In many cases, actin filaments are arranged into bundles and serve as tracks for cytoplasmic streaming in plant cells. We have isolated an actin-filament bundling protein, which is composed of 115-kDa polypeptide (P-115-ABP), from the germinating pollen of lily, Lilium longiflorum [Nakayasu et al. (1998) BIOCHEM: Biophys. Res. Commun. 249: 61]. P-115-ABP shared similar antigenicity with a plant 135-kDa actin-filament bundling protein (P-135-ABP), a plant homologue of villin. A full-length cDNA clone (ABP115; accession no. AB097407) was isolated from an expression cDNA library of lily pollen by immuno-screening using antisera against P-115-ABP and P-135-ABP. The amino acid sequence of P-115-ABP deduced from this clone showed high homology with those of P-135-ABP and four villin isoforms of Arabidopsis thaliana (AtVLN1, AtVLN2, AtVLN3 and AtVLN4), especially AtVLN4, indicating that P-115-ABP can also be classified as a plant villin. The P-115-ABP isolated biochemically from the germinating lily pollen was able to arrange F-actin filaments with uniform polarity into bundles and this bundling activity was suppressed by Ca2+-calmodulin (CaM), similar to the actin-filament bundling properties of P-135-ABP. The P-115-ABP type of plant villin was widely distributed in plant cells, from algae to land plants. In root hair cells of Hydrocharis dubia, this type of plant villin was co-localized with actin-filament bundles in the transvacuolar strands and the sub-cortical regions. Microinjection of the antiserum against P-115-ABP into living root hair cells caused the disappearance of transvaculor strands and alteration of the route of cytoplasmic streaming. In internodal cells of Chara corallina in which the P-135-ABP type of plant villin is lacking, the P-115-ABP type showed co-localization with actin-filament cables anchored on the intracellular surface of chloroplasts. These results indicated that plant villins are widely distributed and involved in the organization of actin filaments into bundles throughout the plant kingdom.

SummaryThe preprophase band (PPB) marks the site on the plant cell cortex where the cell plate will fuse during the final stage of cytokinesis. Recent studies have shown that several cytoskeletal proteins are depleted at the PPB site, but the processes that bring about these changes are still unknown. We have investigated the membrane systems associated with the PPB regions of epidermal cells of onion cotyledons by means of serial thin sections and electron tomograms. In contrast with specimens preserved by chemical fixatives, our highpressure frozen cells demonstrated the presence of large numbers of clathrin-coated pits and vesicles in the PPB regions. The vesicles were of two types: clathrin-coated and structurally related, non-coated vesicles. Quantitative analysis of the data revealed that the number of clathrin-coated pits and vesicles is higher in the PPB regions than outside of these regions. Immunofluorescent microscopy using anti-plant clathrin-antibody confirmed this result. In contrast, no differences in secretory activities were observed. We postulate that the removal of membrane proteins by endocytosis plays a role in the formation of PPB 'memory' structures.

We previously identified a 175 kDa polypeptide in Lilium longiflorum germinating pollen using a monoclonal antibody raised against myosin II heavy chain from Physarum polycephalum. In the present study, the equivalent polypeptide was also found in cultured tobacco BY-2 cells. Analysis of the amino acid sequences revealed that the 175 kDa polypeptide is clathrin heavy chain and not myosin heavy chain. After staining of BY-2 cells, punctate clathrin signals were distributed throughout the cytoplasm at interphase. During mitosis and cytokinesis, clathrin began to accumulate in the spindle and the phragmoplast and then was intensely concentrated in the cell plate. Expression of the C-terminal region of clathrin heavy chain, in which light chain binding and trimerization domains reside, induced the suppression of endocytosis and the formation of an aberrant spindle, phragmoplast, and cell plate, the likely cause of the observed multinucleate cells. These data strongly suggest that clathrin is intimately involved in the formation of the spindle and phragmoplast, as well as in endocytosis.

