Overexpression of cofilin stimulates bundling of actin filaments, membrane ruffling, and cell movement in Dictyostelium.

Cofilin is a low molecular weight actin-modulating protein whose structure and function are conserved among eucaryotes. Cofilin exhibits in vitro both a monomeric actin-sequestering activity and a filamentous actin-severing activity. To investigate in vivo functions of cofilin, cofilin was overexpressed in Dictyostelium discoideum cells. An increase in the content of D. discoideum cofilin (d-cofilin) by sevenfold induced a co-overproduction of actin by threefold. In cells over-expressing d-cofilin, the amount of filamentous actin but not that of monomeric actin was increased. Overexpressed d-cofilin co-sedimented with actin filaments, suggesting that the sequestering activity of d-cofilin is weak in vivo. The overexpression of d-cofilin increased actin bundles just beneath ruffling membranes where d-cofilin was co-localized. The overexpression of d-cofilin also stimulated cell movement as well as membrane ruffling. We have demonstrated in vitro that d-cofilin transformed latticework of actin filaments cross-linked by alpha-actinin into bundles probably by severing the filaments. D. discoideum cofilin may sever actin filaments in vivo and induce bundling of the filaments in the presence of cross-linking proteins so as to generate contractile systems involved in membrane ruffling and cell movement.

Isolation and characterization of a high molecular weight actin-binding protein from Physarum polycephalum plasmodia.

A high molecular weight actin-binding protein was isolated from the Physarum polycephalum plasmodia. The protein ( HMWP ) shares many properties with other high molecular weight actin-binding proteins such as spectrin, actin-binding protein from macrophages, and filamin. It has a potent activity to cross-link F-actin into a gel-like structure. Its cross-linking activity does not depend on calcium concentrations. Hydrodynamic studies have revealed that the protein is in the monomeric state of a polypeptide chain with molecular weight of approximately 230,000 in a high ionic strength solvent, while it self-associates into a dimer under physiological ionic conditions. Electron microscopic examinations of HMWP have shown that the monomer particle observed in a high ionic strength solvent is rod shaped with the two-stranded morphology very similar to that of spectrin. On the other hand, under physiological ionic conditions, the HMWP dimer shows the dumb-bell shape with two globular domains connected with a thin flexible strand.

Dictyostelium IQGAP-related protein specifically involved in the completion of cytokinesis.

The gapA gene encoding a novel RasGTPase-activating protein (RasGAP)-related protein was found to be disrupted in a cytokinesis mutant of Dictyostelium that grows as giant and multinucleate cells in a dish culture. The predicted sequence of the GAPA protein showed considerable homology to those of Gap1/Sar1 from fission yeast and the COOH-terminal half of mammalian IQGAPs, the similarity extending beyond the RasGAP-related domain. In suspension culture, gapA- cells showed normal growth in terms of the increase in cell mass, but cytokinesis inefficiently occurred to produce spherical giant cells. Time-lapse recording of the dynamics of cell division in a dish culture revealed that, in the case of gapA- cells, cytokinesis was very frequently reversed at the step in which the midbody connecting the daughter cells should be severed. Earlier steps of cytokinesis in the gapA- cells seemed to be normal, since myosin II was accumulated at the cleavage furrow. Upon starvation, gapA- cells developed and formed fruiting bodies with viable spores, like the wild-type cells. These results indicate that the GAPA protein is specifically involved in the completion of cytokinesis. Recently, it was reported that IQGAPs are putative effectors for Rac and CDC42, members of the Rho family of GTPases, and participate in reorganization of the actin cytoskeleton. Thus, it is possible that Dictyostelium GAPA participates in the severing of the midbody by regulating the actin cytoskeleton through an interaction with a member of small GTPases.

Characterization of mutant myosins of Dictyostelium discoideum equivalent to human familial hypertrophic cardiomyopathy mutants. Molecular force level of mutant myosins may have a prognostic implication.

