多头绒泡菌核仁骨架的研究

从多头绒泡菌（Ｐｈｙｓａｒｕｍ　ｐｏｌｙｃｅｐｈａｌｕｍ　Ｓｃｈｗ）间期细胞核中分离出核仁,用ＤＮａｓｅ　Ｉ、０．２５ｍｏｌ／Ｌ（ＮＨ４）２ＳＯ４和２ｍｏｌ／ＬＮａＣｌ去除核仁ＤＮＡ和大部分蛋白质,得到核仁骨架。核仁骨架是直径１０３０ｎｍ的纤维组成的网络结构,含有约２０种多肽,其中包括与肌动蛋白电泳迁移率相当的４３ｋＤ左右的多肽。免疫荧光检测结果表明,核仁骨架能与肌动蛋白抗体结合而发出明亮的荧光。免疫斑点印迹结果进一步证实,核仁骨架的蛋白质成分中存在肌动蛋白。免疫电镜结果指出,代表肌动蛋白的金颗粒分布在整个核仁中。     

肌动蛋白是多头绒泡菌细胞核骨架和染色体骨架的组成成分

自多头绒泡菌(Physarum polycephalum Schw.)的原质团中分离细胞核和染色体,分别经DNaseⅠ消化和2 mol/L NaCl抽提后制备成细胞核骨架和染色体骨架。以抗肌动蛋白的抗体作一抗、FITC标记的羊抗兔IgG抗体作二抗进行的间接免疫荧光实验结果显示,细胞核骨架和染色体骨架都分别与抗体呈阳性反应。间接免疫斑点印迹实验结果进一步证实,细胞核骨架和染色体骨架的蛋白质成分中存在与肌动蛋白抗体呈阳性显色反应的抗原。以抗肌动蛋白的抗体作一抗、金颗粒标记的蛋白A作二抗的间接免疫电镜实验结果表明,在实验组间期细胞核的核仁、集缩染色质和核基质以及中期染色体上都有很多金颗粒分布。上述结果证明,肌动蛋白是多头绒泡菌细胞核和染色体及其骨架的组成成分。     

肌动蛋白存在于蚕豆细胞核和染色体中

以兔抗肌动蛋白抗体为一抗,FTTC偶联的羊抗兔IgG抗体为二抗进行间接免疫荧光实验,观察到蚕豆(Vicia faba L.)根端分生组织中完整的细胞核和染色体均有明亮荧光。用抗肌动蛋白抗体和蛋白A-胶体金进行标记的免疫电镜实验结果表明,金颗粒分布在蚕豆细胞核中,集缩染色质和核仁中金颗粒较多。经DNaseI消化和2 mol/L NaCl处理得到去除DNA和组蛋白的细胞核和染色体。免疫荧光实验结果指出,去除DNA和组蛋白的细胞核和染色体与抗肌动蛋白抗体呈阳性反应。上述结果说明,肌动蛋白不仅存在于完整的蚕豆细胞核和染色体中,而且存在于去除DNA和组蛋白的蚕豆细胞核和染色体中。另外,用抗原肌球蛋白抗体所做的免疫荧光标记结果表明,原肌球蛋白也存在于蚕豆细胞核和染色体中。对高等植物细胞核和染色体以及核骨架和染色体骨架是否含有肌动蛋白等问题进行了讨论。     

