Glial fibrillary acidic protein (GFAP) is an intermediate filament protein found predominantly in astrocytes. This specificity has recommended the GFAP gene promoter for targeting transgene expression to astrocytes. Although both we [Brenner et al. J. Neurosci. 14:1030-1037, (1994)] and others [Mucke et al. New Biol. 3:465-474, (1991)] have reported astrocyte specificity for GFAP promoters, we demonstrate here that these DNA sequences can also direct activity in neurons. The pattern of neuronal activity varied with both the nature of the expressed sequence and the transgene insertion site. Specifically, neuronal expression was very high for a protective protein/cathepsin A minigene, moderate for lacZ and undetectable for GFP. These findings, coupled with a survey of the literature, recommend that investigators using GFAP-driven transgenes verify specificity for each line studied, using a detection system whose sensitivity is sufficient to detect a compromising level of misexpression.

Hepatocytes express keratins 8 and 18 (K8/18) as their only cytoskeletal intermediate filament (IF) proteins, and K8/18 mutations predispose their carriers to liver cirrhosis. Transgenic mice that overexpress mutant human K18 (Arg89->>Cys [R89C]) develop mild chronic hepatitis, hepatocyte fragility, keratin filament disruption, and increased susceptibility to drug-induced liver injury. K18 is a major caspase substrate during apoptosis, and K8- or K18-null mice are significantly predisposed to Fas- and possibly tumor necrosis factor (TNF)-mediated apoptosis in the liver. Here we tested the potential role of the K18 R89C mutation on Fas- or TNF-mediated apoptotic liver injury by injecting Fas antibody (Ab) or TNF-alpha plus actinomycin D into mice that overexpress wild-type (WT) human K18 (with intact filament network, termed TG2 mice) or into K18 R89C mice (with disrupted filament network). K18 R89C mice are significantly more susceptible to Fas-mediated liver injury compared with nontransgenic and TG2 mice. This included differences in lethality, histology, apoptosis, and serum transaminase levels. In contrast, K18 WT and R89C mice manifest similar sensitivity to TNF-induced injury. Both Fas- and TNF-induced apoptosis in liver tissues are associated with caspase-mediated K18 degradation and increased keratin phosphorylation on several but not all sites. In conclusion, transgenic mouse K18 mutation and its consequent keratin filament disruption predispose hepatocytes to Fas- but not TNF-mediated apoptotic injury. This supports the association of keratin mutations with cirrhosis in patients with liver disease and suggests that keratins modulate apoptosis induced by Fas but not TNF.

Intercellular junctions which are similar in ultrastructure and protein composition to typical desmosomes have so far only been found in epithelial cells and in heart tissue, specifically in the intercalated disks of cardiac myocytes and at cell boundaries between Purkinje fiber cells. In epithelial cells the cytoplasmic side of desmosomes, the 'desmosomal plaque', represents a specific attachment structure for the anchorage of intermediate filaments (IF) of the cytokeratin type. Cardiac myocytes do not contain cytokeratin filaments. In primary cultures of rat cardiac myocytes, we have examined by immunofluorescence and electron microscopy, using single and double label techniques, whether other types of IF are attached to the desmosomal plaques of the heart. Antibodies to desmoplakin, the major protein of the desmosomal plaque, have been used to label specifically the desmosomal plaques. It is shown that the desmoplakin-containing structures are often associated with IF stained by antibodies to desmin, i.e., the characteristic type of IF present in these cells. Like cytokeratin filaments in epithelial cells, desmin filaments attach laterally to the desmosomal plaque. They also remain attached to these plaques after endocytotic internalization of desmosomal domains by treatment of the cells with EGTA. These desmin filaments do not appear to attach to junctions of the fascia adherens type and to nexuses (gap junctions). These observations show that anchorage at desmosomal plaques is not restricted to IF of the cytokeratin type and that IF composed of either cytokeratin or desmin, specifically attach, in a lateral fashion, to desmoplakin-containing regions of the plasma membrane. We conclude that special domains exist in these two IF proteins that are involved in binding to the desmosomal plaque.

