High Resolution Detection of Mechanical Forces Exerted by Locomoting Fibroblasts on the Substrate

We have developed a new approach to detect mechanical forces exerted by locomoting fibroblasts on the substrate. Cells were cultured on elastic, collagen-coated polyacrylamide sheets embedded with 0.2-μm fluorescent beads. Forces exerted by the cell cause deformation of the substrate and displacement of the beads. By recording the position of beads during cell locomotion and after cell removal, we discovered that most forces were radially distributed, switching direction in the anterior region. Deformations near the leading edge were strong, transient, and variable in magnitude, consistent with active local contractions, whereas those in the posterior region were weaker, more stable, and more uniform, consistent with passive resistance. Treatment of cells with cytochalasin D or myosin II inhibitors caused relaxation of the forces, suggesting that they are generated primarily via actin–myosin II interactions; treatment with nocodazole caused no immediate effect on forces. Immunofluorescence indicated that the frontal region of strong deformation contained many vinculin plaques but no apparent concentration of actin or myosin II filaments. Strong mechanical forces in the anterior region, generated by locally activated myosin II and transmitted through vinculin-rich structures, likely play a major role in cell locomotion and in mechanical signaling with the surrounding environment.

Cell-Matrix Adhesions Differentially Regulate Fascin Phosphorylation

Cell adhesion to individual macromolecules of the extracellular matrix has dramatic effects on the subcellular localization of the actin-bundling protein fascin and on the ability of cells to form stable fascin microspikes. The actin-binding activity of fascin is down-regulated by phosphorylation, and we used two differentiated cell types, C2C12 skeletal myoblasts and LLC-PK1 kidney epithelial cells, to examine the hypothesis that cell adhesion to the matrix components fibronectin, laminin-1, and thrombospondin-1 differentially regulates fascin phosphorylation. In both cell types, treatment with the PKC activator 12-tetradecanoyl phorbol 13-acetate (TPA) or adhesion to fibronectin led to a diffuse distribution of fascin after 1 h. C2C12 cells contain the PKC family members α, γ, and λ, and PKCα localization was altered upon cell adhesion to fibronectin. Two-dimensional isoelectric focusing/SDS-polyacrylamide gels were used to determine that fascin became phosphorylated in cells adherent to fibronectin and was inhibited by the PKC inhibitors calphostin C and chelerythrine chloride. Phosphorylation of fascin was not detected in cells adherent to thrombospondin-1 or to laminin-1. LLC-PK1 cells expressing green fluorescent protein (GFP)-fascin also displayed similar regulation of fascin phosphorylation. LLC-PK1 cells expressing GFP-fascin S39A, a nonphosphorylatable mutant, did not undergo spreading and focal contact organization on fibronectin, whereas cells expressing a GFP-fascin S39D mutant with constitutive negative charge spread more extensively than wild-type cells. In contrast, C2C12 cells coexpressing S39A fascin with endogenous fascin remained competent to form microspikes on thrombospondin-1, and cells that expressed fascin S39D attached to thrombospondin-1 but did not form microspikes. Blockade of PKCα activity by TPA-induced down-regulation led to actin association of wild-type fascin in fibronectin-adherent C2C12 and LLC-PK1 cells but did not alter the distribution of S39A or S39D fascins. The association of fascin with actin in fibronectin-adherent cells was also evident in the presence of an inhibitory antibody to integrin α5 subunit. These novel results establish matrix-initiated PKC-dependent regulation of fascin phosphorylation at serine 39 as a mechanism whereby matrix adhesion is coupled to the organization of cytoskeletal structure.

A terbenzimidazole that preferentially binds and conformationally alters structurally distinct DNA duplex domains: A potential mechanism for topoisomerase I poisoning