Actin is Immunolocalized in the Nuclei and Chromosomes of Vicia faba

Meristematic cells of Vicia faba L. were labeled with rabbit anti-actin antibody and FITC-conjugated goat anti-rabbit lgG antibody and observed with fluorescence microscopy. Both the nuclei and chromosomes sent forth distinctive fluorescence, indicating that actin is present in the nuclei and chromosomes. Sections were reacted with the anti-actin antibody and protein A-colloidal gold and observed with transmission electron microscopy. Gold particles were found over the whole nuclei, and a lot of particles were concentrated in condensed chromatin areas and nucleoli, confirming the observations with the fluorescence microscopy. V. faba nuclei and chromosomes were treated with DNase Ⅰ and 2 mol/L NaC1, and DNA and histone-depleted nuclei and chromosomes were obtained. Indirect immunofluorescence tests showed that the DNA and histone-depleted nuclei and chromosomes reacted positively with the anti-actin antibody. These results demonstrated that actin exists not only in intact nuclei and chromosomes but also in DNA and histone-depleted nuclei and chromosomes of V. faba. In addition, the authors' results indicate that tropomyosin is present in the nuclei and chromosomes of V. faba. Presence of actin in nuclei and chromosomes as well as in DNA and histone-depleted nuclei and chromosomes of higher plants is discussed.     

Anti-actin antibodies generated against profilin:actin distinguish between non-filamentous and filamentous actin, and label cultured cells in a dotted pattern

Actin polymerization is a prominent feature of migrating cells, where it powers the protrusion of the leading edge. Many studies have characterized the well-ordered and dynamic arrangement of filamentous actin in this submembraneous space. However, less is known about the organization of unpolymerized actin. Previously, we reported on the use of covalently coupled profilin:actin to study actin dynamics and presented evidence that profilin-bound actin is a major source of actin for filament growth. To locate profilin:actin in the cell we have now used this non-dissociable complex for antibody generation, and obtained monospecific anti-actin and anti-profilin antibodies from two separate immunizations. Fluorescence microscopy revealed drastic differences in the staining pattern generated by the anti-actin antibody preparations. With one, distinct puncta appeared at the actin-rich leading edge and sometimes aligned with microtubules in the interior of the lamella, while the other displayed typical actin filament staining. Labelling experiments in vitro demonstrated failure of the first antibody to recognize filamentous actin and none of the two bound microtubules. The two anti-profilin antibodies purified in parallel generated a punctated pattern similar to that seen with the first anti-actin antibody. All antibody preparations labelled the nuclei.     

Identification of L-cell growth stimulating antibody as anti-actin

Anti-L-cell antisera having potent cell growth stimulatory properties were shown by Western blotting to have predominant specificity toward a protein with a molecular weight of 42K which we identified as actin. Extractions of L cells, based upon the known insolubility of cytoskeletal proteins (including actin) in Triton X-100 and the solubility of actin in low ionic strength Ca2+ and ATP-containing buffer, led to actin-enriched preparations that retained immunoreactivity with the anti-L-cell antisera. The 42-kDa antigen binds to deoxyribonuclease I, has a pI = 5.2-5.4, and has an amino acid composition, including the presence of 3-methylhistidine, compatible with compositions determined for actins from other sources. Rabbit antiserum specific for this 42-kDa protein, isolated by SDS-PAGE, reproduced the cell growth stimulation by the anti-L-cell antisera and absorption of the antiserum with purified L-cell actin eliminated this stimulation. Moreover, these antibodies bind to the microfilaments of 3T3 fibroblasts. When purified actins were used as soluble antigen inhibitors of the immune reactivity of antiserum to 42-kDa protein with intact L cells, rabbit thymus actin competed with the surface molecules on L cells and reduced the stimulatory effect of the antiserum by 80% at an actin concentration of 150 micrograms/ml. Chicken muscle actin reduced the antibody stimulation effect by only 24% at the same protein concentration, and mouse muscle actin was ineffective as an inhibitor. The F(ab')2 fraction of anti-42K IgG was effective in stimulating L cells, thus documenting the immune nature of the actin-anti-42K interaction. We conclude that anti-actin antibodies, upon binding to actin-like cell surface determinants on L cells, stimulate cellular metabolism.     