Crescentin, which is the founding member of a rapidly growing family of bacterial cytoskeletal proteins, was previously proposed to resemble eukaryotic intermediate filament (IF) proteins based on structural prediction and in vitro polymerization properties. Here, we demonstrate that crescentin also shares in vivo properties of assembly and dynamics with IF proteins by forming stable filamentous structures that continuously incorporate subunits along their length and that grow in a nonpolar fashion. De novo assembly of crescentin is biphasic and involves a cell size-dependent mechanism that controls the length of the structure by favoring lateral insertion of crescentin subunits over bipolar longitudinal extension when the structure ends reach the cell poles. The crescentin structure is stably anchored to the cell envelope, and this cellular organization requires MreB function, identifying a new function for MreB and providing a parallel to the role of actin in IF assembly and organization in metazoan cells. Additionally, analysis of an MreB localization mutant suggests that cell wall insertion during cell elongation normally occurs along two helices of opposite handedness, each counterbalancing the other's torque.

Modulated fringe pattern photobleaching (MFPP) was used to measure the translational diffusion of microinjected fluorescein isothiocyanate (FITC)-labeled proteins of different sizes in the cytoplasm of cultured muscle cells. This technique, which is an extension of the classical fluorescence recovery after photobleaching (FRAP) technique, allows the measurement of the translational diffusion of macromolecules over several microns. Proteins used had molecular masses between 21 and 540 kDa. The results clearly indicated that the diffusivity of the various proteins is a decreasing function of their hydrodynamic radius. This decrease is more rapid with globular proteins than with FITC-labeled dextrans (, Biophys. J. 70:2327-2332), most likely because, unlike globular proteins, dextrans are randomly coiled macromolecules with a flexible structure. These data do not exclude the possibility of a rapid diffusion over a short distance, unobservable with our experimental set-up, which would take place within the first milliseconds after bleaching and would correspond to the diffusion in restricted domains followed by impeded diffusion provoked by the network of microtubules, microfilaments, and intermediate filaments. Thus our results may complement rather than contradict those of Verkman and collaborators (, J. Cell Biol. 138:1-12). The biological consequence of the size-dependent restriction of the mobility of proteins in the cell cytoplasm is that the formation of intracellular complexes with other proteins considerably reduces their mobility.

To study the construction of the ER, we used the microtubule-disrupting drug nocodazole to induce the complete breakdown of ER structure in living cells followed by recovery in drug-free medium, which regenerates the ER network within 15 min. Using the fluorescent dye 3,3'-dihexyloxacarbocyanine iodide to visualize the ER, we have directly observed the network construction process in living cells. In these experiments, the ER network was constructed through an iterative process of extension, branching, and intersection of new ER tubules driven by the ER motility previously described as tubule branching. We have tested the cytoskeletal requirements of this process. We find that newly formed ER tubules are aligned with single microtubules but not actin fibers or vimentin intermediate filaments. Microtubule polymerization preceded the extension of ER tubules and, in experiments with a variety of different drugs, appeared to be a necessary condition for the ER network formation. Furthermore, perturbations of the pattern of microtubule polymerization with microtubule-specific drugs caused exactly correlated perturbations of the pattern of ER construction. Induction of abnormally short, nonintersecting microtubules with 20 microM taxol prevented the ER network formation; ER tubules only extended along the few microtubules contacting the aggregated ER membranes. This requirement for a continuous network of intersecting microtubules indicates that ER network formation takes place through the branching and movement of ER membranes along microtubules. Cytochalasin B had no apparent effect on the construction of the ER network during recovery, despite apparently complete disruption of actin fibers as stained by phalloidin. Blockage of protein synthesis and disorganization of intermediate filaments with cycloheximide pretreatment also failed to perturb ER construction.

We have examined the nuclear localization of isoprenylated proteins in CHO-K1 cells labeled with [14C]mevalonate. Nuclear proteins of 68, 70, and 74 kD, posttranslationally modified by an isoprenoid, are also components of a nuclear matrix-intermediate filament preparation from CHO cells. Furthermore, the 68-, 70-, and 74-kD isoprenylated polypeptides are immunoprecipitated from cell extracts with two different anti-lamin antisera. Based on exact two-dimensional comigration with lamin B, both from rat liver lamin and CHO nuclear matrix-intermediate filament preparations, and its immunoprecipitation with anti-lamin antisera, we conclude that the 68-kD isoprenylated protein found in nuclei from [14C]mevalonate-labeled CHO cells is lamin B. The more basic 74-kD isoprenylated nuclear protein is similar in molecular mass and isoelectric pH variants to the lamin A precursor polypeptide reported by others. Starving cells for mevalonate results in a dramatic accumulation of a polypeptide that comigrates on two-dimensional, non-equilibrium pH gradient electrophoresis (NEPHGE) gels with the 74-kD isoprenylated protein. The 70-kD isoprenylated protein, which is resolved on NEPHGE gels as being higher in molecular mass and slightly more basic than lamin B, has not yet been identified.

