Protein bodies in corn endosperm are enclosed by and enmeshed in F-actin

Summary When corn endosperm is gently homogenized in a microfilament-stabilizing buffer and filtrates centrifuged at very low g forces, abundant protein bodies are present in the pellet. When stained with both rhodamine-phalloidin and fluorescein-S₁-myosin and viewed under epifluorescence, individual protein bodies appear to be enclosed in F-actin and groups of protein bodies appear to be enmeshed within a network of fine actin filaments. The yield of actinenclosed protein bodies is not affected by the addition of non-ionic detergents to the grinding medium, but it is severely decreased with the addition of cytoskeleton-disrupting agents including Tris-HCl, KCl, KI, and ammonium sulphate, and the interconnecting fine filaments are totally abolished. Heparin, which interacts with membrane-cytoskeleton complexes, causes apparent coagulation of the protein bodies. The evidence from fluorescent microscopy suggesting that F-actin surrounds and enmeshes the protein bodies is supported by electron microscopy, gel electrophoresis, and Western blot analysis.Abbreviations BSA bovine serum albumin - DTE dithioerythritol - EGTA ethylehe-glycol-bis(B-aminoethyl ether) N, N, N, N-tetraacetic acid - HEPES (N-[hydroxyethylpiperazine-N-[2-ethanesulfonic acid]) - HMM heavy meromyosin - MSB microfilament-stabilizing buffer - PMSF phenyl-methylsulfonyl fluoride - PTA phospho-tungstic acid - PTE polyoxyethylene-10-tridecyl ether - Tris Tris-(hydroxymethyl) aminomethaneM     

Effect of calcium and F-actin on the rate of SH2 modification in skeletal myosin

The rate constant of modification of a specific thiol group, SH₂, with N-ethylmaleimide (NEM) has been used to estimate the conformational change in the local area containing SH₂ (SH₂ region) of skeletal myosin as a structural probe. The rate of Mg²⁺-ATP-induced SH₂ modification of subfragment-1 (S-l) isozymes was regulated by Ca²⁺ in the pCa range below 6.4 and was not regulated in the pCa range above 6.4. No substantial difference between S-1 containing alkali light chain, A1, (S-1(A1)) and S-1 containing alkali light chain, A2, (S-1(A2)) was observed in the Ca²⁺-dependent rate of SH₂ modification. Due to the presence of this Ca²⁺ regulation in myosin (absence in S-1 isozymes) in the pCa range above 6.4, absence of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) light chain in S-1 isozymes, and high affinity of Ca²⁺ for DTNB light chain, this Ca²⁺ regulation in the pCa range above 6.4 is possibly related to the Ca²⁺ binding to DTNB light chain. F-Actin, which is entirely free from tropomyosin and troponin, enhanced the rate of Mg²⁺-ATP-induced SH₂ modification of S-1 isozymes equally and of myosin, and reduced the Ca²⁺ sensitivity with an increase in F-actin concentration.     

Rhodamine-phalloidin staining of F-actin in pollen after dimethylsulphoxide permeabilization

Summary Bundles of actin filaments were observed in in vitro cultured pollen of Lilium longiflorum and Nicotiana tabacum which had been permeabilized in a buffered medium containing 5% dimethylsulphoxide (DMSO), EGTA, rhodaminephalloidin for F-actin staining, and sucrose as an osmoticum. In imbibed pollen grains, especially those of lily, numerous short bundles and small foci of F-actin were clearly visible. In germinated pollen grains, fine bundles of F-actin could be seen to converge at the aperture of the pollen grain. Along the entire length of the pollen tube, including the extreme tip, a dense three-dimensional netaxial distribution of actin filaments was observed. The F-actin patterns visualized after permeabilization with DMSO are much finer and more detailed than those observed after conventional fixation with formaldehyde.     

