scholarly journals Immunochemical and immunocytochemical identification of a myosin heavy chain polypeptide in Nicotiana pollen tubes

1989 ◽  
Vol 92 (4) ◽  
pp. 569-574
Author(s):  
X.J. Tang ◽  
P.K. Hepler ◽  
S.P. Scordilis
Keyword(s):  
Pollen Tube ◽  
Nuclear Envelope ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Immunofluorescence Microscopy ◽  
Generative Cell ◽  
Pollen Tubes ◽  
Nuclear Surface ◽  
Common Pattern ◽  
Western Blots

A myosin heavy chain polypeptide has been identified and localized in Nicotiana pollen tubes using monoclonal anti-myosin antibodies. The epitopes of these antibodies were found to reside on the myosin heavy chain head and rod portion and were, therefore, designated anti-S-1 (myosin S-1) and anti-LMM (light meromyosin). On Western blots of the total soluble pollen tube proteins, both anti-S-1 and anti-LMM label a polypeptide of approximately 175,000 Mr. Immunofluorescence microscopy shows that both antibodies yield numerous fluorescent spots throughout the whole length of the tube, often with an enrichment in the tube tip. These fluorescent spots are thought to represent vesicles and/or organelles in the pollen tubes. In addition to this common pattern, anti-S-1 stains both the generative cell and the vegetative nuclear envelope. The different staining patterns of the nucleus between anti-S-1 and anti-LMM may be caused by some organization and/or anchorage state of the myosin molecules on the nuclear surface that differs from those on the vesicles and/or organelles.

Citrus pollen tube development in cross-compatible gynoecia, self-incompatible gynoecia, and in vitro

1988 ◽  
Vol 66 (12) ◽  
pp. 2527-2532 ◽  
Author(s):  
Tracy L. Kahn ◽  
Darleen A. DeMason
Keyword(s):  
Pollen Tube ◽  
Generative Cell ◽  
Pollen Tubes ◽  
Citrus Paradisi ◽  
Incompatible Pollination ◽  
Incompatible Cross ◽  
Compatible Pollinations ◽  
Compatible Pollen ◽  
Incompatibility Reaction

The route of 'Orlando' tangelo (Citrus paradisi Macf. × C. reticulata Blanco) pollen tubes was traced and compared in self-incompatible pollinations and cross-compatible pollinations with 'Dancy' tangerine (C. reticulata Blanco). In both crosses, 'Orlando' pollen germinated in the stigmatic exudate and grew between the papillae on the stigma surface and inter-cellularly between the parenchymatous cells until reaching a stylar canal by 3 days. However, in the incompatible pollination, irregular deposition of callose occurred in the pollen tube walls as early as 1 day after pollination. By day 6, pollen tubes were in the upper portion of the ovary in the compatible pollination, whereas most pollen tubes from the incompatible pollination were still in the upper style. 'Orlando' pollen tube growth rate decreased substantially by day 3 in both the self-incompatible pollination and in vitro but increased rapidly after day 3 in the compatible combination. The generative cell divided between 1 and 3 days after pollination in the compatible cross. Generative cell division was observed by day 3 in only a few pollen tubes in the incompatible cross and in cultured tubes. Compatible pollen tubes grew slowly for the first 3 days after pollination, during which time generative cells divided and then grew rapidly until fertilization. In contrast, incompatible pollen tubes showed morphological features indicative of an incompatibility reaction by 1 day after pollination and grew slowly for a period of 6 days, and then ceased growth.

scholarly journals An unconventional myosin heavy chain gene from Drosophila melanogaster.