In most eukaryotic cells, the nucleus is localized to a specific location. This highlight article focuses on recent advances describing the mechanisms of nuclear migration and anchorage. Central to nuclear positioning mechanisms is the communication between the nuclear envelope and the cytoskeleton. All three components of the cytoskeleton-microtubules, actin filaments and intermediate filaments-are involved in nuclear positioning to varying degrees in different cell types. KASH proteins on the outer nuclear membrane connect to SUN proteins on the inner nuclear membrane. Together they transfer forces between the cytoskeleton and the nuclear lamina. Once at the outer nuclear membrane, KASH proteins can interact with the cytoskeleton. Nuclear migrations are a component of many cellular migration events and defects in nuclear positioning lead to human diseases, most notably lissencephaly.

The presence of intermediate filament proteins of cytokeratin/prekeratin type and vimentin type was evaluated in non-neoplastic thyroid glands and in different types of thyroid neoplasms. Follicular epithelium of both normal and goitrous thyroids showed a strong reaction with anticytokeratin antibodies that widely cross-react with various simple epithelia. On the other hand, in normal thyroid, there were only occasionally (in one of 12 cases) solitary cells reacting with antibodies to epidermal prekeratin. In nodular goiters, such cells were often seen (eight of 18), especially among the lining cells of cysts, and in chronic thyroiditis in all (12 of 12) cases. Only the stromal cells and intraluminal macrophages reacted with antibodies to vimentin. Neoplastic cells of papillary carcinomas showed a positive staining reaction both with antibodies to cytokeratins and to epidermal prekeratin. Follicular carcinoma cells, although positive for cytokeratins, could generally not be stained with antibodies to epidermal prekeratin. Medullary carcinoma cells also showed cytokeratin positivity and, only occasionally, positivity for epidermal prekeratin. Anaplastic carcinomas were also reactive with antibodies to cytokeratin but, for the most part, were negative for epidermal prekeratin. Interestingly, some neoplastic cells of all types of thyroid carcinomas also appeared to contain vimentin, as shown with both polyclonal and monoclonal antivimentin antibodies. In contrast to carcinomas, the intermediate filaments of thyroid sarcomas and lymphomas were only of vimentin type. Furthermore, it was found that the papillary structures in benign goiters were only reactive with cytokeratin antibodies and lacked, in contrast to papillary carcinomas, epidermal prekeratin-like immunoreactivity. Hence, the analysis of intermediate filament proteins of thyroid tumors can be utilized to differentiate between papillary and follicular carcinomas and between benign and malignant papillary lesions as well as between anaplastic thyroid carcinomas and sarcomas or lymphomas.

Cells of an established clonal line (RVF-SMC) derived from rat vena cava are described by light and electron microscope methods and biochemical analysis of the major proteins. The cells are flat, and they moderately elongate and form monolayers. They are characterized by prominent cables of microfilaments bundles decoratable with antibodies to actin and alpha-actinin. These bundles contain numerous densely stained bodies and are often flanked by typical rows of surface caveolae and vesicles. The cells are rich in intermediate-sized filaments of the vimentin type but do not show detectable amounts of desmin and cytokeratin filaments. Isoelectric focusing and protein chemical studies have revealed actin heterogeneity. In addition to the two cytoplasmic actins, beta and gamma, common to proliferating cells, two smooth muscle-type actins (an acidic alpha-like and a gamma-like) are found. The major (alpha-type) vascular smooth muscle actin accounts for 28% of the total cellular actin. No skeletal muscle or cardiac muscle actin has been detected. The synthesis of large amounts of actin and vimentin and the presence of at least three actins, including alpha-like actin, have also been demonstrated by in vitro translation of isolated poly(A)+ mRNAs. This is, to our knowledge, the first case of expression of smooth muscle-type actin in a permanently growing cell. We conclude that permanent cell growth and proliferation is compatible with the maintained expression of several characteristic cell features of the differentiated vascular smooth muscle cell including the formation of smooth muscle-type actin.

