Characterization and ultrastructural role of the major components of the intestinal microvillus cytoskeleton.
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Actinin↗
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Mechanism of transformation by Rous sarcoma virus: events within adhesion plaques.
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Animals↗
Antibodies to cytokeratins bind to epitopes in human uterine smooth muscle cells in normal and pathological pregnancies.
Cytokeratin antibodies have been widely used for the identification of trophoblast cells in the placental bed, following their invasion from the developing conceptus. Their identification centres upon the expression of cytokeratin in epithelial cells, from which trophoblast cells are derived. Our recent observations indicate that this strict relationship may be more complex than was thought. Cryostat and paraffin sections of human decidua and myometrium, taken from the placental bed and the uterotomy cut, were examined immunocytochemically for cytokeratins using ten antibody clones selected to identify different cytokeratin proteins and antigenic epitopes. Biopsy specimens were obtained from normal and pathological pregnancies (pre-eclampsia, fetal retardation, amnioninfection, hysterorrhexis, placenta praevia) at the time of caesarean section (26-41 weeks of pregnancy). Antibodies against nine clones, CAM 5.2, MNF 116, AE1/AE3, CK5, KS-B17.2, CY-90, M20, E3, and 34 beta E12 identified, as expected, syncytial giant cells and mononuclear trophoblasts within the placental bed and glandular epithelial cells throughout the uterus. In addition, they stained numerous fusiform cells that were classified by established criteria to represent smooth muscle cells, both within blood vessels and myometrium. No staining differences were observed between normal and pathological disorders. These results indicate that cytokeratin antibodies CAM 5.2, MNF 116 and AE1/AE3, and other antibodies targeting proteins 8 and 18, cross-react with epitopes expressed in cells other than giant trophoblastic cells and mononuclear trophoblasts in the uterus and, thus, caution has to be used when such antibodies are used for the diagnostic characterization of tissues related to the placental bed.
Actins↗
Modulation of dopamine D(2) receptor signaling by actin-binding protein (ABP-280).
Proteins that bind to G protein-coupled receptors have recently been identified as regulators of receptor anchoring and signaling. In this study, actin-binding protein 280 (ABP-280), a widely expressed cytoskeleton-associated protein that plays an important role in regulating cell morphology and motility, was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. The specificity of this interaction was originally identified in a yeast two-hybrid screen and confirmed by protein binding. The functional significance of the D(2) receptor-ABP-280 association was evaluated in human melanoma cells lacking ABP-280. D(2) receptor agonists were less potent in inhibiting forskolin-stimulated cAMP production in these cells. Maximal inhibitory responses of D(2) receptor activation were also reduced. Further yeast two-hybrid experiments showed that ABP-280 association is critically dependent on the carboxyl domain of the D(2) receptor third cytoplasmic loop, where there is a potential serine phosphorylation site (S358). Serine 358 was replaced with aspartic acid to mimic the effects of receptor phosphorylation. This mutant (D(2)S358D) displayed compromised binding to ABP-280 and coupling to adenylate cyclase. PKC activation also generated D(2) receptor signaling attenuation, but only in ABP-containing cells, suggesting a PKC regulatory role in D(2)-ABP association. A mechanism for these results may be derived from a role of ABP-280 in the clustering of D(2) receptors, as determined by immunocytochemical analysis in ABP-deficient and replete cells. Our results suggest a new molecular mechanism of modulating D(2) receptor signaling by cytoskeletal protein interaction.
Adenylyl Cyclases↗
D2 and D3 dopamine receptor cell surface localization mediated by interaction with protein 4.1N.