Apyrase from pea stems: Isolation, purification, characterization and identification of a NTPase from the cytoskeleton fraction of pea stem tissue

The cytoskeleton pellet from the first internode of dark-grown pea stems was disintegrated in a high salt buffer, ultracentrifuged to remove ribosomes and the post-ribosomal supernatant was applied to a heparin affinity column. Significant ATPase activity was present in the cytoskeleton fraction and this was eluted from the column at 0.6–0.7 M KOAc, in the same fractions as a 49-kDa protein (which we called B3). B3 was desalted and further purified by cation exchange column chromatography. Purified B3 catalyzed hydrolysis of ATP, CTP, GTP, TTP, UTP and ADP and thus appears to be an apyrase (ATP diphosphohydrolase, EC 3.6.1.5). Partial amino acid sequences of three major fragments were obtained by digestion of B3 by Staphylococcus aureus V8 protease (EC 3.4.21.19), and all these sequences were consistent with the previously reported amino acid sequences for pea nucleoside triphosphatase (NTPase, EC 3.6.1.15) (PIR S48859), which is thought to be an apyrase.     

Photoinduction of circular F-actin on chloroplast in a fern protonemal cell

Summary Circular F-actin on a photooriented chloroplast was observed by rhodamine-phalloidin staining in the fernAdiantum protonemal cells in which phytochrome- or blue light receptor-mediated intracellular photoorientation of chloroplasts was induced. The circular structure located along the edge of chloroplast on the side facing the plasma membrane but not on the opposite side. Most of the chloroplasts in protonemal cell have dumbbell-shape and the circular ring-like structure was found on each half of the dumbbell. The structure was not observed in the cells which were kept in the dark, indicating the change of F-actin organization by the light condition. Possible role of the structure on the anchorage of chloroplast in its intracellular photoorientation was discussed.     

The organization of F-actin in root tip cells ofAdiantum capillus veneris throughout the cell cycle

Summary The patterns of F-actin in relation to microtubule (Mt) organization in dividing root tip cells ofAdiantum capillus veneris were studied with rhodamine-phalloidin (RP) labelling and tubulin immunofluorescence. Interphase cells display a well organized network of cortical/subcortical, endoplasmic and perinuclear actin filaments (AFs), not particularly related to the interphase Mt arrays. The cortical AFs seem to persist during the cell cycle while the large subcortical AF bundles disappear by preprophase/prophase and reappear after cytokinesis is completed. In some but not all of the preprophase cells the cortical AFs tend to form a band (AF-PPB) coincident with the preprophase band of Mts (Mt-PPB). In metaphase and anaphase cells AFs are localized in the cell cortex, around the spindle and inside it coincidently with kinetochore Mt bundles. During cytokinesis AFs are consistently found in the phragmoplast. In oryzalin treated cells neither Mt-PPBs, spindles and phragmoplasts exist, nor such F-actin structures can be observed. In cells recovering from oryzalin, AF-PPBs, AF kinetochore bundles and AF phragmoplasts reform. They show the same pattern with the reinstating respective Mt arrays. In contrast, in cells treated with cytochalasin B (CB), AFs disappear but all categories of Mt arrays form normally.These observations show that F-actin organization in root tip cells ofA. capillus veneris differs from that of root tip cells of flowering plants examined so far. In addition, Mts seem to be crucial for F-actin organization as far as it concerns the PPB, the mitotic spindle, and the phragmoplast.Abbreviations AF actin filament - CB cytochalasin B - MBS m-male-imidobenzoyl-N-hydroxysuccinimide ester - MSB microtubule stabilizing buffer - Mt microtubule - PBS phosphate buffered saline - PPB preprophase band - RP rhodamine phalloidin     

The myosin ATPase inhibitor 2,3-butanedione-2-monoxime disorganizes microtubules as well as F-actin in Saccharomyces cerevisiae

