Ann L. Cleary

The Tangled1 Gene Is Required for Spatial Control of Cytoskeletal Arrays Associated with Cell Division during Maize Leaf Development

The cytoskeleton plays a major role in the spatial regulation of plant cell division and morphogenesis. Arrays of microtubules and actin filaments present in the cell cortex during prophase mark sites to which phragmoplasts and associated cell plates are guided during cytokinesis. During interphase, cortical microtubules are believed to influence the orientation of cell expansion by guiding the pattern in which cell wall material is laid down. Little is known about the mechanisms that regulate these cytoskeleton-dependent processes critical for plant development. Previous work showed that the Tangled1 (Tan1) gene of maize is required for spatial regulation of cytokinesis during maize leaf development but not for leaf morphogenesis. Here, we examine the cytoskeletal arrays associated with cell division and morphogenesis during the development of tan1 and wild-type leaves. Our analysis leads to the conclusion that Tan1 is required both for the positioning of cytoskeletal arrays that establish planes of cell division during prophase and for spatial guidance of expanding phragmoplasts toward preestablished cortical division sites during cytokinesis. Observations on the organization of interphase cortical microtubules suggest that regional influences may play a role in coordinating cell expansion patterns among groups of cells during leaf morphogenesis.

F-actin redistributions at the division site in livingTradescantia stomatal complexes as revealed by microinjection of rhodamine-phalloidin

SummaryMicroinjection of rhodamine-phalloidin into living cells of isolatedTradescantia leaf epidermis and visualisation by confocal microscopy has extended previous results on the distribution of actin in mitotic cells of higher plants and revealed new aspects of actin arrays in stomatal cells and their initials. Divisions in the stomatal guard mother cells and unspecialised epidermal cells are symmetrical. Asymmetrical divisions occur in guard mother precursor cells and subsidiary mother cells. Each asymmetrical division is preceded by migration of the nucleus and the subsequent accumulation of thick bundles of anticlinally oriented actin filaments localised to the area of the anticlinal wall closest to the polarised nucleus. During prophase, in all cell types, a subset of cortical actin filaments coaligns to form a band, which, like the preprophase band of microtubules, accurately delineates the site of insertion of the future cell wall. Following the breakdown of the nuclear envelope, F-actin in these bands disassembles but persists elsewhere in the cell cortex. Thus, cortical F-actin marks the division site throughout mitosis, firstly as an appropriately positioned band and then by its localised depletion from the same region of the cell cortex. This sequence has been detected in all classes of division inTradescantia leaf epidermis, irrespective of whether the division is asymmetrical or symmetrical, or whether the cell is vacuolate or densely cytoplasmic. Taken together with earlier observations on stamen hair cells and root tip cells it may therefore be a general cytoskeletal feature of division in cells of higher plants.

Cytoskeletal dynamics in living plant cells

Microtubules and microfilaments have been imaged in living plant cells and their dynamic changes recorded during division, growth and development. Carboxyfluorescein labeled brain tubulin has been injected into cells that are maintained in an active state in a culture chamber on the microscope stage. Subsequent imaging with the confocal microscope reveals microtubules in the preprophase band, the mitotic apparatus, the phragmoplast, and the cortical array. The structural changes of these microtubules have been observed during transitional stages. In addition, their dynamic features are demonstrated by depolymerization in elevated calcium, low temperature, and in the drug oryzalin, and by repolymerization when returned to normal conditions. Examination of living Tradescantia stamen hair cells, which have been injected with fluorescent phalloidin to label the actin microfilaments, reveals hitherto undisclosed aspects of the preparation of the division site and dynamics of the phragmoplast cytoskeleton. During prophase microfilaments occur throughout the cell cortex, with those in the region of the preprophase band becoming transversely aligned. At nuclear envelope breakdown, these specifically disassemble, leaving a circumferential zone from which microfilaments remain absent throughout division. During cytokinesis microfilaments arise within the phragmoplast, oriented parallel to the microtubules, but excluded from the zone where the MTs overlap and where cell plate vesicles aggregate. The phragmoplast microfilaments, in a manner similar to microtubules, shorten in length, expand in girth, and eventually disassemble when the cell plate is complete.