We identified protein 4.1N as a D2-like dopamine receptor-interacting protein in a yeast two-hybrid screen. Protein 4.1N is a neuronally enriched member of the 4.1 family of cytoskeletal proteins, which also includes protein 4.1R of erythrocytes and the 4.1G and 4.1B isoforms. The interaction of protein 4.1N was specific for the D2 and D3 dopamine receptors and was independently confirmed in pulldown and coimmunoprecipitation assays. Deletion mapping localized the site of dopamine receptor/protein 4.1N interaction to the N-terminal segment of the third intracellular domain of D2 and D3 receptors and the carboxyl-terminal domain of protein 4.1N. D2 and D3 receptors were also found to interact with the highly conserved carboxyl-terminal domain of proteins 4.1R, 4.1G, and 4.1B. Immunofluorescence studies show that protein 4.1N and D2 and D3 dopamine receptors are expressed at the plasma membrane of transfected human embryonic kidney 293 and mouse neuroblastoma Neuro2A cells. However, expression of D2 or D3 receptors with a protein 4.1N truncation fragment reduces the level of D2 and D3 receptor expression at the plasma membrane. These results suggest that protein 4.1N/dopamine receptor interaction is required for localization or stability of dopamine receptors at the neuronal plasma membrane.
Amino Acid Sequence↗
Disruption of transforming growth factor-beta signaling in ELF beta-spectrin-deficient mice.
Disruption of the adaptor protein ELF, a beta-spectrin, leads to disruption of transforming growth factor-beta (TGF-beta) signaling by Smad proteins in mice. Elf-/- mice exhibit a phenotype similar to smad2+/-/smad3+/- mutant mice of midgestational death due to gastrointestinal, liver, neural, and heart defects. We show that TGF-beta triggers phosphorylation and association of ELF with Smad3 and Smad4, followed by nuclear translocation. ELF deficiency results in mislocalization of Smad3 and Smad4 and loss of the TGF-beta-dependent transcriptional response, which could be rescued by overexpression of the COOH-terminal region of ELF. This study reveals an unexpected molecular link between a major dynamic scaffolding protein and a key signaling pathway.
Abnormalities, Multiple↗
Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus.
Rous sarcoma virus (RSV)-induced transformation is mediated by the action of the viral src gene product pp60src. This transforming protein is found at several cytoplasmic locations, including the adhesion plaques of RSV-transformed cells. In these studies, we have focused on the adhesion plaque location of pp60src and determined whether any of the induced transformation parameters correlate with the presence of pp60src in the adhesion plaques. A series of partial transformation mutants of RSV that induce distinct transformation phenotypes were used, and infected chicken embryo cells were examined for (i) intracellular pp60src location, (ii) vinculin localization, (iii) abundance of phosphotyrosine on vinculin, (iv) integrity of stress fibers, and (v) expression of cell surface fibronectin. The results indicate that, among the limited number of mutants studied here, the presence of pp60src in adhesion plaques is independent of growth in soft agar and the increased phosphorylation of vinculin on tyrosine, but it does correlate with the loss of cell surface fibronectin. An elevated abundance of phosphotyrosine on vinculin is insufficient to cause stress fiber dissolution and is independent of the loss of fibronectin from the extracellular matrix. However, the increased relative amount of phosphotyrosine on vinculin is related to the ability of the cells to grow in soft agar. The adhesion plaque binding and tyrosine-specific kinase activities seem to represent two independent functions of pp60src.
Animals↗
Regulation of MAP kinase signaling modules by scaffold proteins in mammals.
The mitogen-activated protein kinase (MAPK) group of serine/threonine protein kinases mediates the response of cells to many extracellular stimuli such as cytokines and growth factors. These protein kinases include the extracellular signal-regulated protein kinases (ERK) and two stress-activated protein kinases (SAPK), the c-Jun N-terminal kinases (JNK), and the p38 MAPK. The enzymes are evolutionarily conserved and are activated by a common mechanism that involves a protein kinase cascade. Scaffold proteins have been proposed to interact with MAPK pathway components to create a functional signaling module and to control the specificity of signal transduction. Here we critically evaluate the evidence that supports a physiologically relevant role of MAPK scaffold proteins in mammals.
Adaptor Proteins, Signal Transducing↗
Mutations that affect flightin expression in Drosophila alter the viscoelastic properties of flight muscle fibers.