A series of 14 monoclonal antibodies (MAs) has been obtained from a single rat-mouse fusion using gel-excised bovine glial filament (GF) proteins as immunogens. These MAs were characterized by two separate immunochemical assays and by two different immunohistochemical methods. Nine MAs demonstrated specificity for GF proteins. One MA also recognized an epitope shared by intermediate filaments (IF) of the vimentin class (VF). Using the enzyme-linked immunosorbent assay, four of the MAs recognized 200,000, 150,000, and 51,000 dalton proteins, suggesting that these MAs were specific for GF proteins (the 51,000 dalton protein) and neurofilament (NF) proteins (the two high-molecular-weight proteins). However, in both of the immunohistochemical assay systems, these MAs stained neurons and their processes but not astroglial cells. These observations strongly suggest that the 51,000-dalton protein recognized by these four MAs was not derived from GF proteins but instead represents derivatives of NF protein subunits comigrating in gels with GF proteins. These data provide additional information concerning the unique and shared antigenic determinants of the three classes of IF (NF, GF, and VF) of the CNS. In addition, they draw attention to the fact that proteins of certain IF may undergo degradation and comigrate in gels with the proteins of unrelated IF. This emphasizes the need for the use of independent immunochemical and immunohistochemical assays in the characterization of the specificity of MAs.

Desmosomes are major intercellular junctions found in association with intermediate filaments in epithelial, cardiac and arachnoidal tissue. Desmoplakins I and II (DPI and II) are highly related proteins localized in the innermost part of the desmosomal plaque and are candidates for linking intermediate filaments (IF) to the desmosomal complex. While investigators agree that DPI is present in all epithelia, they disagree on the distribution of DPII. Some have reported DPII to be restricted to stratified tissue and have furthermore suggested that the expression of DPII may be linked to stratification. We have compared the expression of DPI and II at the mRNA and protein levels in cell lines derived from simple, transitional and stratified epithelia. Northern blot analysis revealed DPI and II mRNA to be present in all cell lines as well as simple and stratified epithelial tissues. However, DPII mRNA could not be detected in cardiac muscle tissue. Immunoblotting and immunoprecipitation demonstrated the presence of DPI and II in all cell lines at the whole-cell protein level as well as in association with cytoskeletal fractions. Immunofluorescence staining was used to correlate the biochemical findings with the localization of DPI and II. While most cell lines exhibited typical intercellular and in many cases cytoplasmic DP staining, T24 cells exhibited predominantly diffuse and dotty cytoplasmic staining. In addition, we investigated whether changes in DPI and II expression occurred following calcium-induced cell contact formation and stratification in the human pharyngeal cell line, FaDu. No significant changes in mRNA or whole-cell protein levels were observed during a period of 5 days following the calcium switch. However, immunoblotting revealed a significant increase in DPI and II levels in the insoluble protein pool during desmosome formation. These observations indicated a possible recruitment of soluble DPI/II into an insoluble pool after induction of desmosome assembly by the calcium switch, consistent with earlier reports for MDCK cells. In summary, our results suggest that the expression of DPII is not strictly linked to stratification or differentiation; however, the apparent absence of DPII mRNA from cardiac muscle suggests it may not be a constituent of all desmosomes.