Pre‐mitotic nuclear migration in subsidiary mother cells of Tradescantia occurs in G1 of the cell cycle and requires F‐actin

We have studied pre-mitotic nuclear migration in living subsidiary mother cells (SMCs) of Tradescantia virginiana. Divisions in the four SMCs of each stomatal complex are asymmetrical and are preceded by the migration of nuclei from random locations in the cells to positions adjacent to the central guard mother cells (GMCs). In newly polarised SMCs, nuclei display erratic movements which gradually dampen over time. In older complexes, where nuclear migration occurred earlier in the ontogeny of the leaf, nuclei are stably positioned and change in morphology from spherical to dome-shaped. Labelling with bromodeoxyuridine (BrdU) shows that SMCs polarised in G1 of the cell cycle and remain polarised for a minimum of 22 h before entering mitosis, while the inducing GMCs stay in G1. Centrifugation (1,320 g, 15 min) of epidermal peels displaces the majority of nuclei to the centrifugal end of cells, including nuclei in polarised SMCs. After centrifugation, most SMC nuclei return towards the GMCs within 100 min in control and 5 microM oryzalin treated peels. However, nuclei are unable to reposition in the presence of cytochalasin B (5 micrograms/ml). Thus, the signal for SMC polarisation is issued very early in the ontogeny of the cells (G1), is apparently sustained for a prolonged period, and results in the actin-dependent migration of nuclei towards the GMC. Cytological changes and nuclear migrations similar to those occurring in polarising SMCs can be induced by a local application of pressure to the surface of epidermal cells.

Microtubule organization during development of stomatal complexes inLolium rigidum

SummaryMicrotubule (MT) arrays in stomatal complexes ofLolium have been studied using cryosectioning and immunofluorescence microscopy. This in situ analysis reveals that the arrangement of MTs in pairs of guard cells (GCs) or subsidiary cells (SCs) within a complex is very similar, indicating that MT deployment is closely coordinated during development. In premitotic guard mother cells (GMCs), MTs of the transverse interphase MT band (IMB) are reorganized into a longitudinal array via a transitory array in which the MTs appear to radiate from the cell edges towards the centre of the walls. Following the longitudinal division of GMCs, cortical MTs are reinstated in the GCs at the edge of the periclinal and ventral walls. The MTs become organized into arrays which radiate across the periclinal walls, initially from along the length of the ventral wall and later only from the pore site. As the GCs elongate, the organization of MTs and the patterns of wall expansion differ on the internal and external periclinal walls. A final reorientation of MTs from transverse to longitudinal is associated with the elongation and constriction of GCs to produce mature complexes. During cytokinesis in the subsidiary mother cells (SMCs), MTs appear around the reforming nucleus in the daughter epidermal cells but appear in the cortex of the SC once division is complete. Our results are thus consistent with the idea that interphase MTs are nucleated in the cell cortex in all cells of the stomatal complex but not in adjacent epidermal cells.

Plasma membrane-cell wall connections: Roles in mitosis and cytokinesis revealed by plasmolysis of Tradescantia virginiana leaf epidermal cells

SummaryTradescantia virginiana leaf epidermal cells were plasmolysed by sequential treatment with 0.8 M and 0.3 M sucrose. Plasmolysis revealed adhesion of the plasma membrane to the cell wall at sites coinciding with cytoskeletal arrays involved in the polarisation of cells undergoing asymmetric divisions — cortical actin patch — and in the establishment and maintenance of the division site —preprophase band of microtubules and filamentous (F) actin. The majority of cells retained adhesions at the actin patch throughout mitosis. However, only approximately 13% of cells formed or retained attachments at the site of the preprophase band. After the breakdown of the nuclear envelope, plasmolysis had a dramatic effect on spindle orientation, cell plate formation, and the plane of cytokinesis. Spindles were rotated at abnormal angles including tilted into the plane of the epidermis. Cell plates formed but were quickly replaced by vacuole-like intercellular compartments containing no Tinopal-stainable cell wall material. This compartment usually opened to the apoplast at one side, and cytokinesis was completed by the furrow extending across the protoplast. This atypical cytokinesis was facilitated by a phragmoplast containing microtubules and F-actin. Progression of the furrow was unaffected by 25 μg of cytochalasin B per ml but inhibited by 10 μM oryzalin. Phragmoplasts were contorted and misguided and cytokinesis prolonged, indicating severe disruption to the guidance mechanisms controlling phragmoplast expansion. These results are discussed in terms of cytoskeleton-plasma membrane-cell wall connections that could be important to the localisation of plasma membrane molecules defining the cortical division site and hence providing positional information to the cytokinetic apparatus, and/or for providing an anchor for cytoplasmic F-actin necessary to generate tension on the phragmoplast and facilitate its directed, planar expansion.

