Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells.

Transient transformation with Agrobacterium is a widespread tool allowing rapid expression analyses in plants. However, the available methods generate expression in interphase and do not allow the routine analysis of dividing cells. Here, we present a transient transformation method (termed 'TAMBY2') to enable cell biological studies in interphase and cell division. Agrobacterium-mediated transient gene expression in tobacco BY-2 was analysed by Western blotting and quantitative fluorescence microscopy. Time-lapse microscopy of cytoskeletal markers was employed to monitor cell division. Double-labelling in interphase and mitosis enabled localization studies. We found that the transient transformation efficiency was highest when BY-2/Agrobacterium co-cultivation was performed on solid medium. Transformants produced in this way divided at high frequency. We demonstrated the utility of the method by defining the behaviour of a previously uncharacterized microtubule motor, KinG, throughout the cell cycle. Our analyses demonstrated that TAMBY2 provides a flexible tool for the transient transformation of BY-2 with Agrobacterium. Fluorescence double-labelling showed that KinG localizes to microtubules and to F-actin. In interphase, KinG accumulates on microtubule lagging ends, suggesting a minus-end-directed function in vivo. Time-lapse studies of cell division showed that GFP-KinG strongly labels preprophase band and phragmoplast, but not the metaphase spindle.

"Caged cytoskeletons": a rapid method for the isolation of microtubule-associated proteins from synchronized plant suspension cells.

In the cytoskeleton method for isolating microtubule-associated proteins MAP65, DcKRP120-1 and DcKRP120-2, carrot cells are first converted to protoplasts but this method cannot be used to isolate mitotic MAPs as mitotic synchrony is eroded during lengthy cellulase treatment. Anti-microtubule cycle blocks would also be unsuitable. We report here a method for overcoming these problems. Cellulase degradation of tobacco BY-2 cells for only several minutes allows extraction of detergent-soluble proteins, leaving synchronized "caged cytoskeletons" for depolymerization and enabling affinity purification of MAPs on neurotubules. This rapid and simple method should be of general utility: it can be bulked up, avoids anti-microtubule blocks, and is applicable to other cell suspensions. The effectiveness of the caged cytoskeleton method is demonstrated by comparing known MAPs (the 65 kDa structural MAPs and the kinesin-related protein, TKRP125) in synchronized cells taken at the mitotic peak with those in unsynchronized cells.

A new class of microtubule-associated proteins in plants.

In plants there are three microtubule arrays involved in cellular morphogenesis that have no equivalent in animal cells. In animals, microtubules are decorated by another class of proteins - the structural MAPS - which serve to stabilize microtubules and assist in their organization. The best-studied members of this class in plants are the MAP-65 proteins that can be purified together with plant microtubules after several cycles of polymerization and depolymerization. Here we identify three similar MAP-65 complementary DNAs representing a small gene family named NtMAP65-1, which encode a new set of proteins, collectively called NtMAP65-1. We show that NtMAP65-1 protein localizes to areas of overlapping microtubules, indicating that it may function in the behaviour of antiparallel microtubules in the mitotic spindle and the cytokinetic phragmoplast.

Cortical microtubules form a dynamic mechanism that helps regulate the direction of plant growth.

Plants form an axis by controlling the direction of cell expansion; this depends on the way in which cellulose microfibrils in the wall resist stretching in particular directions. In turn, the alignment of cellulose microfibrils correlates strongly with the alignment of plasma membrane-associated microtubules, which therefore seem to act as templates for laying down the wall fibrils. Microtubules are now known to be quite dynamic, and to reorient themselves between transverse and longitudinal alignments. Plants "steer" the direction of growth by reorienting the cellulose/microtubule machinery. For example, the model predicts that a transverse reorientation on one flank of an organ and a longitudinal orientation on the other should lead to bending. This response has recently been observed in living, gravistimulated maize coleoptiles microinjected with fluorescent microtubule protein. This paper reviews the idea of the dynamic microtubule template and discusses possible mechanisms of reorientation. Recent biochemical work has shown that microtubules are decorated with different classes of associated proteins, whose potential roles are outlined.

The 65-kDa carrot microtubule-associated protein forms regularly arranged filamentous cross-bridges between microtubules.