The terbenzimidazoles are a class of synthetic ligands that poison the human topoisomerase I (TOP1) enzyme and promote cancer cell death. It has been proposed that drugs of this class act as TOP1 poisons by binding to the minor groove of the DNA substrate of TOP1 and altering its structure in a manner that results in enzyme-mediated DNA cleavage. To test this hypothesis, we characterize and compare the binding properties of a 5-phenylterbenzimidazole derivative (5PTB) to the d(GA4T4C)2 and d(GT4A4C)2 duplexes. The d(GA4T4C)2 duplex contains an uninterrupted 8-bp A⋅T domain, which, on the basis of x-ray crystallographic data, should induce a highly hydrated “A-tract” conformation. This duplex also exhibits anomalously slow migration in a polyacrylamide gel, a feature characteristic of a noncanonical global conformational state frequently described as “bent.” By contrast, the d(GT4A4C)2 duplex contains two 4-bp A⋅T tracts separated by a TpA dinucleotide step, which should induce a less hydrated “B-like” conformation. This duplex also migrates normally in a polyacrylamide gel, a feature further characteristic of a global, canonical B-form duplex. Our data reveal that, at 20°C, 5PTB exhibits an ≈2.3 kcal/mol greater affinity for the d(GA4T4C)2 duplex than for the d(GT4A4C)2 duplex. Significantly, we find this sequence/conformational binding specificity of 5PTB to be entropic in origin, an observation consistent with a greater degree of drug binding-induced dehydration of the more solvated d(GA4T4C)2 duplex. By contrast with the differential duplex affinity exhibited by 5PTB, netropsin and 4′,6-diamidino-2-phenylindole (DAPI), two AT-specific minor groove binding ligands that are inactive as human TOP1 poisons, bind to both duplexes with similar affinities. The electrophoretic behaviors of the ligand-free and ligand-bound duplexes are consistent with 5PTB-induced bending and/or unwinding of both duplexes, which, for the d(GA4T4C)2 duplex, is synergistic with the endogenous sequence-directed electrophoretic properties of the ligand-free duplex state. By contrast, the binding to either duplex of netropsin or DAPI induces little or no change in the electrophoretic mobilities of the duplexes. Our results demonstrate that the TOP1 poison 5PTB binds differentially to and alters the structures of the two duplexes, in contrast to netropsin and DAPI, which bind with similar affinities to the two duplexes and do not significantly alter their structures. These results are consistent with a mechanism for TOP1 poisoning in which drugs such as 5PTB differentially target conformationally distinct DNA sites and induce structural changes that promote enzyme-mediated DNA cleavage.

Cell locomotion and focal adhesions are regulated by substrate flexibility

Responses of cells to mechanical properties of the adhesion substrate were examined by culturing normal rat kidney epithelial and 3T3 fibroblastic cells on a collagen-coated polyacrylamide substrate that allows the flexibility to be varied while maintaining a constant chemical environment. Compared with cells on rigid substrates, those on flexible substrates showed reduced spreading and increased rates of motility or lamellipodial activity. Microinjection of fluorescent vinculin indicated that focal adhesions on flexible substrates were irregularly shaped and highly dynamic whereas those on firm substrates had a normal morphology and were much more stable. Cells on flexible substrates also contained a reduced amount of phosphotyrosine at adhesion sites. Treatment of these cells with phenylarsine oxide, a tyrosine phosphatase inhibitor, induced the formation of normal, stable focal adhesions similar to those on firm substrates. Conversely, treatment of cells on firm substrates with myosin inhibitors 2,3-butanedione monoxime or KT5926 caused the reduction of both vinculin and phosphotyrosine at adhesion sites. These results demonstrate the ability of cells to survey the mechanical properties of their surrounding environment and suggest the possible involvement of both protein tyrosine phosphorylation and myosin-generated cortical forces in this process. Such response to physical parameters likely represents an important mechanism of cellular interaction with the surrounding environment within a complex organism.

US9, a stable lysine-less herpes simplex virus 1 protein, is ubiquitinated before packaging into virions and associates with proteasomes

The US9 gene of herpes simplex virus 1 encodes a virion tegument protein with a predicted Mr of 10,000. Earlier studies have shown that the gene is not essential for viral replication in cells in culture. We report that (i) US9 forms in denaturing polyacrylamide gels multiple overlapping bands ranging in Mr from 12,000 to 25,000; (ii) the protein recovered from infected cells or purified virions reacts with anti-ubiquitin antibodies; (iii) autoradiographic images of US9 protein immunoprecipitated from cells infected with [35S]methionine-labeled virus indicate that the protein is stable for at least 4 h after entry into cells (the protein was also stable for at least 4 h after a 1-h labeling interval 12 h after infection); (iv) antibody to subunit 12 of proteasomes pulls down US9 protein from herpes simplex virus-infected cell lysates; and (v) the US9 gene is highly conserved among the members of the alpha subfamily of herpes viruses, and the US9 gene product lacks lysines. We conclude that US9 is a lysine-less, ubiquitinated protein that interacts with the ubiquitin-dependent pathway for degradation of proteins and that this function may be initiated at the time of entry of the virus into the cell.

