Susan A. Lancelle

Ultrastructure of the cytoskeleton in freeze-substituted pollen tubes ofNicotiana alata

SummaryThe 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.

A method for rapid freeze fixation of plant cells

SummaryWe 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‡C. The rotary solenoid allows for an adjustable, repeatable immersion rate. Substitution takes place at −80 ‡C in acetone with 2% OsO4 and is followed by en bloc staining in either hafnium tetrachloride or uranyl acetate. We have utilized these techniques on plant cells, for which there has been relatively little published work when compared to other organisms. The results show that, with the versatile specimen holder and rapid, repeatable immersion rates, different cell types, including pollen, stamen hairs, and germinating moss spores, can be rapidly frozen with repeatable success. The improved preservation achieved with rapid freeze fixation over conventional chemical fixation reveals itself particularly in the structure of the plasmamembrane, the cytoskeleton, chromatin, and certain endomembrane systems.

Actin microfilaments do not form a dense meshwork inLilium longiflorum pollen tube tips

SummaryControversy over whether the apical region of a growing pollen tube contains a dense array of actin microfilaments (MFs) was the impetus for the present study. Microinjection of small amounts of fluorescently labeled phalloidin allowed the observation of MF bundles inLilium longiflorum pollen tubes that were growing and functioning normally. The results show that while the pollen tube contains numerous MF bundles arranged axially, the apical region is essentially devoid of them. The MF bundles could be seen shifting and changing in distribution as the cells grew, but they always remained out of the apical regions. Perturbation of normal growth and function by caffeine causes a change in the MF distribution, which returns to normal upon removal of caffeine from the growth medium. The lack of MFs in the apex is confirmed by careful immunogold electron microscopic analysis of thin sections of rapidly frozen and freeze-substituted pollen tubes, in which very fine MF bundles could be seen somewhat closer to the tip than is discernible with fluorescence microscopy. Still, these are very few in number and are basically absent from the very tip. Thus a reassessment of current assumptions about the distribution of actin in the pollen tube apical region is required.

Actin-endoplasmic reticulum complexes inDrosera

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.

Association of actin with cortical microtubules revealed by immunogold localization inNicotiana pollen tubes

SummaryIt 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.

Immunogold labelling of actin on sections of freeze-substituted plant cells

SummaryWe 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.

Vesicle production and fusion during lobe formation inMicrasterias visualized by high-pressure freeze fixation

SummaryTwo different types of Golgi vesicles involved in wall formation can be visualized during lobe growth inMicrasterias when using high-pressure freeze fixation followed by freeze substitution. One type that corresponds to the “dark vesicles” (DV) described in literature seems to arise by a developmental process occurring at the Golgi bodies with the single vesicles being forwarded from one cisterna to the next. The other vesicle type appears to be produced at thetrans Golgi network without any visible precursors at the Golgi cisternae. A third type of vesicle, produced by median andtrans cisternae, contains slime; these are considerably larger than those previously mentioned and they do not participate in wall formation. The distribution of the two types of cell wall vesicles at the cell periphery and their fusion with the plasma membrane are shown for the first time, since chemical fixation is too slow to preserve a sufficient number of vesicles in the cortical cytoplasm. The results indicate that fusions of both types of vesicles with the plasma membrane are possible all over the entire surface of the growing half cell. However, the DVs are much more concentrated at the growing lobes, where they form queues several vesicles deep behind zones on the plasma membrane thought to be specific fusion sites. The structural observations reveal that the regions of enhanced vesicle fusion correspond in general to the sites of calcium accumulation determined in earlier studies. By virtue of the absence of the DVs in the region of cell wall indentations the second type of wall forming vesicle appears prominent; they too fuse with the plasma membrane and discharge their contents to the wall.

Growth inhibition and recovery in freeze-substitutedLilium longiflorum pollen tubes: structural effects of caffeine

SummaryIn 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.

HIGH-PRESSURE FREEZING IMPROVES THE ULTRASTRUCTURAL PRESERVATION OF IN VIVO GROWN LILY POLLEN TUBES

SummaryWe have used high-pressure freezing followed by freeze substitution (HPF/FS) to preserve in vivo grown lily pollen tubes isolated from the style. The results indicated that HPF/FS (i) allows excellent preservation of the pollen tubes, (ii) maintains in situ the stylar matrix secreted by the transmitting tract cells, and (iii) preserves the interactions that exist between pollen tubes. Particular attention has been given to the structure of the pollen tube cell wall and the zone of adhesion. The cell wall is composed of an outer fibrillar layer and an inner layer of material similar in texture and nature to the stylar matrix and that is not callose. The stylar matrix labels strongly for arabinogalactan proteins (AGPs) recognized by monoclonal antibody JIM13. The zone of adhesion between pollen tubes contains distinct matrix components that are not recognized by JIM13, and apparent cross-links between the two cell walls. This study indicates that HPF/FS can be used successfully to preserve in vivo grown pollen tubes for ultrastructural investigations as well as characterization of the interactions between pollen tubes and the stylar matrix.

Sporangial structure inPhytophthora is disrupted after high pressure freezing

SummaryThe 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.