Recent studies have revealed that familial hypertrophic cardiomyopathy (FHC) is caused by missence mutations in myosin heavy chain or other sarcomeric proteins. To investigate the functional impact of FHC mutations in myosin heavy chain, mutants of Dictyostelium discoideum myosin II equivalent to human FHC mutations were generated by site-directed mutagenesis, and their motor function was characterized at the molecular level. These mutants, i.e., R397Q, F506C, G575R, A699R, K703Q, and K703W are respectively equivalent to R403Q, F513C, G584R, G716R, R719Q, and R719W FHC mutants. We measured the force generated by these myosin mutants as well as the sliding velocity and the actin-activated ATPase activity. These measurements showed that the A699R, K703Q, and K703W myosins exhibited unexpectedly weak affinity with actin and the lowest level of force, though their ATPase activity remained rather high. F506C mutant which has been reported to have benign prognosis exhibited the least impairment of the motile and enzymatic activities. The motor functions of R397Q and G575R myosins were classified as intermediate. These results suggest that the force level of mutant myosin molecule may be one of the key factors for pathogenesis which affect the prognosis of human FHC.

Induction of cancer, actinic keratosis, and specific p53 mutations by UVB light in human skin maintained in severe combined immunodeficient mice.

To study the mechanism and risk of human skin cancer from solar light, we exposed human skin transplanted to severe combined immunodeficient mice to daily doses of UVB for periods of approximately 2 years. We have succeeded for the first time in inducing cancer and solar (actinic) keratosis in human skin by UVB. Of 18 normal skins exposed to doses of 7.3 x 10(5) to 1.8 x 10(6) J/m2, 14 actinic keratoses (77.8%) and 3 squamous cell carcinomas (16.7%) developed, whereas neither actinic keratosis nor cancer was observed in 15 human skins not exposed to UVB. Each human skin showed a different susceptibility, and skins sensitive for actinic keratosis were also sensitive for cancer induction. Among p53 mutations at various sites, mutation at codon 242 (C TGC --> C CGC; Cys --> Arg) was specifically observed in both skin cancers and actinic keratoses. Furthermore, double or triple mutations were induced in all UVB-induced skin cancers and in three of eight actinic keratoses. Most of the mutations (17 of 20) occurred at dipyrimidine sites.

Electron microscopic visualization of the N terminus of the myosin heavy chain using a site-directed antibody.

We determined the spatial location of the N terminus of the heavy chain of rabbit skeletal muscle myosin by electron microscopy, using a site-directed antibody raised against its N-terminal eight residues as an electron microscopic probe. By examining rotary-shadowed images of the heavy meromyosin-antibody complex, we measured distances between the head-rod junction and the attachment site of the antibody bound on the head. The average distance was estimated to be about 12 nm. The result indicates that the N terminus of the heavy chain is located at the middle region of the head.

Electron microscopic mappings of myosin head with site-directed antibodies.

Site-directed antibodies were raised against three synthetic peptides whose sequences correspond to a region around the reactive lysine residue and two protease-sensitive regions of subfragment 1 (S1) of skeletal muscle myosin (one at the junction of the 23,000 Mr and 50,000 Mr segments, the J1 junction; and the other at the junction of the 50,000 Mr and 20,000 Mr segments of the heavy chain, the J2 junction). The antisera cross-reacted with intact myosin with titres of 5 x 10(4) (anti-J1 antiserum) and 10(4) (anti-J2 and anti-reactive lysine residue antisera). Site-specific antibodies purified by S1-Sepharose readily bound to myosin. Electron microscopic examinations of antibody-myosin complexes revealed that the J1 and J2 junctions are located 15 nm and 16 nm from the head-rod junction, respectively, while the reactive lysine residue region is 13 nm from the junction.

Electron microscopic visualization of the SH1 thiol of myosin by the use of an avidin-biotin system.