Striated muscles across phyla share a highly conserved sarcomere design yet exhibit broad diversity in contractile velocity, force, power output, and efficiency. Insect asynchronous flight muscles are characterized by high-frequency contraction, endurance, and high-power output. These muscles have evolved an enhanced delayed force response to stretch that is largely responsible for their enhanced oscillatory work and power production. In this study we investigated the contribution of flightin to oscillatory work using sinusoidal analysis of fibers from three flightless mutants affecting flightin expression: 1) fln0, a flightin null mutant, 2) Mhc13, a myosin rod point mutant with reduced levels of flightin, and 3) Mhc6, a second myosin rod point mutant with reduced levels of phosphorylated flightin. Fibers from the three mutants show deficits in their passive and dynamic viscoelastic properties that are commensurate with their effect on flightin expression and result in a significant loss of oscillatory work and power. Passive tension and passive stiffness were significantly reduced in fln0 and Mhc13 but not in Mhc6. The dynamic viscous modulus was significantly reduced in the three mutants, whereas the dynamic elastic modulus was reduced in fln0 and Mhc13 but not in Mhc6. Tension generation under isometric conditions was not impaired in fln0. However, when subjected to sinusoidal length perturbations, work-absorbing processes dominated over work-producing processes, resulting in no net positive work output. We propose that flightin is a major contributor to myofilament stiffness and a key determinant of the enhanced delayed force response to stretch in Drosophila flight muscles.
Animals↗
Is prostate-specific membrane antigen a multifunctional protein?
Prostate-specific membrane antigen (PSMA) is a metallopeptidase expressed predominantly in prostate cancer (PCa) cells. PSMA is considered a biomarker for PCa and is under intense investigation for use as an imaging and therapeutic target. Although the clinical utility of PSMA in the detection and treatment of PCa is evident and is being pursued, very little is known about its basic biological function in PCa cells. The purpose of this review is to highlight the possibility that PSMA might be a multifunctional protein. We suggest that PSMA may function as a receptor internalizing a putative ligand, an enzyme playing a role in nutrient uptake, and a peptidase involved in signal transduction in prostate epithelial cells. Insights into the possible functions of PSMA should improve the diagnostic and therapeutic values of this clinically important molecule.
Antigens, Neoplasm↗
K(+) channel-dependent migration of fibroblasts and human melanoma cells.
Previously, we showed that migration of transformed renal epithelial cells (MDCK-F cells) is a K(+) channel-dependent process [J Clin Invest 1994;93:1631]. In order to determine whether K(+) channel activity is a general requirement for locomotion, we extended our observations to NIH3T3 fibroblasts and human melanoma cells. Migration of both cell types and its dependence on K(+) channel activity was measured at the single cell level by time lapse photography in the absence and presence of the specific K(+) channel blocker charybdotoxin (CTX). Locomotion of both cell types is inhibited by K(+) channel blockade. CTX slows down migration of fibroblasts and of melanoma cells dose-dependently by up to 61 +/- 11%. These findings suggest that K(+) channel activity is a general prerequisite for migration. To determine whether CTX-induced inhibition of migration of fibroblasts and melanoma cells involves quantitative changes of actin filaments, we indirectly measured filamentous actin by quantitating binding of fluorescently labeled phalloidin. Whereas CTX elicits a decrease of bound phalloidin in fibroblasts there is an increase in melanoma cells. Since migration of tumor cells is required for invading surrounding tissue, we developed an assay to test whether CTX-induced inhibition of migration also impairs invasion of melanoma cells. Melanoma cells were seeded on a layer of high resistance renal epithelial cells (MDCK cells clone C7; transepithelial resistance R(te) >3,000 Omegacm(2)) and R(te) was measured daily. R(te) starts to decrease 2 days after seeding of melanoma cells onto MDCK-C7 cells. By day 7, R(te) has dropped to 24 +/- 1.5% of control. K(+) channel blockade with CTX (10 nmol/l) cannot prevent or delay this drop of R(te). R(te) reaches the same level with or without CTX. These results indicate that the disruption of an epithelial layer, unlike migration of melanoma cells, cannot be modulated by K(+) channel blockade.
3T3 Cells↗
Mechanisms and disturbances of neuronal migration.
