Localization of myosin II to chromosome arms and spindle fibers in PtK1 cells: a possible role for an actomyosin system in mitosis

Summary. The enzymes of importance in moving chromosomes are called motor proteins and include dynein, kinesin, and possibly myosin II. These three molecules are all included in the category of ATPases, in that they have the ability to convert chemical energy into mechanical energy. Both dynein and kinesin have been documented as molecules that “walk” along microtubules in the mitotic spindle, carrying cargo such as chromosomes. Myosin II, analogous to the muscle contraction system, transiently interacts along actin filaments and associates with kinetochore microtubules. In this paper we present evidence that a third ATPase, myosin II, may act as a “thruster” to propel chromosomes during the mitotic process. Double-label immunocytochemistry to actin and myosin II shows that myosin II is localized on chromosome arms at the beginning of mitosis and remains localized to the chromosomes throughout mitosis. Specific staining of myosin II is relegated to the outside of chromosomes with the highest density of staining occurring between the spindle poles and the chromosomes. This specific localization could account for the movement of chromosomes during mitosis, since they segregate towards the spindle poles, along kinetochore microtubules containing actin filaments, after aligning at the equatorial region of the cell at metaphase. We conclude from this study that there is an actomyosin system present in the mitotic spindle and that myosin is attached to chromosome arms and may act as a thruster in moving chromosomes during the mitotic process. Correspondence and reprints: Department of Biological Sciences, University of Denver, 2190 E Iliff Avenue, Denver, CO 80208, U.S.A.     

Striated microfilament bundles attaching to the plasma membrane of cytoplasmic bridges connecting spermatogenic cells in the black snail, Semisulcospira libertina (Mollusca, Mesogastropoda)

Striated microfilament bundles attaching to the plasma membrane of cytoplasmic bridges between spermatogenic cells are described in the black snail, Semisulcospira libertina. The bundles were occasionally observed in bridges connecting spermatogonia, spermatocytes and typical spermatids. Relations between bundles and centrioles could not be detected. The bundle had electron dense cross bands with a periodicity of approximately 200 nm, and attached to the membrane with almost right angle at the cross linker level. Phalloidin cytochemistry revealed that the bundle contained F-actin. In a case, a bundle connected two cytoplasmic bridges.     

Actin microfilaments in fungi

The cytoskeletal protein actin is among the most abundant proteins in nature. It is almost ubiquitous, occurring in all eukaryotes and in an ancestral form in prokaryotes. Actin monomers can polymerise to form microfilaments, structures that play a critical role in a number of fundamental cell processes in fungi such as morphogenesis, cytokinesis and the movement of organelles. Microfilaments are extremely dynamic structures and can be rapidly modified through their interactions with a number of actin binding proteins (ABPs). The purpose of the following review is to introduce actin and microfilaments in fungi to a general mycological audience and to provide a basic framework from which further study is possible.     

Sites of microtubule reorganization in tobacco BY-2 cells during cell-cycle progression

Summary The sites of microtubule (MT) reorganization were examined in synchronized tobacco BY-2 cells. The MTs of these cells were completely destroyed by a combined cold and drug treatment at 0 °C with 100 M propyzamide for 3 h. After the cells were washed and cultured at 30 °C, the reorganization of MTs was observed in detail. Sites for MT reorganization at each stage of the cell cycle were identified on the cell cortex and nuclei, the mitotic apparatus, the nuclei (or the nuclei and cell cortex), and the cell cortex in the S-G₂ phase, M phase, M/G₁ interface, and g₁ phase, respectively. The polypeptide synthesis elongation factor (EF)-1 is co-localized with these sites of MT reorganization. At some stages, microfilaments (MFs) were found to be involved in the reorganization of MTs. Based on these results, the mode of MT reorganization during cell cycle progression is discussed.Abbreviations EF-1 elongation factor 1 - MAP microtubule-associated protein - MF microfilament - MIs mitotic indices - MT microtubule     