Interactions between microtubules and filamentous actin (F-actin) are essential to many cellular processes, but their mechanisms are poorly understood. We investigated possible roles of the myosin family of proteins in the interactions between filamentous actin (F-actin) and microtubules of budding yeast Saccharomyces cerevisiae with the general myosin ATPase inhibitor 2,3-butanedione-2-monoxime (BDM). The growth of S. cerevisiae was completely inhibited by BDM at 20 mmol/L and the effect of BDM on cell growth was reversible. In more than 80% of BDM-treated budding yeast cells, the polarized distribution of F-actin was lost and fewer F-actin dots were observed. When cells were synchronized in G₁ with α-factor and released in the presence of BDM, cell number did not increase and cells were mainly arrested in G₁ DNA content without any bud, suggesting that myosin activity is required for new bud formation and the start of a new cell cycle. More than 10% of the BDM-treated cells also revealed defects in nuclear migration to the bud neck as well as in nuclear shape. Consistent with these defects, the orientation of mitotic spindles was random in the 57% of cells treated with 20 mmol/L BDM and immunostained with anti-tubulin antibody. Furthermore, microtubule structures were completely disorganized in most of the cells incubated in 50 mmol/L BDM, while similar amounts of tubulin proteins were present in both BDM-treated and untreated cells. These results show that the general myosin inhibitor BDM disorganizes microtubule structures as well as F-actin, and suggest that BDM-sensitive myosin activities are necessary for the interaction of F-actin and microtubules to coordinate polarized bud growth and the shape and migration of the nucleus in S. cerevisiae. This revised version was published online in July 2006 with corrections to the Cover Date.     

Fluorescence and Electron Microscopic Localization of F-actin in the Ependymocytes

The organization of F-actin in the ventricular system has been reported to display pronounced regional differences with respect to shape, size, and development. However, the real roles played by F-actin in these cells cannot be understood unless the precise localization of F-actin is defined. In the present study, we used double-fluorescence labeling to further examine the localization of F-actin in the ependymocytes and its spatial relation to the other two cytoskeletal components, microtubules and intermediate filaments. Then we converted fluorescence signals for F-actin to peroxidase/DAB reaction products by use of a phalloidin-based FITC-anti-FITC system. This detection technique provided an overview of the distribution of F-actin in the ependymocytes at the ultrastructural level, and has been proven to be helpful in correlating light and electron microscopic investigations. (J Histochem Cytochem 57:741–751, 2009)     

Comparison of F-actin fluorescent labeling methods in pollen tubes of Lilium davidii

Fluorescence labeling of F-actin in pollen tubes by various methods has produced inconsistent results in the literature. Here, we report that EGTA, which was always used in fixative buffers in the past and thought to help cytoskeleton stabilization, can significantly affect F-actin distribution and lead to the formation of thick F-actin bundles at the tip of the pollen tube. We also found that vacuum-infiltration for the first 5 min during pollen tube fixation can better preserve normal cytoplasm structure and F-actin distribution. In contrast, m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) treatment before chemical fixation resulted in a shortening of the free zone of thick F-actin bundles in the pollen tube tip. Taken together, our results suggest that exclusion of EGTA and MBS from the fixative buffer, in combination with vacuum-infiltration in the first 5 min of fixation, can improve F-actin fluorescence labeling in pollen tubes of Lilium davidii.Li Wang and Yi-Min Liu are considered joint first authors     

Coincident filament distribution and histochemical localization of F-actin in the enterocytes of the larval dragonflyAeshna cyanea

Summary The fluorescence pattern of the enterocytes of larvalAeshna cyanea obtained by the incubation of cryosections with rhodaminephalloidin, a histochemical probe for F-actin, exactly coincides with the distribution of 6 nm filaments. These prevail as 1. core bundles in the microvilli 2. terminal web including the rootlets of the core bundles and and filaments associated with the adhesive junctions, 3. intercisternal arrays in the subapical stacks of smooth endoplasmic reticulum, and 4. cortical web underneath the basolateral plasma membranes.Abbreviations EDTA ethylenediaminetetraacetic acid - HMM heavy meromyosin - PBS phosphate-buffered saline - PIPES piperazine-N, N-bis (2-ethanesulfonic acid) - rER and sER rough and smooth endoplasmic reticulum     

Intracellular-free calcium dynamics and F-actin alteration in the formation of macrophage foam cells