多头绒泡菌核仁及其骨架中原肌球蛋白的免疫细胞化学鉴定

分离多头绒泡菌(physarum polycephalum)细胞的核仁,先用Dnase I消化,去除核仁内的DNA;然后用025mol/L (NH4)2ＳＯ４和2mol/L NaCl相继抽提去掉大部分蛋白质,制备成核仁骨架。SDSPAGE分析结果表明,核仁骨架中含有约20种多肽,其中包括37kD左右与原肌球蛋白分子量相当的多肽。以兔抗原肌球蛋白抗体为一抗,FITC标记的羊抗兔IgG抗体为二抗的间接免疫荧光检测结果表明,核仁和核仁骨架样品都能发出明亮的荧光,而对照样品未见明亮的荧光。间接免疫斑点印迹检测结果进一步证明,在核仁骨架的蛋白质成分中存在原肌球蛋白。胶体金免疫电镜检测结果显示,标记原肌球蛋白抗体的标本上有较多的金颗粒,而对照组标本上只有极少的金颗粒。金颗粒在核仁中主要呈散在分布。     

The profilin:actin complex localizes to sites of dynamic actin polymerization at the leading edge of migrating cells and pathogen-induced actin tails

A unique set of affinity-purified anti-profilin and anti-actin antibodies generated against a covalently coupled version of the profilin:actin complex was used to assess the distribution of profilin and non-filamentous actin in mouse melanoma cells. In agreement with the profilin:actin complex being the principal source of actin for filament formation, we observed extensive co-distribution of both antibody preparations with vasodilator-stimulated phosphoprotein (VASP) and the p34 subunit of the Arp2/3 complex, both of which are components of actin polymer-forming protein complexes in the cell. This suggests that the localization of profilin and actin revealed with these antibodies in fact reflects the distribution of the profilin:actin complex rather than the two proteins separately. Significantly, protruding lamellipodia and filopodia showed intensive labeling. The two antibody preparations were also used to stain HeLa cells infected with Listeria monocytogenes or vaccinia virus. In both cases, the pattern of antibody staining of the pathogen-induced microfilament arrangement differed, suggesting a varying accessibility for the antibody-binding epitopes.     

Actin and Arp2/3 localize at the centrosome of interphase cells

Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel (“lower dimer”) actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.     

Composition and structure of nucleolar skeleton (nucleolar matrix)——Actin and fibrillarin are two main protein components of nucleolar skeleton

Purified nucleoli of HeLa cells were treated sequentially with nonionic detergent, nucleic acid enzyme, low salt and high salt. The residual nucleolar structure termed nucleolar skeleton (nucleolar matrix) was shown as a fine network under electron microscope with DGD embedding-unembedding technique. Such structures of BHK-21 cell and mouse liver cell are similar to that of HeLa cell. The protein composition of the nucleolar skeleton of HeLa cells was analyzed. The protein composition of such nucleolar residual shows obvious difference from the compositions of nuclear matrix and chromosome scaffold. The major protein composition of the nucleolar skeleton of HeLa cells contains 6-7 polypeptides. Their molecular weights are about 48, 43, 36 and 33 ku. Further studies show that actin and fib-rillarin are two major protein components of nucleolar skeleton of HeLa cells.     

Composition and structure of nucleolar skeleton (nucleolar matrix)-Actin and fibrillarin are two main protein components of nucleolar skeleton

Purified nucleoli of HeLa cells were treated sequentially with nonionic detergent, nucleic acid enzyme, low salt and high salt. The residual nucleolar structure termed nucleolar skeleton (nucleolar matrix) was shown as a fine network under electron microscope with DGD embedding-unembedding technique. Such structures of BHK-21 cell and mouse liver cell are similar to that of HeLa cell. The protein composition of the nucleolar skeleton of HeLa cells was analyzed. The protein composition of such nucleolar residual shows obvious difference from the compositions of nuclear matrix and chromosome scaffold. The major protein composition of the nucleolar skeleton of HeLa cells contains 6–7 polypeptides. Their molecular weights are about 48, 43, 36 and 33 ku. Further studies show that actin and fibrillarin are two major protein components of nucleolar skeleton of HeLa cells.     

