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  • Freeze substitution  (8)
  • Cell division  (2)
  • Funaria  (2)
  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Protoplasma 131 (1986), S. 153-165 
    ISSN: 1615-6102
    Keywords: Freeze substitution ; Hafnium tetrachloride ; Plant cell ultrastructure ; Rapid freeze fixation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We describe here an apparatus that permits rapid freeze fixation of whole cells, which are then prepared by freeze substitution and resin embedment for examination in the EM. The freezing device utilizes a rotary solenoid that rapidly plunges the specimen holder, a formvar-film-covered thin wire loop, into a well of stirred liquid propane at −180
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Protoplasma 150 (1989), S. 72-74 
    ISSN: 1615-6102
    Keywords: Actin ; Cytoskeleton ; Freeze substitution ; Hyperbaric freezer ; Microfilaments ; Rapid freeze fixation ; Immunogold localization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have successfully combined the superior ultrastructural preservation capabilities of rapid freeze fixation and freeze substitution (RF-FS) with immunogold antibody localization techniques to label microfilament (MF) bundles with monoclonal antibody to actin in two different plant tissues:Nicotiana pollen tubes andDrosera tentacles. We have thus verified that the extensive MF bundles seen in these cells after RF-FS are composed of actin, a protein that is difficult to preserve by conventional fixation methods for electron microscopy.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Protoplasma 131 (1986), S. 211-223 
    ISSN: 1615-6102
    Keywords: Bud formation ; Cytokinin ; Funaria ; Morphometry ; Polarity ; Tip growth
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Cytokinin stimulates caulonemata ofFunaria to undergo an asymmetric division leading to the gametophore. The earliest detectable event is a small protuberance at the distal portion of the cell accompanied by the reorganization of the underlying organelles into a polarized distribution reminiscent of a tip growing cell. Dictyosomes and associated vesicles accumulate in the protuberance directly beneath the plasma membrane with mitochondria subjacent to the vesicular layer. Endoplasmic reticulum lies beneath the mitochondrial zone directly above the large central vacuole, while chloroplasts are outside the bud. As development continues the bud elongates causing the outer cell wall to exfoliate. During the above events the nucleus migrates toward the bud site concomitant with an increase in the number of microtubules between the nucleus and the base of the outgrowth. Nucleoli, extruded from the nucleus during a previous division, persist as diffuse fragments within the protuberance. Upon reaching the bud site, division occurs with the developing phragmoplast being initiated distal to the caulonema tip cell. The former polarized distribution of the cytoplasm is altered as mitochondria, chloroplasts and small vacuoles become evenly dispersed throughout the cytoplasm; dicytosomes and endoplasmic reticulum occupy a cortical position. These events indicate a change from 2-D tip growth to 3-D diffuse growth. To quantify the ultrastructural changes associated with bud formation we performed a morphometric analysis of cells in various stages of budding. The relative volumes of dictyosomes and vesicles adjacent to the bud apex decrease during bud development coincident with an increase in these organelles in lower portions of the cytoplasm. Mitochondria and chloroplasts follow this same pattern although their highest relative volumes initially are 4 μm from the bud apex and outside the bud site, respectively. These data, as well as density profile topographic maps for vesicle fractions, support the contention that cytokinin induces a change in morphological symmetry and polarity in the fine structure ofFunaria.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Protoplasma 201 (1998), S. 158-171 
    ISSN: 1615-6102
    Keywords: Accessible volume ; Calmodulin ; Cell division ; Fixation ; Fluorescent-analog cytochemistry ; Tradescantia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Because the activity of calmodulin (CaM) may be dependent upon its structural distribution, we have examined its spatial localization in living cells. We have focused on cell division and cell plate formation, where conventional immunofluorescence studies report that CaM is specifically associated with microtubules (MTs) of the spindle and the phragmoplast. In dividing stamen hair cells ofTradescantia virginiana that were injected with fluorescently labeled CaM and examined by confocal laser scanning microscopy (CLSM), we found that the labeled protein is uniformly distributed throughout the cell and is not localized with the phragmoplast MTs or any other obvious structure. To explore why these images from live cells differ from those prepared by immunolabeling, we investigated the fate of CaM during fixation and compared it with the localization of fixable dextran and tubulin. The results show that fixation causes severe changes in cell morphology and in the distribution of CaM and dextran in three quarters of the cells. Conversely, injected rhodamine-tubulin did not show redistribution after fixation. We conclude that in the live cell, CaM is largely uniformly distributed throughout the cytoplasm, and secondly that conventional chemical fixation does not preserve CaM, and probably many other soluble proteins, in its in vivo distribution. The role postulated for CaM in mitosis, solely based on indirect immunofluorescence microscopy, has to be re-evaluated.