1992 ◽  
Vol 119 (4) ◽  
pp. 823-834 ◽  
Author(s):  
K A Kellerman ◽  
K G Miller
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Cytoskeletal Proteins ◽  
Protein Isoforms ◽  
Actin Binding ◽  
Chain Gene ◽  
Cdna Clones ◽  
Western Blots ◽  
Unconventional Myosin ◽  
Amino Terminal

As part of a study of cytoskeletal proteins involved in Drosophila embryonic development, we have undertaken the molecular analysis of a 140-kD ATP-sensitive actin-binding protein (Miller, K. G., C. M. Field, and B. M. Alberts. 1989. J. Cell Biol. 109:2963-2975). Analysis of cDNA clones encoding this protein revealed that it represents a new class of unconventional myosin heavy chains. The amino-terminal two thirds of the protein comprises a head domain that is 29-33% identical (60-65% similar) to other myosin heads, and contains ATP-binding, actin-binding and calmodulin/myosin light chain-binding motifs. The carboxy-terminal tail has no significant similarity to other known myosin tails, but does contain a approximately 100-amino acid region that is predicted to form an alpha-helical coiled-coil. Since the unique gene that encodes this protein maps to the polytene map position 95F, we have named the new gene Drosophila 95F myosin heavy chain (95F MHC). The expression profile of the 95F MHC gene is complex. Examination of multiple cDNAs reveals that transcripts are alternatively spliced and encode at least three protein isoforms; in addition, a fourth isoform is detected on Western blots. Developmental Northern and Western blots show that transcripts and protein are present throughout the life cycle, with peak expression occurring during mid-embryogenesis and adulthood. Immunolocalization in early embryos demonstrates that the protein is primarily located in a punctate pattern throughout the peripheral cytoplasm. Most cells maintain a low level of protein expression throughout embryogenesis, but specific tissues appear to contain more protein. We speculate that the 95F MHC protein isoforms are involved in multiple dynamic processes during Drosophila development.

scholarly journals Pollen performance before and during the autotrophic–heterotrophic transition of pollen tube growth

2003 ◽  
Vol 358 (1434) ◽  
pp. 1009-1018 ◽  
Author(s):  
Andrew G. Stephenson ◽  
Steven E. Travers ◽  
Jorge I. Mena-Ali ◽  
James A. Winsor
Keyword(s):  
Pollen Tube ◽  
Pollen Tube Growth ◽  
Generative Cell ◽  
Pollen Tubes ◽  
Tube Growth ◽  
Pollen Competition ◽  
Heritable Variation ◽  
Pollen Selection ◽  
Pollen Performance ◽  
New Findings

For species with bicellular pollen, the attrition of pollen tubes is often greatest where the style narrows at the transition between stigmatic tissue and the transmitting tissue of the style. In this region, the tubes switch from predominantly autotrophic to predominantly heterotrophic growth, the generative cell divides, the first callose plugs are produced, and, in species with RNase–type self–incompatibility (SI), incompatible tubes are arrested. We review the literature and present new findings concerning the genetic, environmental and stylar influences on the performance of pollen before and during the autotrophic–heterotrophic transition of pollen tube growth. We found that the ability of the paternal sporophyte to provision its pollen during development significantly influences pollen performance during the autotrophic growth phase. Consequently, under conditions of pollen competition, pollen selection during the autotrophic phase is acting on the phenotype of the paternal sporophyte. In a field experiment, using Cucurbita pepo , we found broad–sense heritable variation for herbivore–pathogen resistance, and that the most resistant families produced larger and better performing pollen when the paternal sporophytes were not protected by insecticides, indicating that selection during the autotrophic phase can act on traits that are not expressed by the microgametophyte. In a study of a weedy SI species, Solanum carolinense , we found that the ability of the styles to arrest self–pollen tubes at the autotrophic–heterotrophic transition changes with floral age and the presence of developing fruits. These findings have important implications for selection at the level of the microgametophyte and the evolution of mating systems of plants.