Composition and structure of nucleolar skeleton (nucleolar matrix)

Purified nucleoli of HeLa cells were treated sequentially with nonionic detergent, nucleic acid enzyme, low salt and high salt. The residual nucleolar structure termed nucleolar skeleton (nucleolar matrix) was shown as a fine network under electron microscope with DGD embedding-unembedding technique. Such structures of BHK-21 cell and mouse liver cell are similar to that of HeLa cell. The protein composition of the nucleolar skeleton of HeLa cells was analyzed. The protein composition of such nucleolar residual shows obvious difference from the compositions of nuclear matrix and chromosome scaffold. The major protein composition of the nucleolar skeleton of HeLa cells contains 6–7 polypeptides. Their molecular weights are about 48, 43, 36 and 33 ku. Further studies show that actin and fibrillarin are two major protein components of nucleolar skeleton of HeLa cells.     

A simple procedure to produce monospecific polyclonal antibodies of high affinity against actin from muscular sources

A simple and effective technique to produce monospecific polyclonal antibodies of high affinity against actin is described. In this procedure, rabbit skeletal muscle actin in the 1:1 complex with bovine pancreatic deoxyribonuclease I is used as antigen to immunize rabbits. The antisera obtained are shown to contain antibodies against both actin and deoxyribonuclease I. By affinity chromatography the two antibody preparations were separated and characterized. The affinity-purified anti-deoxyribonuclease I and anti-actin do not show cross-reactivity. Thus, anti-deoxyribonuclease I inhibits the enzymic activity of deoxyribonuclease I and stains the enzyme after Western blotting. Affinity-purified anti-actin does not inhibit deoxyribonuclease I activity and stains only actin after Western blotting. The affinity-purified anti-actin can be used in a number of different actin-detecting techniques such as in immunohistochemistry and in immunoblotting techniques. This antibody recognizes only actins from muscular tissues with high affinity. Immunoblots of polyacrylamide gels in the presence of ampholytes (IEF) indicate that this antibody only recognizes the alpha-variants of actin. Thus, the skeletal and cardiac alpha-actins are recognized but not the smooth muscle gamma-isoform and the cytoplasmic actins. Vascular smooth muscle alpha-actin is not recognized when using immunoblotting or enzyme-linked immunosorbent techniques. On frozen sections, however, the anti-actin antibody clearly stained vascular smooth muscle cells. Epitope analysis using actin fragments generated by limited proteolysis and selective cleavage using hydroxylamine indicate that this antibody is directed against a rather limited region within the N-terminus of actin.     

Further evidence that phosphoprotein C23 (110 kD/pI 5.1) is the nucleolar silver staining protein

Previously we demonstrated a similar distribution between nucleolar organizing region-(NOR)-specific silver staining and localization of nucleolar phosphoprotein C23 (MW 110 kD/pI 5.1) [1, 2]. We now report that under fixation conditions which allow for antibody binding and subsequent silver staining, monoclonal antibody against protein C23 blocks NOR silver staining as well as silver staining in interphase nucleoli. Monoclonal antibody against nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1) did not block silver staining in either NORs or interphase nucleoli. These, along with earlier observations, provide evidence that nucleolar phosphoprotein C23 is the major silver staining protein of the nucleolus and that it is directly or indirectly associated with rDNA.     

Mitogen-like monoclonal anti-actin antibodies

Monoclonal antibodies (IgM kappa) have been produced to actin isolated electrophoretically from L cell extracts. These monoclonal anti-actin antibodies bind to intact L cells and modulate DNA synthesis and cell proliferation, much like affinity-purified polyclonal rabbit antibody to the same Mr 42,000 actin. In addition, monoclonal antibodies specific for actin from Entamoeba histolytica also bound to and modulated the growth of L cells. A monoclonal antibody directed against a neuroblastoma surface antigen did not produce stimulation of L cells, and the binding activity of anti-actin monoclonal antibody to L cells was removed by absorption with actin covalently coupled to Sepharose. These observations demonstrate the specificity of interaction between the anti-actin monoclonal antibodies and the surface of intact L cells. We conclude that a surface actin-like molecule on the L cell, when bound by specific monoclonal antibody, initiates a stimulatory signal which results in enhanced cellular metabolism.     