The cytokeratin family of intermediate filament (IF) proteins can be grouped into the epithelial polypeptides ("soft alpha-keratins"), of which at least 19 exist in the various human epithelia, and the hair-type cytokeratins ("hard alpha-keratins"), which are typical of trichocytes, i.e., the living hair-forming cells. We have recently shown [34] that the hair follicles from diverse mammalian species contain a set of eight major cytokeratin polypeptides, four each of the acidic (type I) and the basic (type II) subfamily, which are different from all known epithelial cytokeratins. In addition, we have identified two new minor trichocytic cytokeratin polypeptides, designated Hax (type I) and Hbx (type II). Antibodies against trichocytic cytokeratins that do not crossreact with any of the epithelial cytokeratins have enabled us to study the expression of both kinds of cytokeratin in the various cell types of human and bovine hair follicles. Using immunofluorescence microscopy, we have observed intense reactions of trichocytic cytokeratins only in cells contributing to the forming hairs, i.e., hair shaft, medulla and cuticle, whereas immunostaining of the peribulbar matrix cells was weaker, if at all detectable. In contrast, epithelial cytokeratins were localized in both the inner and outer root sheath epithelia but, surprisingly, also in certain portions of the trichocyte column, notably cells of the cuticle, certain medullary cells, and trichocytes of the basalmost peripapillary cell layers. Cells coexpressing trichocytic and epithelial cytokeratins have been identified by double-label immunofluorescence microscopy. Epithelial cytokeratins of the inner and outer root sheath epithelia include, most remarkably, "simple-epithelium-type" cytokeratins 8, 18, and 19; these occur in certain peribulbar regions, in distinct patterns, but with variable frequencies. The occurrence of simple epithelial cytokeratins in hair follicles has also been confirmed by high-sensitivity immunoblotting of follicular polypeptides separated by gel electrophoresis. Vimentin-positive cells were abundantly interspersed (in some follicles, but not in all) between the trichocytes of the peripapillary cone, most of them probably being melanocytes. The cell-type complexity of the hair follicle and the different patterns of cytoskeletal protein expression in the various hair follicle cells are discussed in relation to the development and growth of this organ.

Previous analyses of the nuclear lamina of mammalian cells have revealed three major protein components (lamins A, B and C) that have been identified by protein sequence homology as members of the intermediate filament (IF) protein family. It has been claimed that mammalian cells contain either all three lamins or lamin B alone. Using monoclonal antibodies specific for B-type lamins and cDNA cloning we identified a second major mammalian B-type lamin (murine lamin B2), thus showing that lamin composition in mammals is more complex than previously thought. Lamin B2 is coexpressed with lamin B1 (formerly termed lamin B) in all somatic cells and mammalian species that we analysed, including a variety of cells currently believed to contain only a single lamin. This suggests that two B-type lamins are necessary to form a functional lamina in mammalian somatic cells. By cDNA cloning we found that Xenopus laevis lamin LII is the amphibian homolog of mammalian lamin B2. Lamin expression during embryogenesis of amphibians and mammals shows striking similarities. The first lamins expressed in the early embryo are the two B-type lamins, while A-type lamins are only detected much later in development. These findings indicate that the genomic differentiation into two B-type lamins occurred early in vertebrate evolution and has been maintained in both their primary structure and pattern of expression.

OBJECTIVE

To deduce the function of the lens-specific cytoskeletal structure, the beaded filament, by blocking expression of the fiber cell-specific beaded filament protein CP49.

METHODS

The first exon of the mouse CP49 gene was deleted by using targeted genomic deletion techniques. Gene deletion was assessed through Southern blot analysis and PCR. Translation and protein expression were characterized by Northern and Western blot analysis of both CP49 and its assembly partner filensin. The architecture of knockout lenses was compared with that of wild-type lenses at the histologic level by light microscopy. Lens clarity was assessed in situ by direct ophthalmic examination and slit lamp microscopy.

RESULTS

Transcription and translation of CP49 were successfully negated in knockout animals. Lenses homozygous for the CP49 deletion showed no obvious changes in lens architecture at the light microscope level. Filensin levels were sharply reduced, although filensin mRNA levels appeared unchanged. Direct examination of lenses showed no obvious loss of lens clarity, but slit lamp examination revealed the emergence of opacification in even the youngest animals. The opacification worsened with age.

CONCLUSIONS

The absence of CP49 causes a subtle loss of optical clarity in the ocular lens, a loss that worsens with age. However, CP49 is not essential for the assumption or maintenance of overall fiber cell shape or long-range order of fiber cells. CP49 appears to regulate the protein levels of its assembly partner filensin, suggesting a mechanism for the regulation of beaded filament protein stoichiometry.

