Spatial aspects of cytokinesis in plant cells.

Plant cytokinesis in a particular orientation and location can be viewed as having several component stages, often beginning with the establishment of division polarity before karyokinesis occurs. Improved methods for preserving the in situ distribution of actin microfilaments and observations of individual live cells during treatment with cytoskeleton-disrupting drugs are making it possible to elucidate the roles of microtubules and microfilaments in cytokinesis. Current evidence points to involvement of the cytoskeleton throughout the stages of preparation for, and execution of, cytokinesis in many types of plant cell division.

Immunofluorescence microscopy of tubulin and microtubule arrays in plant cells. I. Preprophase band development and concomitant appearance of nuclear envelope-associated tubulin.

The development of the preprophase band (PPB) of microtubules (MT) in meristematic plant cells was studied by using antibodies to pig brain tubulin and indirect immunofluorescence microscopy. The PPB is first visible as a wide band of MT that are arranged only slightly more densely than flanking MT of the cortical interphase array. MT progressively become more tightly packed together, and other cortical MT are no longer seen as the PPB matures. The surface of the nuclear envelope (NE) displays no tubulin fluorescence during interphase but begins to fluoresce in the early stages of PPB development, and its intensity progressively increases thereafter. The pattern at the NE is usually diffuse at first, suggesting the presence of nonpolymerized tubulin, but fibers along the NE can be resolved at later stages. MT, arranged either radially or as a meshwork, can occur between the nucleus and cell cortex, and sometimes appear to connect the PPB and NE directly. Isolated preprophase nuclei from cells ruptured during processing often retain the PPB in its normal orientation, indicating stable linkages between the nucleus and PPB. Fluorescent cross-bars perpendicular to the axis of the MT were resolved in some PPB, suggesting lateral linkages. This suggestion is reinforced by the presence of PPB that hold together as a ribbon of MT in certain preparations, allowing PPB to be isolated from the rest of the cytoplasm and the nucleus.

Localization of Ca++-containing antimonate precipitates during mitosis.

Intracellular bound Ca++ has been localized throughout mitosis and cytokinesis in two plant species by means of in situ precipitation with potassium antimonate and electron microscope visualization. Identification of Ca++ as the major cation precipitated was made by comparing solubility properties in water, EDTA, and EGTA of the intracellular deposits with respect to those of K+-, Mg++-, and Ca++-antimonate standards. In spermatogenous cells of the water fern, Marsilea vestita, and stomatal complex cells of barley, Hordeum vulgare, antimonate deposits have been found associated with the endoplasmic reticulum (ER), vacuoles, euchromatin/nucleoplasm, and mitochondria. The last contain a much higher density of precipitates in Marsilea than in Hordeum. Dictyosomes and the nuclear envelope of Marsilea also contain antimonate deposits, as do the plasmalemma, cell wall, and phragmoplast vesicles of Hordeum. Microtubule-organizing centers such as kinetochores and the blepharoplast of Marsilea do not stain. In spite of differences in associated antimonate between certain organelles of the two species, the presence of antimonate aong the ER throughout the cell cycle is common to both. Of particular interest are those precipitates seen along the tubules and cisternae of the extensive smooth ER that surrounds and invades the mitotic spindle in both species. The ability to bind divalent cations makes the mitotic apparatus (MA)-associated ER a likely candidate for regulation of free Ca++ levels in the immediate vicinity of structural components and processes that are Ca++-sensitive and proposed to be Ca++-regulated.

Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells.

Cells were prepared for indirect immunofluorescence microscopy after paraformaldehyde fixation of multicellular root apices and brief incubation in cell wall-digesting enzymes. This allowed subsequent separation of the tissue into individual cells or short files of cells which were put onto coverslips coated with polylysine. Unlike spherical protoplasts made from living tissues, these preparations retain the same polyhedral shape as the cells from which they are derived. Cellular contents, including organized arrays of microtubules, are likewise structurally stabilized. Antibodies to porcine brain tubulin react with all types of microtubule array known to occur in plant meristematic cells, namely, interphase cortical microtubules, pre-prophase bands, the mitotic spindle, and phragmoplast microtubules. The retention of antigenicity in permeabilized, isolated, stabilized cells from typical, wall-enclosed plant cells has much potential for plant immunocytochemistry, and in particular should facilitate work on the role of microtubules in the morphogenesis of organized plant tissues.