The effect of ICK1, a plant cyclin-dependent kinase inhibitor, on mitosis in living plant cells

Abstract. The inhibitory activity of Arabidopsisthaliana ICK1, a plant cyclin-dependent kinase inhibitor, has previously been characterised by its effect on plant cyclin-dependent kinase activity in vitro and its effect on growth in transgenic plants. Herein, we examine cyclin-dependent kinase-driven cell-cycle events, probed by testing the sensitivity of living cells to introduced ICK1 protein. The microinjection of ICK1 into individual Tradescantia virginiana stamen hair cells during late prophase and prometaphase resulted in a clear protein-specific increase in the metaphase transit time (time from nuclear envelope breakdown to the onset of anaphase) in a manner dependent on load and injection time. The results indicate a continuing role for cyclin-dependent kinases in mitotic progression and provide in vivo evidence at the cellular level that ICK1 can restrict growth in the plant by inhibiting cell division.

Organisation of microtubules and actin filaments in the cortex of differentiating Selaginella guard cells

SummaryUsing fluorescent probes and confocal laser scanning microscopy we have examined the organisation of the microtubule and actin components of the cytoskeleton in kidney-shaped guard cells of six species of Selaginella. The stomata of Selaginella exhibit novel cytoskeletal arrangements, and at different developmental stages, display similarities in microtubule organisation to the two major types of stomata: grass (dumbbell-shaped) and non-grass (kidney-shaped). Initially, cortical microtubules and F-actin radiate from the stomatal pore and extend across the external and internal periclinal cell surfaces of the guard cells. As the stomata differentiate, the cytoskeleton reorients only along the internal periclinal walls. Reorganisation is synchronous in guard cells of the same stoma. Microtubules on the inner periclinal walls of the guard cells now emanate from areas of the ventral wall on either side of the pore and form concentric circles around the pore. The rearrangement of F-actin is similar to that of microtubules although F-actin is less well organised. Radial arrays of both microtubules and F-actin are maintained adjacent to the external surfaces. Subsequently, in two of the six species of Selaginella examined, microtubules on both the internal and external walls become oriented longitudinally and exhibit no association with the ventral wall. In the other four species, microtubules adjacent to the internal walls revert to the initial radial alignment. These findings may have implications in the development and evolution of the stomatal complex.

Microtubule arrays during mitosis in monoplastidic root tip cells ofIsoetes

SummaryMitosis in monoplastidic root tip cells ofIsoetes was studied by fluorescence (confocal laser scanning microscopy) and transmission electron microscopy. The two major components of division polarity, spindle axis and division site, are established more or less simultaneously in preprophase. Morphogenetic plastid migration results in positioning of a daughter plastid at each pole of the future spindle. Concomitant with establishment of the spindle axis, the division site is marked by a girdling band of mirotubules (preprophase band). Endoplasmic microtubules interconnect the polar plastids and preprophase band suggesting a mechanism for communication and final alignment of spindle axis and division site. As in other monoplastidic cells, the spindle appears to emanate from the plastids. In telophase, the focus of microtubules shifts to the reforming nuclei as the phragmoplast is initiated. Microtubules which continue to emanate from plastids are incorporated into the phragmoplast as it expands beyond the interzonal region. Cortical microtubules are restored in a random fashion before assuming a transverse arrangement in interphase.

Actin in Formation of Stomatal Complexes

Stomata have long been recognised as a model system in which to study regulated division in plant cells because the precursor cells divide in predictable sequences and planes. The formation of functional stomatal complexes, whether a solitary guard cell pair or guard cells complexed with multiple subsidiary cells, requires precisely oriented cell divisions reliant on the correct functioning of the cytoskeleton. In particular, many stage-specific configurations of cortical actin form during the development of stomatal complexes. These may be critical to normal stomatal development and function. The distribution and function of actin during stomatogenesis is discussed.