Effect of unloading on type I myosin heavy chain gene regulation in rat soleus muscle

2005 ◽  
Vol 98 (4) ◽  
pp. 1185-1194 ◽  
Author(s):  
Julia M. Giger ◽  
Fadia Haddad ◽  
Anqi X. Qin ◽  
Ming Zeng ◽  
Kenneth M. Baldwin
Keyword(s):  
Myosin Heavy Chain ◽  
Soleus Muscle ◽  
Heavy Chain ◽  
Regulatory Elements ◽  
Hindlimb Suspension ◽  
Type I ◽  
Transcriptional Enhancer ◽  
Western Blots ◽  
E Box ◽  
Enhancer Factor

Slow-twitch soleus, a weight-bearing hindlimb muscle, predominantly expresses the type I myosin heavy chain (MHC) isoform. However, under unloading conditions, a transition in MHC expression occurs from slow type I toward the fast-type isoforms. Transcriptional processes are believed to be involved in this adaptation. To test the hypothesis that the downregulation of MHC1 in soleus muscle following unloading is controlled through cis element(s) in the proximal region of the promoter, the MHC1 promoter was injected into soleus muscles of control rats and those subjected to 7 days of hindlimb suspension. Mutation analyses of six putative regulatory elements within the −408-bp region demonstrated that three elements, an A/T-rich, the proximal muscle-type CAT (βe3), and an E-box (−63 bp), play an important role in the basal level of MHC1 gene activity in the control soleus and function as unloading-responsive elements. Gel mobility shift assays revealed a diminished level of complex formation of the βe3 and E-box probes with nuclear extract from hindlimb suspension soleus compared with control soleus. Supershift assays indicated that transcriptional enhancer factor 1 and myogenin factors bind the βe3 and E-box elements, respectively, in the control soleus. Western blots showed that the relative concentrations of the transcriptional enhancer factor 1 and myogenin factors were significantly attenuated in the unloaded soleus compared with the control muscle. We conclude that the downregulation of MHC1 in response to unloading is due, in part, to a significant decrease in the concentration of these transcription factors available for binding the positive regulatory elements.

Heterogeneity of myosin heavy-chain expression in fast-twitch fiber types of mature avian pectoralis muscle

1996 ◽  
Vol 74 (5) ◽  
pp. 715-728 ◽  
Author(s):  
B. W. C. Rosser ◽  
D. M. Waldbillig ◽  
M. Wick ◽  
E. Bandman
Keyword(s):  
Monoclonal Antibodies ◽  
Myosin Heavy Chain ◽  
Muscle Fiber ◽  
Heavy Chain ◽  
Fiber Types ◽  
Pectoralis Muscle ◽  
Western Blots ◽  
Myhc Isoforms ◽  
Fast Twitch ◽  
Myhc Expression

The aims of this study are to investigate the diversity of myosin heavy-chain (MyHC) expression among avian fast-twitch fibers, and to test the hypothesis that dissimilar MyHC isoforms are found in each of the principal avian fast-twitch fiber types. MyHCs within the muscle fibers of the pectoralis of 31 species of bird are characterized using immunocytochemical methods. A library of 11 monoclonal antibodies previously produced against chicken MyHCs is used. The specificity of these antibodies for MyHCs in each of the muscles studied is confirmed by Western blots. The results show that avian fast-twitch glycolytic fibers and fast-twitch oxidative-gylcolytic fibers can contain different MyHCs. Among the species studied, there is also a conspicuous variety of MyHC isoforms expressed. In addition, the results suggest that two epitopes are restricted to chickens and closely allied gallinaceous birds. There are no apparent correlations between MyHC epitope and presupposed contractile properties. However, the presence of different isoforms in different fast-twitch fiber types suggests a correlation between isoform and contractile function.Key words: muscle, fiber, myosin, avian.

scholarly journals A myosin heavy-chain-like polypeptide is associated with the nuclear envelope in higher eukaryotic cells.

1986 ◽  
Vol 103 (3) ◽  
pp. 711-724 ◽  
Author(s):  
M Berrios ◽  
P A Fisher
Keyword(s):  
Nuclear Envelope ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Nuclear Periphery ◽  
Cell Nuclei ◽  
Higher Plant ◽  
Atpase Subunit ◽  
Nuclear Lamins

