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  • Freeze substitution  (8)
  • High pressure freezing  (2)
  • 1
    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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  • 2
    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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  • 3
    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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  • 4
    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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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Protoplasma 167 (1992), S. 215-230 
    ISSN: 1615-6102
    Keywords: Lilium ; Freeze substitution ; Pollen tubes ; Rapid freeze fixation ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In view of the importance of the lily pollen tube as an experimental model and the improvements in ultrastructural detail that can now be attained by the use of rapid freeze fixation and freeze substitution (RF-FS), we have reexamined the ultrastructure of these cells in material prepared by RF-FS. Several previously unreported details have been revealed: (1) the cytoplasm is organized into axial “slow” and “fast” lanes, each with a distinct structure; (2) long, straight microtubule (MT) and microfilament (MF) bundles occur in the cytoplasm of the fast lanes and are coaligned with every organelle present; (3) the cortical cytoplasm contains complexes of coaligned MTs, MFs, and endoplasmic reticulum (ER); (4) the cortical ER is arranged in a tight hexagonal pattern and individual elements are closely appressed to the plasma membrane with no space between; (5) mitochondria and ER extend into the extreme apex along the flanks of the pollen tube, and vesicles and ER are packed into an inverted cone-shaped area at the center of the apex; (6) MF bundles in the tip region are fewer, finer, and in random orientation in comparison to those of the fast lanes; (7) the generative cell (GC) cell wall complex contains patches of plasmodesmata; (8) The GC cytoplasm contains groups of spiny vesicles that are closely associated with and seem to be fusing with or pinching off from mitochondria, and (9) the vegetative nucleus (VN) contains internal MT-like structures as well as numerous cytoplasmic MTs associated with its membrane and also located between the VN and GC.
    Type of Medium: Electronic Resource
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  • 6
    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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  • 7
    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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  • 8
    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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