One of the reactive thiols in the myosin head, SH1, was covalently labeled with a biotin derivative, N-iodoacetyl-N'-biotinylhexylenediamine. When 50% of the SH1 thiol was modified with the biotin reagent as judged from measurements of ATPase activities, the biotinylated myosin bound one mole of avidin per mole of myosin at the saturating level. The avidin-myosin complex was readily formed in the presence of MgADP or MgATP. Peptide maps of the biotinylated myosin revealed that SH1 is actually the site of biotinylation with N-iodoacetyl-N'-biotinylhexylenediamine. Electron microscopic examination of the avidin-myosin complex showed that the attachment site of avidin on the myosin head is 130 A from the head-rod junction, indicating that the SH1 thiol is located there.

Location of the SH group of the alkali light chain on the myosin head as revealed by electron microscopy.

The location of the single cysteinyl residue of the alkali light chain on the myosin head was determined by electron microscopy. The cysteinyl residue of isolated alkali light chain 2 was biotinylated and the light chain was exchanged with that of heavy meromyosin in 4.7 M-NH4Cl. Avidin was attached to the biotin in the heavy meromyosin and the complex was rotary shadowed and observed in the electron microscope. The distance from the head-rod junction to the centre of avidin was 8(+/- 3) nm (mean value +/- standard deviation: n = 105).

Structural unit of the erythrocyte cytoskeleton. Isolation and electron microscopic examination.

We isolated a protein complex containing major cytoskeletal components from the Triton shell of bovine erythrocytes. This protein complex, which we called the 26-S complex, consisted of three major components, spectrin, band-4.1 protein and actin, and one minor component, band-4.9 protein. The molar ratio of spectrin heterodimer:band 4.1:actin was determined by sodium dodecyl sulfate (SDS) gel electrophoresis to be about 1:2:2, approximately the same as that for the Triton shell. By electron microscopic examinations of rotary-shadowed specimens, it was revealed that the 26-S complex had a "spider-like" morphology with a central core and several spectrin heterodimers radiating from it. The number of spectrin arms in the complex was not constant but was in the range between 3 and 6. The complexes with five spectrin heterodimers were the most numerous. The results showed that the 26-S complex contained on the average five spectrin heterodimers, ten band-4.1 polypeptides and ten actin monomers. As judged from the formation of oligomeric 26-S complexes through spectrin arms, the central core of the complex presumably contains band 4.1 and actin. Supporting this conclusion, the central core acted as a nucleus for actin polymerization when the 26-S complex was mixed with G-actin under an actin-polymerizing condition. The 26-S complex could form large aggregates under a certain condition that spectrin was promoted to associate from dimer to tetramer. We conclude that the 26-S complex is the structural unit of the erythrocyte cytoskeleton.

Spatial relationship between SH1 and the actin binding site on myosin subfragment-1 surface.

To examine the spatial relationship between SH1 thiol and actin binding site on subfragment-1 surface, we studied the interaction with actin of subfragment-1 whose SH1 was labeled with an iodoacetate derivative of biotin and covered with avidin. Subfragment-1--avidin complex bound F-actin and its Mg2+ ATPase activity was activated by actin. Considering the size and the location of biotin binding site on avidin, our results suggest that SH1 is separated from the actin binding site on subfragment-1 surface by at least 17-20 A.

Novel Dictyostelium unconventional myosin, MyoM, has a putative RhoGEF domain.

We have cloned a novel unconventional myosin gene myoM in Dictyostelium. Phylogenetic analysis of the motor domain indicated that MyoM does not belong to any known subclass of the myosin superfamily. Following the motor domain, two calmodulin-binding IQ motifs, a putative coiled-coil region, and a Pro, Ser and Thr-rich domain, lies a combination of dbl homology and pleckstrin homology domains. These are conserved in Rho GDP/GTP exchange factors (RhoGEFs). We have identified for the first time the RhoGEF domain in the myosin sequences. The growth and terminal developmental phenotype of Dictyostelium cells were not affected by the myoM(-) mutation. Green fluorescent protein-tagged MyoM, however, accumulated at crown-shaped projections and membranes of phase lucent vesicles in growing cells, suggesting its possible roles in macropinocytosis.