The formation of macrophage foam cells, which is the key event in atherosclerosis, occurs by the uptake of oxidized low-density lipoprotein (Ox-LDL) via the scavenger receptor (CD36) pathway. Ca(2+) plays an important role in atherosclerosis. However, in the spatiotemporal view, the correlation between kinetic changes of intracellular-free calcium ([Ca(2+)](i)) and the cellular dysfunctions in the formation of macrophage foam cells has not yet been studied in detail. By the use of confocal laser scanning microscope and flow cytometer, we have detected Ca(2+) dynamics, the assembly of F-actin, and the expression of CD36 under the exposure of U937-derived macrophages to Ox-LDL. The uptake of Ox-LDL significantly increased [Ca(2+)](i) in U937-derived macrophages in both acute and chronic treatments (P<0.01). In particular, the increases of the induced [Ca(2+)](i) were different in the presence or absence of extracellular Ca(2+) under acute exposure. A time-dependent rise in F-actin assembly and CD36 expression at 12 and 24h was induced, respectively, by Ox-LDL. The spatiotemporal increases of [Ca(2+)](i) induced by Ox-LDL probably have the key effect on the early phrase in the formation of macrophage foam cells.     

C-terminal fragments of thymopoietin favor formation of F-actin bundles: Electron microscopic data

Electron microscopy showed that PEP₃₃, a synthetic peptide corresponding to the C-terminal part of the thymic hormone thymopoietin, favors bundling of F-actin filaments in the presence of 0.1 M KCl. The structure of PEP₃₃ aggregates located within bundles between actin filaments is very similar to the structure of aggregates visible in preparations of pure PEP₃₃. No changes were observed in the structure of G-actin in the presence of PEP₃₃. A similar though weaker bundling effect was also detected for PEP₅, or thymopentin, a fragment of PEP₃₃. This peptide favors the formation of bundles of actin filaments of small size. The possible role of aggregation of actin filaments under the action of thymopoietin peptides is discussed in the light of the fact that the systematic release of thymopoietin from thymus leads to a phenomenon characteristic of the serious neuromuscular disease myasthenia gravis.     

Caldesmon restricts the movement of both C- and N-termini of tropomyosin on F-actin in ghost fibers during the actomyosin ATPase cycle

New data on the movements of tropomyosin singly labeled at alpha- or beta-chain during the ATP hydrolysis cycle in reconstituted ghost fibers have been obtained by using the polarized fluorescence technique which allowed us following the azimuthal movements of tropomyosin on actin filaments. Pronounced structural changes in tropomyosin evoked by myosin heads suggested the "rolling" of the tropomyosin molecule on F-actin surface during the ATP hydrolysis cycle. The movements of actin-bound tropomyosin correlated to the strength of S1 to actin binding. Weak binding of myosin to actin led to an increase in the affinity of the tropomyosin N-terminus to actin with simultaneous decrease in the affinity of the C-terminus. On the contrary, strong binding of myosin to actin resulted in the opposite changes of the affinity to actin of both ends of the tropomyosin molecule. Caldesmon inhibited the "rolling" of tropomyosin on the surface of the thin filament during the ATP hydrolysis cycle, drastically decreased the affinity of the whole tropomyosin molecule to actin, and "freezed" tropomyosin in the position characteristic of the weak binding of myosin to actin.     

F肌动蛋白作为胞间连丝组分的结构与生理学证据

以蒜(Allium sativum L.)瓣鞘外表皮为材料,利用荧光特异探针与共焦镜检术,结合透射电镜与免疫金标记对表皮细胞间胞间联络的性质、结构进行了系统观察.结果表明,加厚壁上的通道是由狭长的管状胞质和初生纹孔场上成束的胞间连丝衔接组成,前者实为原生质体的一部分.单个胞间连丝的孔径为60～70 nm,属正常胞间连丝范围,它们乃相邻细胞间共质联系的所在.荧光探针TRITC-Phalloidin (TRITC-Ph)标记的结果显示,整个通道上呈现红色荧光的纤索在接近初生纹孔场处明显变窄,与超微结构观察中所见的结构特点相吻合,共焦镜下观察到的初生壁上的荧光亮斑乃初生纹孔场中成束胞间连丝被标记的反映,从而有效地证实了F肌动蛋白在常态胞间连丝上的存在.免疫金标记实验显示在管状胞质中和胞间连丝上有金颗粒分布,这一结果为证实荧光标记物具肌动蛋白性质提供了有说服力的补充.     