Determination of the binding constants of the centromere protein Cbf1 to all 16 centromere DNAs of Saccharomyces cerevisiae

Cbf1p is a Saccharomyces cerevisiae chromatin protein belonging to the basic region helix–loop–helix leucine zipper (bHLHzip) family of DNA binding proteins. Cbf1p binds to a conserved element in the 5′-flanking region of methionine biosynthetic genes and to centromere DNA element I (CDEI) of S.cerevisiae centromeric DNA. We have determined the apparent equilibrium dissociation constants of Cbf1p binding to all 16 CDEI DNAs in gel retardation assays. Binding constants of full-length Cbf1p vary between 1.7 and 3.8 nM. However, the dissociation constants of a Cbf1p deletion variant that has been shown to be fully sufficient for Cbf1p function in vivo vary in a range between 3.2 and 12 nM. In addition, native polyacrylamide gel electrophoresis revealed distinct changes in the 3D structure of the Cbf1p/CEN complexes. We also show that the previously reported DNA binding stimulation activity of the centromere protein p64 functions on both the Cbf1 full-length protein and a deletion variant containing only the bHLHzip domain of Cbf1p. Our results suggest that centromeric DNA outside the consensus CDEI sequence and interaction of Cbf1p with adjacent centromere proteins contribute to the complex formation between Cbf1p and CEN DNA.

Antifreeze Proteins in Winter Rye Leaves Form Oligomeric Complexes1

Antifreeze proteins (AFPs) similar to three pathogenesis-related proteins, a glucanase-like protein (GLP), a chitinase-like protein (CLP), and a thaumatin-like protein (TLP), accumulate during cold acclimation in winter rye (Secale cereale) leaves, where they are thought to modify the growth of intercellular ice during freezing. The objective of this study was to characterize the rye AFPs in their native forms, and our results show that these proteins form oligomeric complexes in vivo. Nine proteins were separated by native-polyacrylamide gel electrophoresis from apoplastic extracts of cold-acclimated winter rye leaves. Seven of these proteins exhibited multiple polypeptides when denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After isolation of the individual proteins, six were shown by immunoblotting to contain various combinations of GLP, CLP, and TLP in addition to other unidentified proteins. Antisera produced against individual cold-induced winter rye GLP, CLP, and TLP all dramatically inhibited glucanase activity in apoplastic extracts from cold-acclimated winter rye leaves, and each antiserum precipitated all three proteins. These results indicate that each of the polypeptides may be exposed on the surface of the protein complexes. By forming oligomeric complexes, AFPs may form larger surfaces to interact with ice, or they may simply increase the mass of the protein bound to ice. In either case, the complexes of AFPs may inhibit ice growth and recrystallization more effectively than the individual polypeptides.

Characterization of Two Novel Type I Ribosome-Inactivating Proteins from the Storage Roots of the Andean Crop Mirabilis expansa1

Two novel type I ribosome-inactivating proteins (RIPs) were found in the storage roots of Mirabilis expansa, an underutilized Andean root crop. The two RIPs, named ME1 and ME2, were purified to homogeneity by ammonium sulfate precipitation, cation-exchange perfusion chromatography, and C4 reverse-phase chromatography. The two proteins were found to be similar in size (27 and 27.5 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their isoelectric points were determined to be greater than pH 10.0. Amino acid N-terminal sequencing revealed that both ME1 and ME2 had conserved residues characteristic of RIPs. Amino acid composition and western-blot analysis further suggested a structural similarity between ME1 and ME2. ME2 showed high similarity to the Mirabilis jalapa antiviral protein, a type I RIP. Depurination of yeast 26S rRNA by ME1 and ME2 demonstrated their ribosome-inactivating activity. Because these two proteins were isolated from roots, their antimicrobial activity was tested against root-rot microorganisms, among others. ME1 and ME2 were active against several fungi, including Pythium irregulare, Fusarium oxysporum solani, Alternaria solani, Trichoderma reesei, and Trichoderma harzianum, and an additive antifungal effect of ME1 and ME2 was observed. Antibacterial activity of both ME1 and ME2 was observed against Pseudomonas syringae, Agrobacterium tumefaciens, Agrobacterium radiobacter, and others.