Actin filaments are integral components of the plasma membrane-associated cytoskeleton (membrane skeleton) and are believed to play important roles in the determination of cell polarity, shape, and membrane mechanical properties, however the roles of actin regulatory proteins in controlling the assembly, stability, and organization of actin filaments in the membrane skeleton are not well understood. Tropomodulin is a tropomyosin and actin-binding protein that stabilizes tropomyosin-actin filaments by capping their pointed ends and is associated with the spectrin-actin membrane skeleton in erythrocytes, skeletal muscle cells, and lens fiber cells, a specialized epithelial cell type. In this study, we have investigated the role of tropomodulin and other membrane skeleton components in lens fiber cell differentiation and maturation. Our results demonstrate that tropomodulin is expressed concomitantly with lens fiber cell differentiation and assembles onto the plasma membrane only after fiber cells have begun to elongate and form apical-apical contacts with the undifferentiated epithelium. In contrast, other membrane skeleton components, spectrin, actin, and tropomyosin, are constitutively expressed and assembled on the plasma membranes of both undifferentiated and differentiated fiber cells. Tropomodulin, but not other membrane skeleton components, is also enriched at a novel structure at the apical and basal ends of newly elongated fiber cells at the fiber cell-epithelium and fiber cell-capsule interface, respectively. Once assembled, tropomodulin and its binding partners, tropomyosin and actin, remain membrane-associated and are not proteolyzed during fiber cell maturation and aging, despite proteolysis of alpha-spectrin and other cytoskeletal filament systems such as microtubules and intermediate filaments. We propose that actin filament stabilization by tropomodulin, coupled with partial proteolysis of other cytoskeletal components, represents a programmed remodeling of the lens membrane skeleton that may be essential to maintain plasma membrane integrity and transparency of the extremely elongated, long-lived cells of the lens. The unique localization of tropomodulin at fiber cell tips further suggests a new role for tropomodulin at cell-cell and cell-substratum contacts; this may be important for cell migration and/or adhesion during differentiation and morphogenesis.

The effects of Clostridium botulinum C3 ADP-ribosyltransferase and of Clostridium botulinum C2 toxin were studied on the cytoskeleton of rat hepatoma FAO and human glioma U333 cells. After treatment of these cells for 24 to 48 h with C3 (3-30 micrograms/ml), the actin microfilaments disappeared, and the intermediate filament network was found to collapse, while microtubules remained intact. Similar alterations of the cytoskeletal filaments without affecting microtubules were induced by the actin-ADP-ribosylating C2 toxin. In FAO cells, C3 caused the rounding up of cells. Concomitantly, cytosolic 22 to 24 kDa proteins were ADP-ribosylated in a guanine nucleotide-dependent manner. Rounding up of cells and ADP-ribosylation of proteins in intact cells were observed at similar concentration of the transferase. These data suggest a role of the protein substrates of C3 in the regulation of the cytoskeletal integrity.

Differential screening of a cDNA library from the PC12 rat pheochromocytoma cell line previously revealed a clone, clone 73, whose corresponding mRNA is induced by nerve growth factor (NGF). Induction parallels NGF-stimulated PC12 differentiation from a chromaffinlike phenotype to a sympathetic neuronlike phenotype. We report that DNA sequence analysis reveals that clone 73 mRNA encodes an intermediate filament (IF) protein whose predicted amino acid sequence is distinct from the known sequences of other members of the IF protein family. The sequence has highest homology with desmin and vimentin and includes the highly conserved central alpha-helical rod domain with the characteristic heptad repeat of hydrophobic residues, but has lower homology in the amino-terminal head and carboxyl-terminal tail domains. The head domain contains a large number of serine residues which are potential phosphorylation sites. The expression of clone 73 in vivo in the nervous system of the adult rat was investigated by in situ hybridization of clone 73 probes to tissue sections. The mRNA is expressed at high levels in ganglia of the peripheral nervous system, including the superior cervical ganglion (sympathetic), ciliary ganglion (parasympathetic), and dorsal root ganglion (sensory). In the central nervous system, motor nuclei of cranial nerves III, IV, V, VI, VII, X, and XII as well as ventral horn motor neurons and a restricted set of other central nervous system nuclei express the clone 73 mRNA. Tissues apart from those of the nervous system did not in general express the mRNA, with only very low levels detected in adrenal gland. We discuss the implications of these results for the mechanism of NGF-induced PC12 cell differentiation, the pathways of neuronal development in vivo, and the possible function of the clone 73 IF protein and its relationship to other IF proteins.