The role of the cytoskeleton in the formation of gap junctions by Connexin 30

Mutations in the genes that encode Connexin 26 (GJB2) and Connexin 30 (GJB6) are the most common known cause of hereditary nonsyndromic sensorineural deafness. Cx26 and Cx30 share a similar protein structure, as well as the same expression distribution pattern in the cochlea. Cx26 has different intracellular trafficking properties compared to those of Cx43 and Cx32, whose trafficking manner is consistent with the classical membrane protein secretory pathway. Until now, however, the trafficking patterns of Cx30 have not been studied. By means of an immunofluorescence staining approach, we found that the targeting of Cx30 to gap junctions in transfected HeLa cells is not affected by brefeldin A, suggesting a Golgi-independent feature, similar to Cx26. Nocodazole had a minimal effect on assembly and distribution of Cx30 gap junctions. Cytochalasin B-induced actin filament depolymerization, however, affected both the pattern and the distribution of Cx30 gap junctions. Co-localization with and/or interaction between Cx30 and microtubules and cortical actin filaments, but not with the tight/adherens junction protein ZO-1, was confirmed by immunofluorescence and/or immunoprecipitation methods. The results suggest that the cytoskeleton, and especially actin filaments, are important components in the processes of assembly, trafficking and stabilization of Cx30 gap junctions.     

Actin cytoskeleton in intact and wounded coenocytic green algae

Summary The subcellular distribution of actin was investigated in two related species of coenocytic green algae, with immunofluorescence microscopy. Either no, or fine punctate fluorescence was detected in intact cells of Ernodesmis verticillata (Kützing) Børgesen and Boergesenia forbesii (Harvey) Feldmann. A reticulate pattern of fluorescence appears throughout the cortical cytoplasm of Ernodesmis cells shortly after wounding; this silhouettes chloroplasts and small vacuoles. Slender, longitudinal bundles of actin become evident in contracting regions of the cell, superimposed over the reticulum. Thicker portions of the bundles were observed in well-contracted regions, and the highly-convoluted appearance of nearby cortical microtubules indicates contraction of the bundles in these thicker areas. Bundles are no longer evident after healing; only the reticulum remains. In Boergesenia, a wider-mesh reticulum of actin develops in the cortex of wounded cells, which widens further as contractions continue. Cells wounded in Ca²⁺-free medium do not contract, and although the actin reticulum is apparent, no actin bundles were ever observed in these cells. Exogenously applied cytochalasins have no effect on contractions of cut cells or extruded cytoplasm, and normal actin-bundle formation occurs in treated cells. In contrast, erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) completely inhibits longitudinal contractions in wounded cells, and few uniformly slender actin bundles develop in inhibited cells. These results indicate that wounding stimulates a Ca²⁺-dependent, hierarchical assembly of actin into bundles, whose assembly and functioning are inhibited by EHNA. Contraction of the bundles and concomitant wound healing are followed by cessation of motility and disassembly of the bundles. The spatial and temporal association of the bundles with regions of cytoplasmic contraction, indicates that the actin bundles are directly involved in wound-induced cytoplasmic motility in these algae.Abbreviations EHNA erythro-9-[3-(2-hydroxynonyl)]adenine - MT(s) microtubule(s)     

Nitric oxide blocks the cell cycle of mouse macrophage-like cells in the early G2+M phase

The effects of nitric oxide produced by macrophage-like cells (Mml) on the cell cycle were investigated. Mml cells lost proliferative activity in the presence of interleukin-6 (IL-6) and a subpopulation accumulated in the G₂+ M phase. This level increased in proportion to the incubation time. The DNA content of the cells was slightly lower than that of Mml cells treated with vinbrastine or demecolcine, drugs which block the cell cycle in the M phase. The peak of the early G₂+M phase was reduced by treatment with N^G-mono-methyl- -arginine. However, after treatment with exogenous nitric oxide or sodium nitroprusside, the G₀/G₁ phase increased, but the early-G₂+M and the S phase decreased. The flow cytometry pattern in IL-6-treated Mml was the same as that of cytochalasin B-treated Mml. These data suggest that endogenous nitric oxide affects the microfilament system of IL-6-treated Mml cells and blocks the cell cycle in the early G₂+M phase.     