Inhibitory Regulation of Higher-Plant Myosin by Ca2+ Ions1

Myosin isolated from the pollen tubes of lily (Lilium longiflorum) is composed of a 170-kD heavy chain (E. Yokota and T. Shimmen [1994] Protoplasma 177: 153–162). Both the motile activity in vitro and the F-actin-stimulated ATPase activity of this myosin were inhibited by Ca2+ at concentrations higher than 10−6 m. In the Ca2+ range between 10−6 and 10−5 m, inhibition of the motile activity was reversible. In contrast, inhibition by more than 10−5 m Ca2+ was not reversible upon Ca2+ removal. An 18-kD polypeptide that showed the same mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as that of spinach calmodulin (CaM) was present in this myosin fraction. This polypeptide showed a mobility shift in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a Ca2+-dependent manner. Furthermore, this polypeptide was recognized by antiserum against spinach CaM. By immunoprecipitation using antiserum against the 170-kD heavy chain, the 18-kD polypeptide was coprecipitated with the 170-kD heavy chain, provided that the Ca2+ concentration was low, indicating that this 18-kD polypeptide is bound to the 170-kD myosin heavy chain. However, the 18-kD polypeptide was dissociated from the 170-kD heavy chain at high Ca2+ concentrations, which irreversibly inhibited the motile activity of this myosin. From these results, it is suggested that the 18-kD polypeptide, which is likely to be CaM, is associated with the 170-kD heavy chain as a light chain. It is also suggested that this polypeptide is involved in the regulation of this myosin by Ca2+. This is the first biochemical basis, to our knowledge, for Ca2+ regulation of cytoplasmic streaming in higher plants.

The Glycosylphosphatidylinositol-Anchored Phosphatase from Spirodela oligorrhiza Is a Purple Acid Phosphatase1

We recently presented clear evidence that the major low-phosphate-inducible phosphatase of the duckweed Spirodela oligorrhiza is a glycosylphosphatidylinositol (GPI)-anchored protein, and, to our knowledge, is the first described from higher plants (N. Morita, H. Nakazato, H. Okuyama, Y. Kim, G.A. Thompson, Jr. [1996] Biochim Biophys Acta 1290: 53–62). In this report the purified 57-kD phosphatase is shown to be a purple metalloenzyme containing Fe and Mn atoms and having an absorption maximum at 556 nm. The phosphatase activity was only slightly inhibited by tartrate, as expected for a purple acid phosphatase (PAP). Furthermore, the protein cross-reacted with an anti-Arabidopsis PAP antibody on immunoblots. The N-terminal amino acid sequence of the phosphatase was very similar to those of Arabidopsis, red kidney bean (Phaseolus vulgaris), and soybean (Glycine max) PAP. Extracts of S. oligorrhiza plants incubated with the GPI-specific precursor [3H]ethanolamine were treated with antibodies raised against the purified S. oligorrhiza phosphatase. Radioactivity from the resulting immunoprecipitates was specifically associated with a 57-kD band on sodium dodecyl sulfate-polyacrylamide gels. These results, together with previous findings, strongly indicate that the GPI-anchored phosphatase of S. oligorrhiza is a PAP.

A Chloroplast DNA Helicase II from Pea That Prefers Fork-Like Replication Structures

A DNA helicase, called chloroplast DNA (ctDNA) helicase II, was purified to apparent homogeneity from pea (Pisum sativum). The enzyme contained intrinsic, single-stranded, DNA-dependent ATPase activity and an apparent molecular mass of 78 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The DNA helicase was markedly stimulated by DNA substrates with fork-like replication structures. A 5′-tailed fork was more active than the 3′-tailed fork, which itself was more active than substrates without a fork. The direction of unwinding was 3′ to 5′ along the bound strand, and it failed to unwind blunt-ended duplex DNA. DNA helicase activity required only ATP or dATP hydrolysis. The enzyme also required a divalent cation (Mg2+>Mn2+>Ca2+) for its unwinding activity and was inhibited at 200 mm KCl or NaCl. This enzyme could be involved in the replication of ctDNA. The DNA major groove-intercalating ligands nogalamycin and daunorubicin were inhibitory to unwinding (Ki approximately 0.85 μm and 2.2 μm, respectively) and ATPase (Ki approximately 1.3 μm and 3.0 μm, respectively) activities of pea ctDNA helicase II, whereas ellipticine, etoposide (VP-16), and camptothecin had no effect on the enzyme activity. These ligands may be useful in further studies of the mechanisms of chloroplast helicase activities.