In plants, cortical microtubules (MTs) occur in characteristically parallel groups maintained up to one microtubule diameter apart by fine filamentous cross-bridges. However, none of the plant microtubule-associated proteins (MAPs) so far purified accounts for the observed separation between MTs in cells. We previously isolated from carrot cytoskeletons a MAP fraction including 120- and 65-kDa MAPs and have now separated the 65-kDa carrot MAP by sucrose density centrifugation. MAP65 does not induce tubulin polymerization but induces the formation of bundles of parallel MTs in a nucleotide-insensitive manner. The bundling effect is inhibited by porcine MAP2, but, unlike MAP2, MAP65 is heat-labile. In the electron microscope, MAP65 appears as filamentous cross-bridges, maintaining an intermicrotubule spacing of 25-30 nm. Microdensitometer-computer correlation analysis reveals that the cross-bridges are regularly spaced, showing a regular axial spacing that is compatible with a symmetrical helical superlattice for 13 protofilament MTs. Because MAP65 maintains in vitro the inter-MT spacing observed in plants and is shown to decorate cortical MTs, it is proposed that this MAP is important for the organization of the cortical array in vivo.

Gibberellic acid stabilises microtubules in maize suspension cells to cold and stimulates acetylation of alpha-tubulin.

Gibberellic acid is known to stabilise microtubules in plant organs against depolymerisation. We have now devised a simplified cell system for studying this. Pretreatment of a maize cell suspension with gibberellic acid for just 3 h stabilised protoplast microtubules against depolymerisation on ice. In other eukaryotes, acetylation of alpha-tubulin is known to correlate with microtubule stabilisation but this is not established in plants. By isolating the polymeric tubulin fraction from maize cytoskeletons and immunoblotting with the antibody 6-11B-1, we have demonstrated that gibberellic acid stimulates the acetylation of alpha-tubulin. This is the first demonstrated link between microtubule stabilisation and tubulin acetylation in higher plants.

Gravity-induced reorientation of cortical microtubules observed in vivo.

Cortical microtubules play an important role during morphogenesis by determining the direction of cellulose deposition. Although many triggers are known that can induce the reorientation of cortical plant microtubules, the reorientation mechanism has remained obscure. In our approach, we used gravitropic stimulation which is a strong trigger for microtubule reorientation in epidermal cells of maize coleoptiles. To visualize the gravitropically induced microtubule reorientation in living cells, we injected rhodamine-conjugated tubulin into epidermal cells of intact maize coleoptiles that were exposed to gravitropic stimulation. From these in vivo observations, we propose a reorientation mechanism consisting of four different stages: (1) a transitional stage with randomly organized microtubules; (2) emergence of a few microtubules in a slightly oblique orientation; (3) co-alignment: neighbouring microtubules adopt the oblique orientation resulting in parallel organized microtubules; and (4) the angle of these parallel, organized microtubules increases gradually. Thus, the overall reorientation process could include selective stabilization/ disassembly of microtubules (stage 2) as well as movement of individual microtubules (stages 3 and 4).

A 60-kDa plant microtubule-associated protein promotes the growth and stabilization of neurotubules in vitro.

The search for microtubule-associated proteins (MAPs) in plants is relatively recent. In particular, the "classical MAPs," which stimulate the polymerization and stabilization of microtubules, have only been examined in heterogeneous fractions. As a first step in dissecting the role of individual MAPs, we have chromatographically purified a single 60-kDa protein from a carrot MAP fraction and analyzed its effects on tubulin assembly. MAP60 promoted the formation of long, morphologically regular brain microtubules in vitro, an effect inhibited by preincubation of the MAP with affinity-purified antibodies against this protein. MAP60 also increased the stability of microtubules to dilution and significantly enhanced cold stability to the normally cold-sensitive neurotubules. These in vitro properties are consistent with a role for MAP60 in regulating the turnover/assembly of dynamic plant microtubules in vivo.

Dynamic microtubules under the radial and outer tangential walls of microinjected pea epidermal cells observed by computer reconstruction.