Reorganization of the cortical cytoskeleton in tip-growing fern protonemal cells during phytochrome-mediated phototropism and blue light-induced apical swelling

Summary Circular arrays of cortical microtubules (MTs) and microfilaments (MFs) are found in the subapical region of tip-growing protonemal cells of the fernAdiantum capillus-veneris. Reorganization of the two cytoskeletal structures during phytochrome-mediated phototropism and blue light-induced apical swelling was investigated by double-staining of MTs and MFs with rhodaminephalloidin and an indirect immunofluorescence method with tubulinspecific antibody. Before any growth responses were detectable, the MF and MT structures were reorganized according to similar patterns in both photoresponses, that is, oblique orientation and transient disappearance of the structures occurred during the phototropic response, and the disappearance of the structures occurred during apical swelling. The reorganization of MF structures clearly preceded that of the MT structures in the phototropic response. In the case of apical swelling, both types of circular array disappeared with an almost identical time course.These results provide evidence for the significant role of the circular organization of MFs as well as of MTs, in the light-induced growth responses of tip-growing fern protonemal cells. Possible roles of the circular array of MFs in the regulation of tip growth are discussed.Abbreviations DMSO dimethylsulfoxide - PIPES piperazine-N,N-bis(2-ethane-sulfonic acid) - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetraacetic acid - PMSF phenylmethylsulfonyl fluoride - MF microfilament - MT microtubule - Rh-Phal rhodaminelabeled phalloidin     

细胞松驰素B对牛传染性鼻气管炎病毒感染与复制的影响

体外培养的牛肾细胞,经浓度为50μg/ml的细胞松弛素B(CB)处理2小时后,牛传染性鼻气管炎病毒(IBRV)仍能侵入细胞,繁殖的子代病毒数量不受影响。然而当同样浓度的CB在病毒的整个繁殖期中持续存在时,病毒的繁殖即完全被抑制。说明细胞的吞噬作用至少不是这种病毒进入细胞的唯一方式,可能病毒囊膜与细胞膜的融合是病毒进入细胞的主要方式;而在侵入细胞后的病毒繁殖过程中,细胞微丝和其它对CB敏感的系统起着不可缺少的作用。降解微丝对IBRV的早期发生有明显的影响,其效应强度与CB的浓度有关。还观察到CB处理对病毒形态发生方式有明显影响。     

Localization of a 170 kDa myosin heavy chain in plant cells

Summary A polyclonal antibody directed against a 170 kDa myosin heavy chain from lily pollen tubes was employed to (a) assess the cellular distribution of the polypeptide using immunofluorescence methods, and (b) ascertain if similar polypeptides are present in pollen tubes and somatic cells of other species. Fluorescence is associated with particles of various size as well as an amorphous component, and is concentrated in the apical cytoplasm of lily and tobacco pollen tubes. Apical fluorescence is more extensive in lily than in tobacco, which may be related to different streaming patterns and apical zonation seen at the ultrastructural level. In suspension cells of tobacco andArabidopsis, fluorescence is concentrated around the nuclei. Dual localizations indicate that anti-myosin fluorescence may be associated with the presence of actin. Little or no staining was seen in controls consisting of either pre-immune serum or mono-specific IgG that had been preadsorbed with the 170 kDa polypeptide. Immunoblots show that a 170 kDa immunoreactive polypeptide is present in pollen tubes of tobacco andTradescantia virginiana in addition to lily, and in suspension culture cells of tobacco andArabidopsis and extracts of wholeArabidopsis seedlings. Our results show that a conserved 170 kDa myosin heavy chain is present in a variety of monocot and dicot cells. They are also consistent with the presence of multiple myosins in plants in general and pollen tubes in particular.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - Mf microfilament - Mt microtubule - PBS phosphate-buffered saline - PME 50 mM Pipes, 5mM EGTA - 2mM MgSO₄, pH6.9.