Characterization of a C-terminal-type kinesin-related protein from Dictyostelium discoideum.

We have determined the full sequence of K2, a kinesin-related protein (KRP) in Dictyostelium discoideum. Sequence homology and domain organization placed K2 in the ncd/Kar3 subfamily of the C-terminal-type KRPs. Bacterially expressed, truncated K2 showed ATP-dependent binding to microtubules and microtubule-stimulated ATPase activity. K2-null cells grew and developed normally, suggesting overlapping functions of K2 with other microtubule motor(s). Overexpression of K2 caused partial mitotic arrest. Green fluorescent protein-tagged full-length K2 localized in the nucleus at the interphase and on the mitotic spindle during mitosis. These results suggest that K2 is a microtubule-dependent motor which may play some roles in mitotic spindles.

High incidence of esophageal cancer in esophageal achalasia by the oral administration of N-amyl-N-methylnitrosamine and its prevention by nicardipine hydrochloride in mice.

Esophageal achalasia (EA) is a rare disease in man and animals and there are many discussions on its higher risk of esophageal cancer. N-Amyl-N-methylnitrosamine (AMN) which specifically induces esophageal tumors in mice and rats was given to three mutant mouse strains, i.e. 101/N, STX/Le and BXH-8, which develop a high incidence of EA. The incidence of EA in 101/N, STX/Le, BXH-8 and normal C57BL/6J mice was 38.5% (110/286), 30.1% (43/143), 91.8% (190/207) and 0% (0/167), respectively. The average numbers of AMN-induced esophageal tumors in EA(+) were significantly higher than those of EA(-) in all of the 101/N, STX/Le and BXH-8 mice. Furthermore, significantly larger size tumors and invasive squamous cell carcinomas were found in EA(+) mice than in EA(-) mice. These results indicate the higher sensitivity of EA for both tumor induction and promotion, possibly due to the longer retention of AMN. In fact, relaxation of the lower esophagus by a smooth muscle relaxing calcium-channel blocker, nicardipine hydrochloride, significantly prevented the induction of esophageal tumors.

Morphology and function of human benign tumors and normal thyroid tissues maintained in severe combined immunodeficient mice.

In the improved SCID (severe combined immunodeficient) mice, various human benign tumors of the head and neck region were well maintained morphologically and functionally for 3 years until the experiments were terminated, e.g. transplanted parathyroid adenoma secreted parathyroid hormone (PTH) in the SCID mice for more than 1 year. Normal human thyroid tissue was also well maintained in the SCID mice for 3 years. Rapid and high uptake of radioiodine into the transplanted human thyroid tissue was observed. Furthermore, transplanted human thyroid tissue secreted thyroid hormone (T3) and T3 secretion was stimulated by the injection of human thyroid stimulating hormone (TSH). These findings suggest that the improved SCID mice will provide an invaluable experimental system for investigating the function of normal human tissues and the influence of endogenous and exogenous factors on human tissues.

The sites on calmodulin cross-linked to myosin light chain kinase and troponin I with water-soluble carbodiimide.

To elucidate the interaction of calmodulin with calmodulin binding proteins, we studied the location of the interaction sites on calmodulin by using a chemical cross-linking reagent. Calmodulin prepared from wheat germ was cross-linked to myosin light chain kinase and troponin-I with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linked products were cleaved partially with cyanogen bromide and cross-linked sites were determined by peptide mapping analysis using SDS-urea polyacrylamide gel electrophoresis. Peptides which contain the cross-linked site were displaced from their position because of the attached fragments of myosin light chain kinase or troponin I. The peptide of calmodulin from the N-terminal to Met-73 in the cross-linked product with myosin light chain kinase had the same mobility as that of uncross-linked calmodulin on the map though the amount of the peptide was decreased in the cross-linked product. The peptide from the N-terminal to Met-110 in the cross-linked product was displaced from its position. Similar change in the mobility of the calmodulin peptides was also observed in the cross-linked products with troponin I. It was concluded, therefore, that at least one cross-linked site for myosin light chain kinase and one for troponin I were located between Met-73 and Met-110 of the wheat germ calmodulin.