Neuronal migration appears as a complex ontogenic step occurring early during embryonic and fetal development. Control of neuronal migration involves different cell populations including Cajal-Retzius neurons, subplate neurons, neuronal precursors or radial glia. The integrity of multiple molecular mechanisms, such as cell cycle control, cell-cell adhesion, interaction with extracellular matrix protein, neurotransmitter release, growth factor availability, platelet-activating factor degradation or transduction pathways seems to be critical for normal neuronal migration. The complexity and the multiplicity of these mechanisms probably explain the clinical, radiologic and genetic heterogeneity of human disorders of neuronal migration. The present review will be focused on mechanisms and disturbances of migration of neurons destined to the neocortex. New insights gained from the analysis of animal models as well as from the study of human diseases will be included.
1-Alkyl-2-acetylglycerophosphocholine Esterase↗
Functional imaging in PNH caused by a new FilaminA mutation.
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Brain↗
Germline and mosaic mutations of FLN1 in men with periventricular heterotopia.
OBJECTIVE: To describe the phenotypic spectrum and genetics of periventricular nodular heterotopia (PNH) caused by FLN1 mutations in four men. BACKGROUND: X-linked PNH caused by FLN1 mutations (MIM #300049) implies prenatal or early postnatal lethality in boys and 50% recurrence risk in daughters of affected women. METHODS: Clinical examination, cognitive testing, MRI, and mutation analysis (denaturing high-performance liquid chromatography and direct sequencing) on blood lymphocytes and single hair roots were performed for nine affected individuals, including three men. Neuropathologic study of the brain was performed for an affected boy. RESULTS: In two families, missense mutations were transmitted from mother to son (Met102Val) and from father to daughter (Ser149Phe), causing mild phenotypes in both genders, including unilateral PNH. In a third family, a man was mosaic for an A>G substitution (intron 11 acceptor splice site) on leukocyte DNA and hair roots (mutant = 42% and 69%). Single hair root analysis confirmed that the mutation was not present in all ectodermal derivative cells. A healthy daughter had inherited the X chromosome from her father's wild-type germinal cell population. In the fourth family, an eight-base deletion (AGGAGGTG, intron 25 donor splice site) led to early deaths of boys. Postmortem study in a newborn boy revealed PNH and cardiovascular, genitourinary, and gut malformations. CONCLUSIONS: Periventricular nodular heterotopia caused by FLN1 mutations in men has a wide clinical spectrum and is caused by different genetic mechanisms, including somatic mosaicism. Mutation analysis of FLN1 should support genetic counseling in men with periventricular nodular heterotopia.
Abnormalities, Multiple↗
Antifilamin, antivinculin, and antitropomyosin antibodies in myasthenia gravis.
Antifilamin, antivinculin, and antitropomyosin antibody activities were investigated in sera from 43 patients with myasthenia gravis (MG). Antifilamin and antivinculin antibody activities are significantly higher in MG patients compared with normal controls and patients with Duchenne muscular dystrophy (DMD), Parkinson's disease, and spinocerebellar degeneration. The antifilamin antibody was highly positive (100%) in ocular myasthenia. Antitropomyosin antibody activity was similar to that in DMD patients. No correlation was observed between these antibody activities and the antiacetylcholine receptor antibody titers, the duration of the disease, and thymic pathology.
Antibodies↗
Myofilin, a protein in the thick filaments of insect muscle.