Stable compositions and geometrical structures of vanadium oxide cluster ions, VmOn(±), were investigated by ion mobility mass spectrometry (IM-MS). The most stable compositions of vanadium oxide cluster cations were (V2O4)(V2O5)(m-2)/2(+) and (VO2)(V2O5)(m-1)/2(+), depending on the clusters with even and odd numbers of vanadium atoms. Compositions one-oxygen richer than the cations, such as (V2O5)m/2(-) and (VO3)(V2O5)(m-1)/2(-), were predominantly observed for cluster anions. Assignments of these stable cluster ion compositions, which were determined as a result of collision-induced dissociations in IM-MS, can partly be explained with consideration of spin density distribution. By comparing the experimental collision cross sections (CCSs) obtained from ion mobility measurement with CCSs of the theoretically calculated structures, we confirmed the patterned growth of geometrical structures partially discussed in previous theoretical and spectroscopic studies. We showed that even sized (V2O5)m/2(±) where m = 6-12 had right polygonal prism structures except for the anionic V12O30(-), and for the clusters of odd numbers of vanadium m, cations and anions can either have bridged or pyramid structures. Both of the odd sized structures proposed were derivatives from the even sized right polygonal prism structures. The exception, V12O30(-), which had a CCS almost equal to that of the neighboring smaller V11O28(-), should have a structure of higher density than the right hexagonal prism, in which it was proposed to be a captured pyramid structure, derived from V11O28(-).

ABSTRACT:In order to elucidate the interaction between blood platelets and polymer surfaces of microdomain structure, block copolymers consisting of hydrophilic chains of 2-hydroxyethyl methacrylate (HEMA) and hydrophobic chains of dimethylsiloxane (DMS) were synthesized. Films of the HEMA-DMS block copolymers exhibiting hydrophilic-hydrophobic microphase-separated structures were found to enhance platelet adhesion more than homogeneous surfaces of poly(HEMA) or poly(DMS). Platelet adhesion to the block copolymer increased with morphological changes in microdomains so long as the HEMA composition of the block copolymer · was 0.90 to 0.58. Platelet adhesion also varied with morphological change in the microdomains caused by changing the casting solvents, though an identical copolymer was employed. These results indicated that platelet adhesion to the HEMA-DMS block copolymer was influenced by domain morphology rather than HEMA composition of the copolymers. In spite of the enhancement of platelet adhesion, the HEMA-DMS block copolymers effectively suppressed shape change and aggregation of adhered platelets. The HEMA-DMS block copolymers were considered to have good antithrombogenecity on the basis of inhibition of activation and aggregation of adhered platelets. KEY WORDS 2-Hydroxyethyl Methacrylate-Dimethylsiloxane Block Copolymer / Microdomain Structure / Platelet Adhesion / Antithrombogenecity / The antithrombogenic property of synthetic polymers is influenced remarkably by the balance between hydrophilicity and hydrophobicity of polymer surface. 1 We propose that the microdomain structure of polymer surfaces constructed of hydrophilic and hydrophobic sites of orderly assembled marcomolecules is the most important factor for designing effective antithrombogenic polymers. 2 We synthesized new amphiphilic block copolymers and comb type graft compolymers consisting of a hydrophilic monomer, 2-hydroxyethyl methacrylate (HEMA), and a hydrophobic monomer, styrene (St). 3 Films of HEMA-St block copolymers formed hydrophilic-hydrophobic microdomain structures on their surfaces. Platelet adhesion, an important process in the initial stage of thrombus formation, was studied in relation to block copolymer surfaces. The amount and deformation of adhered platelets were effectively suppressed on the surface of HEMA-St block and graft copolymers. This suppressing effect was found to be influenced by the shape and size of the microdomain rather than by the composition of the copolymers. 2 • 4 The copolymers having microdomain structures of alternate lemellae with about 0.05 µm in width exhibited 649