Identification and possible roles of three types of endopeptidase from germinated wheat seeds.

Little or no endopeptidase activity was detected in extracts of dry mature wheat seeds, but when they were allowed to imbibe water in darkness, the activity expressed per seedling increased notably after d 1, reached a maximum on d 3 and then decreased. Two major endopeptidases, named WEP-1 and WEP-2, were present in the 50-70% saturated ammonium sulfate fraction of d-3 seedlings, and could be separated by hydrophobic column chromatography. WEP-1 was further purified and identified as a 31-kDa polypeptide that was immunoreactive to antiserum raised against REP-1, a major rice cysteine endopeptidase. Experiments with proteinase inhibitors revealed that WEP-1 and WEP-2 are cysteine and serine endopeptidases, respectively. The two enzymes differed in substrate specificity, pH dependence, and the ability to digest major wheat seed proteins. Determination of its amino-terminal amino acid sequence indicated the similarity of WEP-1 to other cereal cysteine endopeptidases which are involved in the digestion of seed storage proteins. The expression of WEP-1 in de-embryonated seeds was induced in the presence of gibberellic acid and its effect was eliminated by abscisic acid. In addition to WEP-1 and WEP-2, a legumain-like asparaginyl endopeptidase was identified in the extract of seedlings on hydrophobic chromatography. The asparaginyl endopeptidase may function in the early step of mobilization of wheat storage proteins in germinated seeds.

Purification of cofilin, a 21,000 molecular weight actin-binding protein, from porcine kidney and identification of the cofilin-binding site in the actin sequence.

Cofilin, a 21,000 molecular weight protein originally purified from porcine brain that is capable of binding to actin filaments in a molar ratio of the protein to actin monomer of 1:1 in the filament (Nishida et al. (1984) Biochemistry 23, 5307-5313), was purified from porcine kidney in the present study. The two cofilins from brain and kidney were indistinguishable from each other with respect to the mobility on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the one-dimensional peptide map, and the mode of interaction with actin. Treatment of the actin-cofilin complex with a zero-length cross-linker, 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide (EDC), generated a cross-linked product with an apparent molecular weight of 63,000. Analysis of this product by peptide mapping (Sutoh (1982) Biochemistry 21, 3654-3661) showed that cofilin was cross-linked with the N-terminal segment of actin containing residues 1-12.

Nordihydrocapsiate, a new capsinoid from the fruits of a nonpungent pepper, capsicum annuum

A new capsiate-like substance, named nordihydrocapsiate (1), has been isolated from the fruits of a nonpungent cultivar, CH-19 Sweet, of pepper (Capsicum annuum). The structure of 1 was determined to be 4-hydroxy-3-methoxybenzyl 7-methyloctanoate by spectroscopic methods.

Stability of capsinoid in various solvents.

To investigate the stability of capsinoid in solvents, the quantitative change of vanillyl nonanoate, a synthetic model capsinoid, in various solvents was measured by HPLC. Vanillyl nonanoate was stable in nonpolar solvents, whereas it was labile in polar solvents. In particular, vanillyl nonanoate tended to decompose in protic solvents such as alcohol and water. Structures of the decomposition products from vanillyl nonanoate in methanol and ethanol were determined to be methyl and ethyl vanillyl ethers, respectively. To clarify the decomposition mechanism of capsinoid, six analogues of vanillyl nonanoate were tested. The stability of the analogues in organic solvents suggested that the hydroxyl group in the para-position of the benzene ring largely contributes to the decomposition of capsinoid.