Thick filaments in striated muscle are myosin polymers with a length and diameter that depend on the fibre type. In invertebrates, the length of the thick filaments varies widely in different muscles and additional proteins control filament assembly. Thick filaments in asynchronous insect flight muscle have an extremely regular structure, which is likely to be essential for the oscillatory contraction of these muscles. The factors controlling the assembly of thick filaments in insect flight muscle are not known. We previously identified a thick filament core protein, zeelin 1, in Lethocerus flight and non-flight muscles. This has been sequenced, and the corresponding proteins in Drosophila and Anopheles have been identified. The protein has been re-named myofilin. Zeelin 2, which is on the outside of Lethocerus flight muscle thick filaments, has been sequenced and because of the similarity to Drosophila flightin, is re-named flightin. In Drosophila flight muscle, myofilin has a molecular weight of 20 kDa and is one of five isoforms produced from a single gene. In situ hybridisation of Drosophila embryos showed that myofilin RNA is first expressed late in embryogenesis at stage 15, a little later than myosin. Antibody to myofilin labelled the entire A-band, except for the H-zone, in cryosections of flight and non-flight muscle. The periodicity of myofilin in Drosophila flight muscle thick filaments was found to be 30 nm by measuring the spacing of gold particles in labelled cryosections; this is about twice the 14.5 nm spacing of myosin molecules. The molar ratio of myofilin to myosin in indirect flight muscle is 1:2, which is the same as that of flightin. We propose a model for the association of these proteins in thick filaments, which is consistent with the periodicity and stoichiometry. Myofilin is probably needed for filament assembly in all muscles, and flightin for stability of flight muscle thick filaments in adult flies.
Alternative Splicing↗
Flight muscle properties and aerodynamic performance of Drosophila expressing a flightin transgene.
Flightin is a multiply phosphorylated, myosin-binding protein found specifically in indirect flight muscles (IFM) of Drosophila. A null mutation in the flightin gene (fln(0)) compromises thick filament assembly and muscle integrity resulting in muscle degeneration and lost of flight ability. Using P-element-mediated transformation with the full-length flightin gene driven by the Actin88F promoter, we have achieved rescue of all fln(0)-related ultrastructural and functional defects of the IFM. Transgenic P{fln(+)}fln(0) 'rescued' flies have fewer thick filaments per myofbril than wild-type flies (782+/-13 vs 945+/-9) but have otherwise normal IFM. Transgenic P{fln(+)}fln(+) 'tetraploid' flies have a normal number of thick filaments. The flightin protein levels in both transgenic strains are similar to wild type. By contrast, flightin levels are reduced in a myosin heavy chain tetraploid strain that produces excess myosin and excess thick filaments. These results suggest that regulation of flightin protein level is independent of gene copy number and that the number of thick filaments assembled per myofibril is influenced independently by myosin and flightin expression. We measured mechanical properties of IFM skinned fibers by sinusoidal analysis and found no significant differences in active viscoelastic properties of flightin-rescued and tetraploid transgenic flies vs wild type. The ability of the fln(+) transgene to overcome deficits in dynamic stiffness and power output in fln(0) suggest that the flightin protein contributes directly to fiber stiffness and stretch activation. However, flight parameters at maximum locomotor capacity, measured in a virtual reality flight simulator, are slightly compromised for both transgenic strains. P{fln(+)}fln(0) and P{fln(+)}fln(+) flies generated enough flight force to sustain hovering flight but showed reduced capability to produce forces in excess of hovering flight force. Both strains showed reductions in stroke frequency but only P{fln(+)}fln(+) showed reductions in stroke amplitude. Muscle and aerodynamic efficiency are similar among the two transgenic strains and wild type. These results illustrate the importance of flightin in flight muscle development and function.
Animals↗
A genetic deficiency that spans the flightin gene of Drosophila melanogaster affects the ultrastructure and function of the flight muscles.
We have developed a reverse-genetic approach to study the function of flightin, a unique protein of the flight muscle myofibril of Drosophila melanogaster. We describe the generation and characterization of Df(3L)fln1, a lethal genetic deficiency in the 76BE region of the third chromosome which deletes several genes, including the gene for flightin. We show that heterozygous flies harboring the Df(3L)fln1 mutation exhibit both impaired flight and ultrastructural defects in their flight muscle myofibrils. We found that the mutation does not interfere with assembly of the myofibril but leads to disorganization of peripheral myofilaments in adult myofibrils. Most myofibrils, nevertheless, retain an intact core that represents approximately 80 % of the normal lattice diameter. Mechanical analysis of single skinned flight muscle fibers demonstrates that the mutation has no significant effect on net power output but increases the frequency at which maximum power is delivered to the wings, potentially reducing the overall performance of the flight system. The results suggest that flightin is an indispensable part of the flight muscle contractile mechanism.
Animals↗