A high molecular weight polypeptide, identified as an ATPase subunit by direct ultraviolet photoaffinity labeling, has been shown to be a component of nuclear envelope-enriched fractions prepared from a variety of higher eukaryotes (Berrios, M., G. Blobel, and P. A. Fisher, 1983, J. Biol. Chem., 258:4548-4555). In rat liver as well as Drosophila melanogaster embryos, this polypeptide appears to be a form of myosin heavy chain. This conclusion is based on both immunochemical and immunocytochemical data, as well as on the results of CNBr and chymotryptic peptide map analyses. In Drosophila, the identification of this myosin heavy chain-like polypeptide as a nuclear envelope component has been corroborated in situ by indirect immunofluorescence analyses using permeabilized whole cells, mechanically extruded nuclei, and cryosections obtained from a number of larval tissues. Localization appears to be restricted to the nuclear periphery in a manner similar to that observed for the nuclear lamins and the pore complex glycoprotein. Antibodies directed against the Drosophila nuclear envelope ATPase have also been shown to decorate mammalian and higher plant cell nuclei in situ. Implications for intracellular nuclear mobility and for nucleocytoplasmic exchange of macromolecules in vivo are discussed.

Identification and localization of three classes of myosins in pollen tubes of Lilium longiflorum and Nicotiana alata

1995 ◽  
Vol 108 (7) ◽  
pp. 2549-2563
Author(s):  
D.D. Miller ◽  
S.P. Scordilis ◽  
P.K. Hepler
Keyword(s):  
Pollen Tube ◽  
Myosin Ii ◽  
Generative Cell ◽  
Freeze Substitution ◽  
Lilium Longiflorum ◽  
Pollen Tubes ◽  
Vegetative Nucleus ◽  
Nicotiana Alata ◽  
Myosin V ◽  
Myosin I

The presence and localization of actin and myosin have been examined in pollen tubes of Lilium longiflorum and Nicotiana alata. Immunoblot analysis of pollen tube extracts with antibodies to actin, myosins IA and IB, myosin II, and myosin V reveals the presence of these contractile proteins. Immunofluorescence microscopy using various methods to preserve the pollen tubes; chemical fixation, rapid freeze fixation and freeze substitution (RF-FS) followed by rehydration or by embeddment in a methacrylate mixture, was performed to optimize preservation. Immunocytochemistry reaffirmed that actin is localized longitudinally in the active streaming lanes and near the cortical surface of the pollen tube. Myosin I was localized to the plasma membrane, larger organelles, the surface of the generative cell and the vegetative nucleus, whereas, myosin V was found in the vegetative cytoplasm in a punctate fashion representing smaller organelles. Myosin II subfragment 1 and light meromyosin were localized in a punctate fashion on the larger organelles throughout the vegetative cytoplasm. In addition, isolated generative cells and vegetative nuclei labeled only with the myosin I antibody. Competition studies indicated the specificity of the heterologous antibodies utilized in this study suggesting the presence of three classes of myosins in pollen. These results lead to the following hypothesis: Myosin I may move the generative cell and vegetative nucleus unidirectionally through the pollen tube to the tip, while myosin V moves the smaller organelles and myosins I and II move the larger organelles (bidirectionally) that are involved in growth.

scholarly journals Localization of actin in pollen tubes of Ornithogalum virens L.

2014 ◽  
Vol 68 (2) ◽  
pp. 97-102
Author(s):  
Małgorzata Stępka ◽  
Fabricio Ciampolini ◽  
Mauro Cresti ◽  
Maria Charzyńska
Keyword(s):  
Pollen Tube ◽  
Actin Filaments ◽  
Laser Scanning ◽  
Higher Plants ◽  
Generative Cell ◽  
Pollen Tubes ◽  
Confocal Laser ◽  
Vegetative Nucleus ◽  
Ornithogalum Virens

The germinating pollen grain (in vivo on the stigma or in vitro in germination medium) forms a pollen tube which transports the vegetative nucleus and generative cell/two sperm cells participating in the process of double fertilization. The growth of the tube and the transport of organelles and the cells occur due to two major motor systems existing in the pollen tubes of higher plants: the tubuline-dynein/kinesin and the actin-myosin system. In pollen tubes of <em>Ornithogalum virens</em> the actin filaments were labelled with TRITC-phalloidin (2 µg/ml) in the PIPES buffer and the 10% sucrose, without the fixative and DMSO. Omission of the fixative and permeabilizing agent (DMSO) allowed better preservation of the structure, and the "fluorescence" of actin was observed in living pollen tubes. Observations in CLSM (confocal laser scanning microscope) showed that actin is distributed in the vicinity of the cell membrane. This could support the view that actin filaments and the plasmalemma form the pollen tube cortex along which the cytoplasmic movement of organelles, and cell transport occurs.