Use of the lipid emulsion system and Salmonella typhimurium mitogen adjuvant to stimulate IgG production in chickens.

Chickens were immunized with the lipid emulsion system and Salmonella typhimurium mitogen adjuvant for fowl plus synthetic peptides corresponding to the divergent amino acid sequences at the carboxyl terminus of each of two different alpha-tubulin isoforms from Arabidopsis thaliana. Antibodies were extracted from egg yolks and used in immunoblot assays to determine the time course of antibody production in two chickens. Specific immunoglobulin production increased 10-14 days after the primary injection in both chickens and reached peak levels shortly thereafter. Booster injections helped maintain IgG production, which eventually dropped off after the final injection.

Adhesion-to-skin performance of a new transdermal nitroglycerin adhesive patch.

Six men and six women participated in a study designed to compare adhesion-to-skin measurements of three transdermal nitroglycerin patch formulations (Minitran, Transderm-Nitro 5, and Nitro-Dur II) with an occlusive surgical tape control. After 24 hours of wear, Minitran was found to be superior to the other three products in skin adhesion. The force required to remove Minitran was significantly higher than that required to remove the other products. Maintaining good skin contact over the entire application period is essential for consistent drug delivery. Improved skin contact can increase transdermal drug delivery. This increase in delivery of nitroglycerin may result in the development of smaller patches rated to deliver the same quantity of nitroglycerin over a 24-hour period.

Immunofluorescence microscopy of tubulin and microtubule arrays in plant cells. III. Transition between mitotic/cytokinetic and interphase microtubule arrays.

Immunofluorescence microscopy of flowering plant root cells indicates that the earliest interphase microtubules appear during cytokinesis, radiating from the former spindle poles and subsequently from the nuclear envelope. They form networks that have microtubule focal points in the cortex underlying cell faces and in the cytoplasm between the nucleus and cortex. Cortical networks are rapidly replaced by the highly aligned array normally associated with interphase. An antibody that in animal cells identifies the location of pericentriolar material, the site of microtubule initiation, is also localized around the plant cell nuclear envelope at the time that putative early interphase microtubule networks are seen.

Preprophase bands, phragmoplasts, and spatial control of cytokinesis.

Features of preprophase bands (PPBs) of microtubules (MTs), and the spatial relationship between phragmosomes, PPB sites, and developing phragmoplasts during cytokinesis, are reviewed, setting new observations in the context of current knowledge. PPBs in onion root tip cells are present by the beginning of the G2 period of the cell cycle. They are at first wide, but later become more compact, narrower bands. MTs traverse the cytoplasm between the band at the cell cortex and the nuclear envelope. This whole assemblage of nucleus, PPB and intervening MTs remains together when the cell is ruptured during preparation for examination by immunofluorescence microscopy. Double bands are occasionally seen in early stages of PPB development, perhaps as a consequence of double induction from neighbouring cells. Calmodulin is not present in PPBs at a higher concentration than in the general cytoplasm, but it is more abundant in parts of the spindle and in the phragmoplast. The PPB MTs disappear at prophase, but nevertheless the new cell plate fuses with the parental cell walls at the PPB site. This spatial relationship can be disrupted by treatment with CIPC. Another experimental disruption of the relationship, accomplished by making minute wounds in the PPB site of mitotic cells in Tradescantia stamen hairs, is described. In other experiments on these cells the phragmoplast is shown to become tethered to the PPB site when the cell plate is half to three-quarters developed, although the telophase nuclei are free to move. Rhodamine-labelled phalloidin reveals putative F-actin in the phragmoplast of Tradescantia, but not in the gap between the extending phragmoplast and the PPB site. Rhodamine-labelled phalloidin also stains cytoplasmic strands that exist when cytoplasmic streaming occurs before and after (but not during) mitosis. Cytochalasin B treatment blocks incorporation of actin into the phragmoplast, which, however, can still develop, though usually abnormally. The F-actin of the phragmoplast may function in consolidation of the cell plate, rather than in spatial guidance of its growth toward the PPB site at the cell surface.

The use of freeze-substitution and LR gold in the study of rye grass (Lolium perenne) pollen.