F-actin is a Nuclear and Chromosomal Component of the Meristematic Cells of Allium sativum

It is known that actin functionates in the form of F-actin. However, the presence of Factin in eukaryotic nuclei and chromosomes has not been well established. The authors labeled meristematic cells of Allium sativum L. with rabbit anti-chicken actin antibody and FITC-conjugated goat anti-rabbit IgG antibody and observed with fluorescence microscopy. Both the nuclei and chromosomes showed prominent yellow-green fluorescence, indicating the presence of actin in them. Fluorescence examination with TR1TC-conjugated phalloidin demonstrated prominent red fluorescence in the intact interphase cells, cytoplasm-free interphase nuclei, prophase and metaphase chromosomes as well as the daughter nuclei at telophase indicating the presence of F-actin; but the fluorescence was absent or very weak in the cells exposed to cytochalasin D before fixation. When double labeling of the anti-actin antibody and phalloidin was applied, the same nuclei and chromosomes were found to emanate yellow-green fluorescence representing actin at the excitation wavelength of F1TC, and red fluorescence representing F-actin at the excitation wavelength of TRITC, respectively. The FITC fluorescence and TRITC fluorescence shared the same distribution among the nuclei and chromosomes. These results indicate that F-actin is a component of the nuclei and chromosomes of the meristematic cells of A. sativum. It also suggests that F-actin may be the major existing form of actin in them.     

Actin is a Constituent of the Nuclear Matrix and Chromosome Scaffold in Physarum poly cephalum

Nuclear matrices and chromosome scaffolds were obtained by digesting and extracting, respectively with DNase Ⅰ and 2 mol/L NaC1, the nuclei and chromosomes isolated from the plasmedia of Physarum polycephalum Schw. The results of the indirect immunofluorescence of tacit antiactin antibody as immunomarker indicated that the nuclear matrices and chromosome scaffolds both had positive reaction with the antibody. The results of the indirect immunodotting experiment further verified the presence of actin antibody in their constituent. Protein A-colloidal gold immunoelectron microscopy technique revealed that gold particles were distributed in the interphase nuclei and metaphase chromosomes. The above results showed that actin is a constituent of the nuclear matrix and chromosome scaffold of P. polycephalum.     

Immunological characterization of an anti-actin antibody specific for cytoplasmic actins and its use for the immunocytological localization of actin in Aplysia nervous tissue

Previous immunochemical and immunocytochemical studies have shown that an antibody to actin prepared from body wall muscle of the marine mollusc Aplysia californica is specific for vertebrate cytoplasmic actins. The ability of this anti-actin to distinguish between different forms of actin most likely reflects the recognition of amino acid sequences unique to cytoplasmic actins. We have confirmed the specificity of this antibody for cytoplasmic actins using nervous tissue as a source of cytoplasmic actin in further immunochemical studies. In addition to binding cytoplasmic actin in purified preparations, the antibody removed actin selectively from crude extracts of nervous tissue of some but not all of the species tested. Our results also suggest that tissue-specific differences in the distribution of cytoplasmic actins may exist. Immunofluorescence studies of Aplysia nervous tissue stained with anti-actin revealed that actin is present in the cell body and axonal processes of Aplysia neurons. Although the function of actin in nerve cells is not understood, the observed pattern of immunofluorescence staining is consistent with the idea that actin may be involved in movement within the axoplasm.     

Mycoplasma and bacterial proteins resembling contractile proteins: a review

The basis of gliding motility in prokaryotes including certain mycoplasmas and the ability of mycoplasmas to retain their characteristic cell shapes in the absence of a supporting cell wall is unexplained. This review examines the available studies describing proteins resembling contractile proteins and cytoskeletal proteins in prokaryotes. Proteins with a significant degree of amino acid sequence homology to the myofibrillar proteins actin and myosin Al light chain and to tropomyosin have been described in prokaryotes. In addition, protein preparations from Mycoplasma pneumoniae have been shown to bind heavy meromyosin fragments, anti-actin antibody, and phalloidin; however, it remains to be proved that proteins in these preparations sharing properties with actin are synthesized by the mycoplasma.