Superoxide Dismutase in Arabidopsis: An Eclectic Enzyme Family with Disparate Regulation and Protein Localization1

A number of environmental stresses can lead to enhanced production of superoxide within plant tissues, and plants are believed to rely on the enzyme superoxide dismutase (SOD) to detoxify this reactive oxygen species. We have identified seven cDNAs and genes for SOD in Arabidopsis. These consist of three CuZnSODs (CSD1, CSD2, and CSD3), three FeSODs (FSD1, FSD2, and FSD3), and one MnSOD (MSD1). The chromosomal location of these seven SOD genes has been established. To study this enzyme family, antibodies were generated against five proteins: CSD1, CSD2, CSD3, FSD1, and MSD1. Using these antisera and nondenaturing-polyacrylamide gel electrophoresis enzyme assays, we identified protein and activity for two CuZnSODs and for FeSOD and MnSOD in Arabidopsis rosette tissue. Additionally, subcellular fractionation studies revealed the presence of CSD2 and FeSOD protein within Arabidopsis chloroplasts. The seven SOD mRNAs and the four proteins identified were differentially regulated in response to various light regimes, ozone fumigation, and ultraviolet-B irradiation. To our knowledge, this is the first report of a large-scale analysis of the regulation of multiple SOD proteins in a plant species.

d-Ribulose-5-Phosphate 3-Epimerase: Cloning and Heterologous Expression of the Spinach Gene, and Purification and Characterization of the Recombinant Enzyme1

We have achieved, to our knowledge, the first high-level heterologous expression of the gene encoding d-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by dl-α-glycerophosphate or ethanol and destabilized by d-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.

Characterization of Two Glutamate Decarboxylase cDNA Clones from Arabidopsis

Two distinct cDNA clones encoding for the glutamate decarboxylase (GAD) isoenzymes GAD1 and GAD2 from Arabidopsis (L.) Heynh. were characterized. The open reading frames for GAD1 and GAD2 were expressed in Escherichia coli and the recombinant proteins were purified by affinity chromatography. Analysis of the recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis suggest that GAD1 and GAD2 encode for 58- and 56-kD peptides, respectively. The enzymatic activities of the pure recombinant GAD1 and GAD2 proteins were stimulated 35- and 13-fold, respectively, by Ca2+/calmodulin but not by Ca2+ or calmodulin alone. Southern-blot analysis of genomic DNA suggests that there is only one copy of each gene in Arabidopsis. The GAD1 transcript and a corresponding 58-kD peptide were detected in roots only. Conversely, the GAD2 transcript and a corresponding 56-kD peptide were detected in all organs tested. The specific activity, GAD2 transcript, and 56-kD peptide increased in leaves of plants treated with 10 mm NH4Cl, 5 mm NH4NO3, 5 mm glutamic acid, or 5 mm glutamine as the sole nitrogen source compared with samples from plants treated with 10 mm KNO3. The results from these experiments suggest that in leaves GAD activity is partially controlled by gene expression or RNA stability. Results from preliminary analyses of different tissues imply that these tendencies were not the same in flower stalks and flowers, suggesting that other factors may control GAD activity in these organs. The results from this investigation demonstrate that GAD activity in leaves is altered by different nitrogen treatments, suggesting that GAD2 may play a unique role in nitrogen metabolism.

Characterization of Spermidine Binding to Solubilized Plasma Membrane Proteins from Zucchini Hypocotyls1

In this work [14C]spermidine binding to total proteins solubilized from plasma membrane purified from zucchini (Cucurbita pepo L.) hypocotyls was investigated. Proteins were solubilized using octyl glucoside as a detergent. Specific polyamine binding was thermolabile, reversible, pH dependent with an optimum at pH 8.0, and had a Kd value of 5 μm, as determined by glass-fiber-filter assays. Sephadex G-25 M gel-filtration assays confirmed the presence of a spermidine-protein(s) complex with a specific binding activity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native polyacrylamide gel electrophoresis of collected fractions having the highest specific spermidine-binding activity, several protein bands (113, 75, 66, and 44 kD) were identified. The specificity of spermidine binding was examined by gel-filtration competition experiments performed using other polyamines and compounds structurally related to spermidine. Partial purification on Sephadex G-200 led to the identification of 66- and 44-kD protein bands, which may represent the putative spermidine-binding protein(s) on the plasmalemma.