Pollen grains of Lolium perenne (rye grass) were prepared for transmission electron microscopy by rapid freezing in liquid propane, substitution in acetone, methanol or diethyl ether, and embedment in the acrylic resin London Resin gold. These were compared to pollen chemically fixed (CF) in aldehyde/osmium tetroxide and embedded in the epoxy resin Quetol 651. Ultrastructural preservation was superior in freeze-substituted (FS) pollen, particularly with the use of acetone or methanol. Optimally preserved FS pollen displayed a homogeneous aspect of the cytoplasm and nucleoplasm, and smooth, uninterrupted contour or organelles. A striking difference was also seen in the preservation of inclusions in the intine. Varied forms and sizes of intine inclusions were evident in FS pollen but these were not discernible in the CF image. The FS scheme studied here presents enormous potential for both ultrastructural and immunolabelling studies in rye grass pollen. Problems discussed include artifacts associated with each of the substitution solvents used, and a gradient of freezing damage observed within the pollen grain.

Preprophase bands of microtubules and the cell cycle: Kinetics and experimental uncoupling of their formation from the nuclear cycle in onion root-tip cells.

We have studied the timing of preprophase band (PPB) development in the division cycle of onion (Allium cepa L.) root-tip cells by combinations of immunofluorescence microscopy of microtubules, microspectrophotometry of nuclear DNA, and autoradiography of [(3)H]thymidine incorporation during pulse-chase experiments. In normally grown onion root tips, every cell with a PPB had the G2 level of nuclear DNA. Some were in interphase, prior to chromatin condensation, and some had varying degrees of chromatin condensation, up to the stage of prophase at which the PPB-prophase spindle transition occurs. In addition, autoradiography showed that PPBs can be formed in cells which have just finished their S phase, and microspectrophotometry enabled us to detect a population of cells in G2 which had no PPBs, these presumably including cells which had left the division cycle. The effects of inhibitors of DNA synthesis showed that the formation of PPBs is not fully coupled to events of the nuclear cycle. Although the mitotic index decreased 6-10-fold to less than 0.5% when roots were kept in 20 µg·ml(-1) aphidicolin for more than 8 h, the percentage of cells containing PPBs did not decrease in proportion: the number of cells in interphase with PPBs increased while the number in prophase decreased. Almost the same phenomena were observed in the presence of 100 µg·ml(-1) 5-aminouracil and 40 µg·ml(-1) hydroxyurea. In controls, all cells with PPBs were in G2 or prophase, but in the presence of aphidicolin, 5-aminouracil or hydroxyurea, some of the interphase cells with PPBs were in the S phase or even in the G1 phase. We conclude that PPB formation normally occurs in G2 (in at least some cases very early in G2) and that this timing can be experimentally uncoupled from the timing of DNA duplication in the cell-division cycle. The result accords with other evidence indicating that the cytoplasmic events of cytokinesis are controlled in parallel to the nuclear cycle, rather than in an obligatorily coupled sequence.

Organized F-actin is essential for normal trichome morphogenesis in Arabidopsis.

Actin microfilaments form a three-dimensional cytoskeletal network throughout the cell and constitute an essential throughway for organelle and vesicle transport. Development of Arabidopsis trichomes, unicellular structures derived from the epidermis, is being used as a genetic system in which to study actin-dependent growth in plant cells. The present study indicates that filamentous actin (F-actin) plays an important role during Arabidopsis trichome morphogenesis. For example, immunolocalization of actin filaments during trichome morphogenesis identified rearrangements of the cytoskeletal structure during the development of the mature cell. Moreover, pharmacological experiments indicate that there are distinct requirements for actin- and microtubule-dependent function during trichome morphogenesis. The F-actin-disrupting drug cytochalasin D does not affect the establishment of polarity during trichome development; however, maintenance and coordination of the normal pattern of cell growth are very sensitive to this drug. In contrast, oryzalin, an agent that depolymerizes microtubules, severely inhibits cell polarization. Furthermore, cytochalasin D treatment phenocopies a known class of mutations that cause distorted trichome morphology. Results of an analysis of cell shape and microfilament structure in wild-type, mutant, and drug-treated trichomes are consistent with a role for actin in the maintenance and coordination of an established growth pattern.