Light-enhanced perception of gravity in stems of intact pea seedlings

Dark-grown, 6-d-old pea seedlings (Pisum sativum L. cv. Alaska) do not respond gravitropically to brief (approx. 3 min) horizontal presentations, but seedlings given a pulse of red light (R) 16–24 h earlier respond to such stimuli by vigorous curvature of the epicotyl. With continuous horizontal stimulation (approx. 100 min), the kinetics and extent of the gravitropic response are almost identical in irradiated and dark-control plants. Prior R thus increases graviperception without altering the rate-limiting steps underlying the generation of curvature. This effect of R on graviperception develops slowly; seedlings studied only a few hours after R show differences in the kinetics of the gravitropic response, but not in presentation time. Neither the kinetics nor the extent of gravitropic curvature should be used as criteria for establishing changes in primary processes in gravitropism.     

Effect of dietary cholesterol on the pattern of osmium deposition in the symbiote-containing cells of the pea aphid

Summary Pea aphids left for 48 h in unbuffered osmium tetroxide show heavy staining of many organelles in the symbiote-containing cells (mycetocytes and sheath), embryos and oenocytes very similar to that characteristic of mammalian sterol-synthesizing cells. However, the staining of the pea-aphid cells is, to a large extent, dependent on the presence of cholesterol benzoate, or free cholesterol, in the aphid's diet. In aphids cultured in vitro with ³H mevalonate in the presence of added cholesterol, the incorporation of label into the cholesterol and lanosterol fractions is significantly reduced. If the dietary cholesterol effects a similar inhibition in vivo, the cholesterol-dependent osmium staining could be due to precursors(s) of cholesterol accumulating in the intracellular sites described.There is also osmium staining of large (normally electron-transparent) vacuoles in mycetocytes, gut and fat body, irrespective of dietary cholesterol.Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, and by a research grant (PCM 74-2401A01) from the National Science FoundationThe authors are grateful to Dr. E.J. Houk and Dr. G.A. DeZoeten for valuable suggestions, and to Mr. Gary Gaard for technical assistance     

Isolation of cytochrome bc 1 complexes from the photosynthetic bacteria Rhodopseudomonas viridis and Rhodospirillum rubrum

Cytochrome bc ₁ complexes have been isolated from wild type Rhodopseudomonas viridis and Rhodospirillum rubrum and purified by affinity chromatography on cytochrome c-Sepharose 4B. Both complexes are largely free of bacteriochlorophyll and carotenoids and contain cytochromes b and c ₁ in a 2:1 molar ratio. For the Rps. viridis complex, evidence has been obtained for two spectrally distinct b-cytochromes. The R. rubrum complex contains a Rieske iron-sulfur protein (present in approximately 1:1 molar ratio to cytochrome c ₁) and catalyzes an antimycin A- and myxothiazol-sensitive electron transfer from duroquinol to equine cytochrome c or R. rubrum cytochrome c ₂. Although an attempt to prepare a cytochrome bc ₁ complex from the gliding green bacterium Chloroflexus aurantiacus was not successful, membranes isolated from phototrophically grown Cfl. aurantiacus were shown to contain a Rieske iron-sulfur protein and protoheme (the prosthetic group of b-type cytochromes).Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

The presence of ring formed actin filaments in plant cells

Summary Ring formed actin filaments were observed in tobacco BY-2 cells. The change of this structure during culture was followed by fluorescence microscopy.     

Isolation and characterization of pathogens attacking Pomacea canaliculata

Seven microorganisms capable of killing Pomacea canaliculata were isolated from soil samples obtained from various agricultural areas of Thailand. The identification of these microorganisms was performed using microscopic examination and biochemical tests. Five strains were identified as Pseudomonas aeruginosa and were designated P. aeruginosa 19.1, 21.2.1, B1.1, P1 and P2. The other two strains were identified as Pseudomonas fluorescens and were designated P. fluorescens 13.1 and Ct1. Pathogenicity studies of these microorganisms to P. canaliculata (Lamarck) were performed and characterized by LC₅₀ levels. The LC₅₀ levels of non-autoclave-treated and autoclave-treated cell suspensions to P. canaliculata were found to be 3.56 × 10⁴–1.35 × 10⁶ c.f.u./ml and 3.09 × 10⁴ to 1.23 × 10⁶ c.f.u./ml, respectively.