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Protoplasma 155 (1990), S. 116-126 
    ISSN: 1615-6102
    Keywords: Actin ; Cytoplasmic streaming ; Drosera ; Endoplasmic reticulum ; Freeze fixation ; Freeze substitution ; Hyperbaric freezing ; High pressure freezing ; Immunogold localization ; Microfilaments ; Plasmalemma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In epidermal cells ofDrosera tentacles that have been preserved for ultrastructural analysis through high pressure freeze fixation and freeze substitution we describe the frequent occurrence of microfilament (MF)-endoplasmic reticulum (ER) complexes. These are found throughout the cytoplasm where they are observed in close association with the plasmalemma (PL), the tonoplast, nuclei, mitochondria, chloroplasts, and microbodies. The MF component of the complexes is identified as actin based on immunogold labelling with actin antibodies. The actin-ER complexes are prominent in the cortical cytoplasm. In this region a network of predominantly tubular ER occupies an intermediary position in which it associates closely with both the PL and the actin MFs. We suggest that the ER, especially those elements adjacent to the PL in the cortical cytoplasm, stabilizes the actin MFs and provides the necessary anchor against which the forces for cytoplasmic streaming are generated.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Protoplasma 169 (1992), S. 168-178 
    ISSN: 1615-6102
    Keywords: Cortical ultrastructure ; Cytoskeleton ; Freeze-substitution ; Endoplasmic reticulum ; Funaria
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The ultrastructural organization of the cortical cytoplasm has been examined in caulonemata, branches and buds of the mossFunaria hygrometrica, which were prepared by rapid freeze-fixation and freeze-substitution (FS). The same structural components occur in the cortex of all three cell types: microtubules (MTs), endoplasmic reticulum (ER), coated and uncoated vesicles, coated pits, and dictyosomes. However, the configuration and density of the cortical ER varies between the three. Caulonemata have an open, polygonal network of ER associated with long MTs oriented mostly parallel to the length of the cell. Lamellar ER, covered with polysomes, is interspersed in the network. Branches have a more tightly arranged ER network, at places occurring in a thick layer, and occasional polysome-decorated lamellae. MTs, which extend to the tip of the branch, are oriented mainly parallel to the cell's long axis and are associated with the cortical ER. Buds have the tightest ER network, which is frequently arranged in a thick layer. Tubules in the polygonal ER of buds are densely covered with ribosomes, whereas tubules in the ER network of caulonemata and branches range from nearly smooth to moderately rough. Closely-spaced ER lamellae, with many polysomes, occur in some buds. The MTs of buds extend into the apical dome and are associated with the cortical ER, but are more randomly oriented than in caulonemata or branches. Close appositions between the ER and PM are observed in all three cells, but are more frequent in buds.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1615-6102
    Keywords: Actin ; Cytoskeleton ; Freeze substitution ; Immunogold labeling ; Microfilaments ; Microtubules ; Pollen tubes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary It is well known that an extensive array of actin microfilament (MF) bundles exists in the cytoplasm of pollen tubes and that it plays an important role in cytoplasmic streaming in these cells. Less well documented or understood is a cortical MF system, which occurs in two forms: single fine filaments running the length of the cortical microtubules (MTs), and MF bundles. In the present study we have utilized double immunogold labeling of tubulin and actin in rapidly frozen and freeze-substituted pollen tubes ofNicotiana alata in an attempt to clarify the distribution and association of these cytoskeletal proteins. We find that both antibodies bind to antigens associated with cortical MTs, while generative cell MTs label only with the tubulin antibody. Bundles of MFs that show a clear reaction with anti-actin are often seen associated with the cortical MTs, but it remains unclear if the single MT-associated MFs are labeled, and thus, if they are composed of actin. Nevertheless, a majority of cortical MTs show a close association with actin and it is possible that these MTs act as guide elements for MF bundles.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1615-6102