Twenty-two benign pleomorphic adenomas of the major salivary glands were studied by transmission electron microscopy and immunohistochemical techniques (three cases) in order to characterize the cell types comprising the epithelial and so-called mesenchymal regions of the tumors. Light- and electron-microscopic studies showed the tumors to consist of variable mixtures of neoplastic ductular epithelial cells, rare acinar cells, and metaplastic myoepithelial cells. Many of the loosely organized "stromal cells" contained structures indicative of their myoepithelial origin, e.g., perinuclear tonofilaments, ectoplasmic actin microfilaments, and remnants of basement membrane. Polyclonal antikeratin antisera strongly stained ductular epithelial and myoepithelial cells, squamoid cell nests, and periductular myoepithelial cells, whereas myxoid and chondroid cells were less intensely stained. Monoclonal cytokeratin antibody AE1 stained only the ductular epithelial cells in both the normal glands and tumors. In contrast, S-100 protein, which is present only in scattered acinar cells and myoepithelial cells in the normal parotid gland, was found in the ductular and periductular myoepithelial cells, isolated myxoid cells, and chondroid and cartilagenous cells in the tumors. Actin was found in all the cell types of the tumor but staining was strongest in the ducts. Double immunofluorescence staining for cytokeratin and vimentin revealed coexpression of both types of intermediate filaments in occasional normal acinar and intercalated duct myoepithelial cells, and in some cells in the myxoid and chondroid regions of the tumors. In the tumors, vimentin was present in occasional periductular myoepithelial cells, stellate myxoid cells, and especially in chondroid cells and chondrocytes. Our findings indicate that benign pleomorphic adenomas of the major salivary glands are pure epithelial cell tumors. The histologic complexity of these neoplasms is due to the ability of the neoplastic ductular myoepithelial cell to modulate its morphologic appearance and intermediate filament composition, and to produce large amounts of matrix substances. We further postulate that these tumors arise from neoplastically transformed intercalated ducts.

Treatment of bovine tongue mucosa with 1 M KCl induced a split in the lamina densa of the basement membrane zone (BMZ). The epithelium was then separated from the underlying connective tissue. Electron microscopic analysis of the stripped epithelium revealed that hemidesmosomes and their associated intermediate filaments (IF) remain along the basal surface of the epithelium. This surface was solubilized in an SDS/urea-containing buffer. Characterization of components of this protein mixture was undertaken using human autoantibodies from bullous pemphigoid (BP) patients that have been shown to recognize hemidesmosomal plaque elements (Mutasim, D. F., Y. Takahashi, R. S. Labib, G. J. Anhalt, H. P. Patel, and L. A. Diaz. 1985. J. Invest. Dermatol. 84:47-53) and by production of mAbs. Affinity-purified autoantibodies directed against 180- and 240-kD polypeptides present in the protein preparation generated strong immunofluorescence staining patterns along the BMZ of bovine tongue mucosa. Furthermore, immunogold localization revealed that these two polypeptides are associated with the hemidesmosomal plaque. A mAb preparation directed against a 125-kD polypeptide present in this same protein mixture lamina lucida side of the hemidesmosome. Autoantibodies in BP serum samples, affinity-purified 180-kD autoantibodies and the mAb preparation generated a punctate stain along the substratum attached surface of epithelial cells maintained on glass substrata for approximately 1 wk. The spots appeared to be associated with bundles of IF in cultured mouse keratinocytes. These monospecific antibody probes should prove invaluable for the study of hemidesmosome structure, assembly, and function.

Patients with temporal lobe epilepsy (TLE) often have a shrunken hippocampus that is known to be the location in which seizures originate. The role of the sclerotic hippocampus in the causation and maintenance of seizures in temporal lobe epilepsy (TLE) has remained incompletely understood despite extensive neuropathological investigations of this substrate. To gain new insights and develop new testable hypotheses on the role of sclerosis in the pathophysiology of TLE, the differential gene expression profile was studied. To this end, DNA microarray analysis was used to compare gene expression profiles in sclerotic and non-sclerotic hippocampi surgically removed from TLE patients. Sclerotic hippocampi had transcriptional signatures that were different from non-sclerotic hippocampi. The differentially expressed gene set in sclerotic hippocampi revealed changes in several molecular signaling pathways, which included the increased expression of genes associated with astrocyte structure (glial fibrillary acidic protein, ezrin-moesin-radixin, palladin), calcium regulation (S100 calcium binding protein beta, chemokine (C-X-C motif) receptor 4) and blood-brain barrier function (Aquaaporin 4, Chemokine (C-C- motif) ligand 2, Chemokine (C-C- motif) ligand 3, Plectin 1, intermediate filament binding protein 55kDa) and inflammatory responses. Immunohistochemical localization studies show that there is altered distribution of the gene-associated proteins in astrocytes from sclerotic foci compared with non-sclerotic foci. It is hypothesized that the astrocytes in sclerotic tissue have activated molecular pathways that could lead to enhanced release of glutamate by these cells. Such glutamate release may excite surrounding neurons and elicit seizure activity.