scholarly journals Nonmuscle Myosin Heavy Chain ⅡA-Mediated Exosome Release via Regulation of the Rho-Associated Kinase 1/Myosin Light Chains/Actin Pathway

2020 ◽  
Vol 11 ◽  
Author(s):  
Yanni Lv ◽  
Jin Chen ◽  
Jinfang Hu ◽  
Yisong Qian ◽  
Ying Kong ◽  
...  
Keyword(s):  
Protein Expression ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Molecular Motors ◽  
Molecular Motor ◽  
Microglial Cells ◽  
Potential Mechanism ◽  
Nonmuscle Myosin ◽  
Western Blots ◽  
Positive Rate

Nonmuscle myosin ⅡA, a kind of ATP-dependent molecular motor, binds actin to form the molecular motors of the cell. We found that interfering with nonmuscle myosin heavy chain (NMMHC) ⅡA could affect the exosome release from microglial cells stimulated by LPS. LPS could enhance exosome release from microglial cells by increasing exosome concentration, elevating the rate of positively labeled CD9 and CD81 proteins and protein expression. The myosin inhibitor, blebbistatin, could decrease the concentration of released exosome and reduce CD9 and CD81 protein expression on the exosome surface compared with that in the LPS group. To further determine the exact subtype of myosin Ⅱ responsible for these effects, we transfected microglial cells with siRNA for MYH9, MYH10, and MYH14. The data showed that only the transfection of siRNA-MYH9, but not MYH10 or MYH14 could decrease the released exosome concentration and particle size compared with those in the LPS group. siRNA-MYH9 would also weaken the CD9 and CD81 protein positive rate and protein expression compared with that in the LPS group by the quantification of CD9 and CD81 fluorescence intensities and by western blotting. Western blots and immunofluorescence assays indicated that NMMHC ⅡA might trigger the ROCK1/MLC/actin signaling pathway of microglial cells upon stimulation by LPS, which might be the potential mechanism of exosome release. These observations demonstrated that NMMHC ⅡA might be the potential target required for exosome release.

Detection and mapping of interactions between chromatin and the nuclear envelope in drosophila embryos

1995 ◽  
Vol 53 ◽  
pp. 764-765
Author(s):  
W.F. Marshall ◽  
A.F. Dernburg ◽  
B. Harmon ◽  
J.W. Sedat
Keyword(s):  
Nuclear Envelope ◽  
Chromatin Condensation ◽  
Three Dimensional ◽  
Physiological Role ◽  
Nuclear Surface ◽  
Intact Cells ◽  
Molecular Determinants ◽  
Surface Harmonic ◽  
Drosophila Embryos

Interactions between chromatin and nuclear envelope (NE) have been implicated in chromatin condensation, gene regulation, nuclear reassembly, and organization of chromosomes within the nucleus. To further investigate the physiological role played by such interactions, it will be necessary to determine which loci specifically interact with the nuclear envelope. This will not only facilitate identification of the molecular determinants of this interaction, but will also allow manipulation of the pattern of chromatin-NE interactions to probe possible functions. We have developed a microscopic approach to detect and map chromatin-NE interactions inside intact cells.Fluorescence in situ hybridization (FISH) is used to localize specific chromosomal regions within the nucleus of Drosophila embryos and anti-lamin immunofluorescence is used to detect the nuclear envelope. Widefield deconvolution microscopy is then used to obtain a three-dimensional image of the sample (Fig. 1). The nuclear surface is represented by a surface-harmonic expansion (Fig 2). A statistical test for association of the FISH spot with the surface is then performed.

Sign in / Sign up

Export Citation Format

Share Document