By microinjecting rhodamine-conjugated pig brain tubulin into living pea stem epidermal cells it has been possible to follow cortical microtubules beneath the outer tangential wall (OTW) as they re-orientate from a transverse to a longitudinal alignment. Earlier immunofluorescence studies on fixed material have shown that parallel cortical microtubules circumnavigate the cell forming apparently continuous arrays which are transverse, oblique or longitudinal to the cell's long axis. If the array re-orientates as a whole then microtubules along the radial walls would be expected to share the alignment of those on the tangential walls. There are, however, reports that microtubules beneath the outer tangential wall have a different orientation from microtubules at the radial cell walls, raising important questions about the construction and behaviour of the array. Using computer-rotated stacks of optical sections collected by confocal scanning laser microscopy it has been possible to display the microtubules along radial as well as tangential walls of the same microinjected cells. These observations demonstrate for living epidermal cells that when microtubules are aligned longitudinally at the outer epidermal wall they remain oblique or transverse at the radial walls. The array may not therefore re-orientate as a whole but seems to undergo re-organization on only one cell face. However, despite the differing angles between the OTW and radial walls microtubules still form patterns which at the level of the confocal microscope are continuous from one cell face to another, around the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a phosphatidylinositol 3-hydroxy kinase in plant cells: association with the cytoskeleton.

Radiolabelling experiments have revealed that plant cells contain the two 3-phosphorylated phosphoinositides: PtdIns3P and PtdIns(3,4)P2 [Brearley and Hanke (1992) Biochem. J. 283, 255-260]. However, nothing is known about the enzymes involved in the metabolism of these plant 3-phosphorylated phosphoinositides. In this study we demonstrate the presence of a PtdIns 3-hydroxy kinase(s) in plant cells. This activity was enriched in the cytoskeletal fraction whereas only low levels of phosphoinositide 3-hydroxy kinase could be detected in plasma membranes and microsomal preparations. This cytoskeletal phosphoinositide 3-hydroxy kinase was found to be wortmannin insensitive and thus resembles PtdIns-specific 3-hydroxy kinases of which vps34p is one example.

Dynamic reorientation of cortical microtubules, from transverse to longitudinal, in living plant cells.

The direction in which plant tissue cells expand is reflected in the alignment of microtubules in the cortical array. When microtubules and coaligned wall microfibrils are arranged transversely around the cell, turgor pressure is chaneled into cell elongation. However, various agents (such as wounding, ethylene, abscisic acid) can cause the microtubules to reorientate by 90 degrees so that they become aligned parallel to the cell's long axis, allowing lateral expansion instead of elongation. The mechanism by which microtubules undergo rapid shifts of alignment is crucial to understanding growth control in plants, but because current models are derived from studies on fixed cells, nothing is known about the dynamics of converting one microtubule alignment to another. Cells tend to have one predominant microtubule alignment--transverse, oblique, or longitudinal--but it is not established whether each represents a stable independent set that only changes by rounds of complete de- and repolymerization, or whether reorientation is a more continuous process involving movement of stable or dynamic microtubules. By microinjecting pea (Pisum sativum) epidermal cells with rhodamine-conjugated brain tubulin and optically sectioning them by confocal laser scanning microscopy, we could follow labeled microtubules for up to 2 hr as they reorientate. Reorientation does not occur by complete depolymerization of microtubules in one orientation followed by polymerization of a new array in another orientation. Instead, increased numbers of discordant microtubules in nontransverse alignment appear in particular locations. Neighboring microtubules then adopt the new alignment, so that there is a stage during which different alignments coexist before the array on the outer tangential cell face finally adopts a uniform steeply oblique/longitudinal configuration. Rapid fluorescence recovery after photobleaching confirms that bundles of cortical microtubules are not stable but exhibit properties consistent with dynamic instability. Dynamic microtubules offer a mechanism for rapid growth responses to a range of physiological stimuli.

Microinjected profilin affects cytoplasmic streaming in plant cells by rapidly depolymerizing actin microfilaments.

BACKGROUND: Cytoplasmic streaming is a conspicuous feature of plant cell behaviour, in which organelles and vesicles shuttle along cytoplasmic strands that contain actin filaments. The mechanisms that regulate streaming and the formation of actin filament networks are largely unknown, but in all likelihood involve actin-binding proteins. The monomeric actin-binding protein, profilin, is a key regulator of actin-filament dynamics in animal cells and it has recently been identified in plants as a pollen allergen. We set out to determine whether plant profilin can act as a monomeric actin-binding protein and influence actin dynamics in plant cells in vivo. RESULTS: Recombinant birch-pollen profilin was purified by polyproline affinity chromatography and microinjected into Tradescantia blossfeldiana stamen hair cells. After profilin injection, a rapid and irreversible change in cellular organization and streaming was observed: within 1-3 minutes the transvacuolar cytoplasmic strands became thinner and snapped, and cytoplasmic streaming ceased. Fluorescein-labelled-phalloidin staining confirmed that this was due to depolymerization of actin filaments. To confirm that the effects observed were due to sequestration of monomeric actin, another monomeric actin-binding protein, DNase I, was injected and found to produce comparable results. CONCLUSIONS: Profilin can act as a potent regulator of actin organization in living plant cells. Its rapid effect on the integrity of cytoplasmic strands and cytoplasmic streaming supports a model in which organelle movements depend upon microfilaments that exist in dynamic equilibrium with the pool of monomeric actin.

