shape, and displayed a dense, fine fibrillar appearance. IF and microfilaments (MF) were distinguished by size and by decoration of MF with myosin subfragment-1. Other IF-MF interactions were seen in cells of all stages: IF were observed to loop through stress fibers, most frequently at the cell margins. In colchicine-treated cells, IF became redistributed into cables that often ran parallel and appeared to merge with stress fibers. Cytochalasin D-treated CEF exhibited loose aggregates of actin-containing material that appeared to be associated with IF. These results suggest the possibility of an interaction between actin-containing structures and IF, particularly during cell spreading in cultured fibroblasts.
Actin Cytoskeleton↗
Is the hemidesmosome a half desmosome? An immunological comparison of mammalian desmosomes and hemidesmosomes.
Although the mammalian epidermal basal cell hemidesmosome bears some superficial resemblance to one half of a desmosome at the ultrastructural level, examination of the structure of the electron-dense submembranous plaques of the hemidesmosome and desmosome reveals that they differ with respect to their overall morphology and dimensions. Based on these findings, we wondered whether components of the desmosome are present in the hemidesmosome. In order to determine this we prepared a number of stratified squamous epithelial tissues for indirect immunofluorescence using antibody preparations directed against known desmosome components including desmoplakin and certain glycoproteins. These antibody preparations do not show reaction with hemidesmosomes by indirect immunofluorescence criteria. We have also utilized bullous pemphigoid (BP) autoantibodies that have been shown to recognize hemidesmosomes in mammalian skin cells [Mutasim et al., J. Invest. Derm., 84:47-53, 1985]. Double label indirect immunofluorescence observations of neonatal mouse skin prepared using desmoplakin antibodies and BP autoantibodies reveal that hemidesmosomes that are stained by the BP autoantibodies are not recognized by the desmoplakin antibodies. We confirmed these findings at the ultrastructural level by indirect immunogold localization of desmoplakin antibodies and BP autoantibodies. Therefore, the hemidesmosome does not appear to be one half of a desmosome and may possess a very different molecular organization relative to the desmosome. We raise the possibility that the variability between the hemidesmosome and desmosome that we detect at the morphological and immunological level may reflect the functional differences of these two types of junctions.
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