    Keywords: High pressure freezing ; Plunge freezing ; Freeze substitution ; Phytophthora ; Sporangia ; Immunogold labelling
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The effects of high pressure on the ultrastructure of sporangia ofPhytophthora cinnamomi andP. palmivora have been examined by comparing sporangia frozen in a Balzers hyperbaric freezer or pressurized in a French pressure cell with sporangia plunge frozen at ambient pressure. Both freeze fixation methods provided excellent preservation of most cell structures, but one organelle type seen in plunge frozen material, the large peripheral vesicle (LPV), was not observed in high pressure frozen sporangia. Instead, these sporangia contained large irregularly shaped structures which exhibit the patterns of spatial distribution and, forP. cinnamomi, the monoclonal antibody binding characteristic of LPVs. These findings suggest that some factor of the hyperbaric freezing process causes LPVs to be degraded. Sporangia ofP. cinnamomi that had been pressurized in a French pressure cell also exhibited large structures with the spatial distribution and monoclonal antibody binding characteristic of LPVs. The apparent expansion of LPVs that follows from both pressurizing treatments causes considerable passive disruption of sporangial structure. This is the first report of a major disturbance of cell structure from use of the Balzers hyperbaric freezer, and reflects the lability, noted in previous work, of LPVs inPhytophthora.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1615-6102
    Keywords: Caffeine ; Freeze substitution ; Lilium ; Pollen tubes ; Rapid freeze fixation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In an attempt to correlate structural effects with the known dissipation of the tip-focused Ca2+ gradient caused by caffeine, we have examined the ultrastructure of caffeine-treated lily pollen tubes prepared by rapid freeze fixation and freeze substitution. We show that treatment with caffeine results in a rapid rearrangement of secretory vesicles at the pollen tube tip; the normal cone-shaped array of vesicles is rapidly dispersed. In addition, microfilament bundles appear in the tip region, where they had previously been excluded. Delocalized vesicle fusion continues in the presence of caffeine but tube extension ceases. Removal of caffeine from the growth medium initially causes tip swelling, delocalized vesicle fusion and presence of microfilaments well into the tip before normal structure and growth resume, concurrent with the previously reported return to a normal Ca2+ gradient.
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Protoplasma 140 (1987), S. 141-150 
    ISSN: 1615-6102
    Keywords: Cytoskeleton ; Freeze substitution ; Nicotiana ; Pollen tubes ; Rapid freeze fixation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The ultrastructure of the cytoskeleton inNicotiana alata pollen tubes grownin vitro has been examined after rapid freeze fixation and freeze substitution (RF-FS). Whereas cytoplasmic microtubules (MTs) and especially microfilaments (MFs) are infrequently observed after conventional chemical fixation, they occur in all samples prepared by RF-FS. Cortical MTs are oriented parallel to the long axis of the pollen tube and usually appear evenly spaced around the circumference of the cell. They are always observed with other components in a structural complex that includes the following: 1. a system of MFs, in which individual elements are aligned along the sides of the MTs and crossbridged to them; 2. a system of cooriented tubular endoplasmic reticulum (ER) lying beneath the MTs, and 3. the plasma membrane (PM) to which the MTs appear to be extensively linked. The cortical cytoskeleton is thus structurally complex, and contains elements such as MFs and ER that must be considered together with the MTs in any attempt to elucidate cytoskeletal function. MTs are also observed within the vegetative cytoplasm either singly or in small groups. Observations reveal that some of these may be closely associated with the envelope of the vegetative nucleus. MTs of the generative cell, in contrast to those of the vegetative cytoplasm, occur tightly clustered in bundles and show extensive cross-bridging. These bundles, especially in the distal tail of the generative cell, are markedly undulated. MFs are observed commonly in the cytoplasm of the vegetative cell. They occur in bundles oriented predominantly parallel to the pollen tube axis. Although proof is not provided, we suggest that they are composed of actin and are responsible for generating the vigorous cytoplasmic streaming characteristic of living pollen tubes.
    Type of Medium: Electronic Resource
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