The profilin multigene family of maize: differential expression of three isoforms.

Profilin is a small (12-15 kDa) actin- and phospholipid-binding protein previously known only from studies on animals and lower eukaryotes but recently identified as a birch pollen allergen. Here we have identified and characterized three members of the profilin multigene family from the plant Zea mays. Two cDNAs isolated from a maize pollen library (ZmPRO 1 and ZmPRO 3) each have a single, large open reading frame encoding a putative polypeptide 131 amino acids long with a predicted molecular weight of approximately 14 kDa. A third maize pollen cDNA (ZmPRO 2) has two in-frame translation initiation codons. Use of the first ATG would result in a polypeptide 137 amino acids long with a molecular weight of 14.8 kDa. The three maize profilins are highly homologous to each other (> 90% nucleotide and amino acid sequence identity) as well as other plant profilins but show far less similarity (30-40% amino acid sequence identity) to animal and lower eukaryote profilins. Multiple sequence alignments indicate that only nine residues are shared by all eukaryotic profilins examined. However, limited comparisons reveal domains in the NH2 and COOH termini that have a high degree of similarity suggesting functional conservation. The maize gene family size is estimated to contain three to six members based on Southern blot experiments with gene-specific and coding region probes. Northern blot analysis demonstrates that the three maize profilin cDNAs characterized here are utilized in a tissue-specific manner and are anther or pollen specific.

Simulation of blood flow in microgravity.

Knowledge of venous, capillary, and arterial blood flow in microgravity is required to modify hemostatic techniques for control of bleeding in traumatic injuries or surgical procedures in space. To simulate human arterial, venous, and capillary bleeding, fresh whole bovine blood was injected by two operators at calculated flow rates (3.5, 7, and 14 mL for venous and 14 and 28 mL for arterial) in 10 seconds with empirical controls in a lucent glove box during zero gravity parabolic flight on NASA's KC-135 aircraft. A pig's foot was used to mimic capillary bleeding. Hemostasis with sponges and laerdal suction was evaluated by video and still photography. Evaluations of the arterial and venous bleeding were conducted at 3 rates x 3 parabolas, and capillary bleeding was evaluated with 5 parabolas x 2 methods (pig's foot and sponge). Influenced by surface tension, the slow venous bleeding coated syringe surfaces and formed a dome over the skin laceration bleeding site. Arterial and venous bleeders broke into uniform spheres with low-velocity spheres bouncing off an absorbent pad and suction tip. Conventional dabbing with gauze fragmented blood into small spheres. Capillary oozing was better controlled by "wicking" up blood with gauze. Repeated arterial bleeding opacified the glove box wall. This stimulation demonstrated unique characteristics of extracorporeal blood flow and inadequacies of common methods of hemostasis in microgravity.

Association of Phosphatidylinositol 4-Kinase with the Plant Cytoskeleton.