Some of the key steps in cancer metastasis are the migration and invasion of tumor cells; these processes require rearrangement of the cytoskeleton. Actin filaments, microtubules, and intermediate filaments involved in the formation of cytoskeletal structures, such as stress fibers and pseudopodia, promote the invasion and metastasis of tumor cells. Therefore, it is important to explore the mechanisms underlying cytoskeletal regulation. The ras homolog family (Rho) and Rho-associated coiled-coil containing protein serine/threonine kinase (ROCK) signaling pathway is involved in the regulation of the cytoskeleton. Moreover, long noncoding RNAs (lncRNAs) have essential roles in tumor migration and guide gene regulation during cancer progression. LncRNAs can regulate the cytoskeleton directly or may influence the cytoskeleton via Rho/ROCK signaling during tumor migration. In this review, we focus on the regulatory association between lncRNAs and the cytoskeleton and discuss the pathways and mechanisms involved in the regulation of cancer metastasis.

Cell division in bacteria is facilitated by a polymeric ring structure, the Z ring, composed of tubulin-like FtsZ protofilaments. Recently it has been shown that in Bacillus subtilis, the Z ring forms through the cell cycle-mediated remodelling of a helical FtsZ polymer. To investigate how this occurs in vivo, we have exploited a unique temperature-sensitive strain of B. subtilis expressing the mutant protein FtsZ(Ts1). FtsZ(Ts1) is unable to complete Z ring assembly at 49 degrees C, becoming trapped at an intermediate stage in the helix-to-ring progression. To determine why this is the case, we used a combination of methods to identify the specific defect of the FtsZ(Ts1) protein in vivo. Our results indicate that while FtsZ(Ts1) is able to polymerize normally into protofilaments, it is defective in the ability to support lateral associations between these filaments at high temperatures. This strongly suggests that lateral FtsZ association plays a crucial role in the polymer transitions that lead to the formation of the Z ring in the cell. In addition, we show that the FtsZ-binding protein ZapA, when overproduced, can rescue the FtsZ(Ts1) defect in vivo. This suggests that ZapA functions to promote the helix-to-ring transition of FtsZ by stimulating lateral FtsZ association.

The effect of infectious bursal disease virus (IBDV) infection on cellular protein expression is essential for viral pathogenesis. To characterize the cellular response to IBDV infection, the differential proteomes of chicken embryo fibroblasts, with and without IBDV infection, were analyzed at different time points with two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF/TOF identification. Comparative analysis of multiple 2-DE gels revealed that the majority of protein expression changes appeared at 48 and 96 h after IBDV infection. Mass spectrometry identified 51 altered cellular proteins, including 13 up-regulated proteins and 38 down-regulated proteins 12-96 h after infection. Notably 2-DE analysis revealed that IBDV infection induced the increased expression of polyubiquitin, apolipoprotein A-I, heat shock 27-kDa protein 1, actins, tubulins, eukaryotic translation initiation factor 4A isoform 2, acidic ribosomal phosphoprotein, and ribosomal protein SA isoform 2. In addition, IBDV infection considerably suppressed those cellular proteins involved in ubiquitin-mediated protein degradation, energy metabolism, intermediate filaments, host translational apparatus, and signal transduction. Moreover 38 corresponding genes of the differentially expressed proteins were quantitated by real time RT-PCR to examine the transcriptional profiles between infected and uninfected chicken embryo fibroblasts. Western blot further confirmed the inhibition of Rho protein GDP dissociation inhibitor expression and the induction of polyubiquitin during IBDV infection. Subcellular distribution analysis of the cytoskeletal proteins vimentin and beta-tubulin clearly demonstrated that IBDV infection induced the disruption of the vimentin network and microtubules late in IBDV infection. Thus, this work effectively provides useful dynamic protein-related information to facilitate further investigation of the underlying mechanism of IBDV infection and pathogenesis.