In eukaryotic cells, phosphatidylinositol 4-hydroxy kinase and phosphatidylinositol-4-phosphate 5-hydroxy kinase are responsible for the formation of the two second messenger precursors phosphatidylinositol-4-phosphate (Ptdlns(4)P) and phosphatidylinositol-4,5-bisphosphate (Ptdlns(4,5)P2). In plant cells, these kinases have been considered to be exclusively membrane associated, with the majority of activity residing in the inner leaflet of the plasmalemma. By sequentially extracting carrot protoplasts with the detergent Nonidet P-40 then more rigorously with Triton X-100, we were able to remove the activity of three separate plasma membrane marker enzymes and to demonstrate that a significant proportion of cellular Ptdlns 4-kinase is associated with the cytoskeleton. When only endogenous substrates were present, Nonidet P-40-permeabilized protoplasts and Nonidet P-40-extracted cytoskeletons displayed a pattern of lipid phosphorylation similar to that obtained with isolated plant membranes or permeabilized cells, whereas the Triton X-100-extracted cytoskeletons showed little or no activity. In contrast, when exogenous substrates were added, a major proportion of PtdlnsP formed was due to kinase activity associated with the cytoskeleton as well as nuclei. However, by subtracting the activity of isolated nuclei, it could be demonstrated that a significant proportion of the detergent-resistant Ptdlns kinase activity resides with the cytoskeletal fraction. These findings suggest that the pathways of polyphosphoinositide biosynthesis in plant cells should be reevaluated to take account of the cytoskeleton and that Ptdlns(4)P itself may play a unique role in modulation of plant cytoskeletal integrity and cellular signal transduction.

The plant cytoskeleton.

Significant progress has been made in four areas: in appreciating the speed with which cortical microtubules reorient in response to environmental signals; in a consolidated understanding of the cytoskeletal nature of the phragmosome--the device that predicts and structures the division plane in vacuolated cells; in the description of new cytoskeletal proteins; and in reports that herald an attack on the cell cycle control of cytoskeletal organization.

Nucleus-associated microtubules help determine the division plane of plant epidermal cells: avoidance of four-way junctions and the role of cell geometry.

To investigate the spatial relationship between the nucleus and the cortical division site, epidermal cells were selected in which the separation between these two areas is large. Avoiding enzyme treatment and air drying, Datura stramonium cells were labeled with antitubulin antibodies and the three-dimensional aspect of the cytoskeletons was reconstructed using computer-aided optical sectioning. In vacuolated cells preparing for division, the nucleus migrates into the center of the cell, suspended by transvacuolar strands. These strands are now shown to contain continuous bundles of microtubules which bridge the nucleus to the cortex. These nucleus-radiating microtubules adopt different configurations in cells of different shape. In elongated cells with more or less parallel side walls, oblique strands radiating from the nucleus to the long side walls are presumably unstable, for they are progressively realigned into a transverse disc (the phragmosome) as broad, cortical, preprophase bands (PPBs) become tighter. The phragmosome and the PPB are both known predictors of the division plane and our observations indicate that they align simultaneously in elongated epidermal cells. These observations suggest another hypothesis: that the PPB may contain microtubules polymerized from the nuclear surface. In elongated cells, the majority of the radiating microtubules, therefore, come to anchor the nucleus in the transverse plane, consistent with the observed tendency of such cells to divide perpendicular to the long axis. In nonrectangular isodiametric epidermal cells, which approximate regular hexagons in section, the radial microtubular strands emanating from the nucleus tend to remain associated with the middle of each subtending cell wall. The strands are not reorganized into a single dominant transverse bar, but remain as a starlike array until mitosis. PPBs in these cells are not as tight; they may only be a sparse accumulation of microtubules, even forming along non-diametrical radii. This arrangement is consistent with the irregular division patterns observed in epidermal mosaics of isodiametric D. stramonium cells. The various conformations of the radial strands can be modeled by springs held in two-dimensional hexagonal frames, and by soap bubbles in three-dimensional hexagonal frames, suggesting that the division plane may, by analogy, be selected by minimal path criteria. Such behavior offers a cytoplasmic explanation of long-standing empirically derived "rules" which state that the new cell wall tends to meet the maternal wall at right angles. The radial premitotic strands and their analogues avoid taking the longer path to the vertex of an angle where a cross wall is already present between neighboring cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Reconstitution of intermediate filaments from a higher plant.

Immunological studies have shown that plants contain intermediate-filament antigens, but it is not known whether these proteins are capable in themselves of forming filaments. To address this problem, a detergent-resistant and high-salt-insoluble fraction from carrot (Daucus carota L.) suspension cells was solubilized with 9 M-urea and then subjected to a two-step dialysis procedure, devised for the reconstitution of animal intermediate filaments. This induced the self-assembly of 10 nm filaments and large bundles of filaments. The predominant components of reconstituted material were polypeptides with apparent molecular masses between 58 and 62 kDa. These polypeptides immunoblotted with two monoclonal antibodies known to show broad cross-reactivity with intermediate filaments across the phylogenetic spectrum. This establishes that the antigens are able to self-assemble into intermediate-sized filaments.