DAVID J. FLANDERS, DAVID J. RAWLINS, PETER J. SHAW and CLIVE W. LLOYD*. Department of Cell Biology, John Innes Institute, John Innes Centre for ...
897
Development 110, 897-904 (1990) Printed in Great Britain © The Company of Biologists Limited 1990
Re-establishment of the interphase microtubule array in vacuolated plant cells, studied by confocal microscopy and 3-D imaging
DAVID J. FLANDERS, DAVID J. RAWLINS, PETER J. SHAW and CLIVE W. LLOYD* Department of Cell Biology, John Innes Institute, John Innes Centre for Plant Science Research, Colney Lane, Norwich NR4 7UH, UK
* Author for correspondence and reprints
Summary There are two conflicting ideas about the site of reassembly of cortical microtubules following cytokinesis. Some observations indicate that microtubules (MTs) radiate from the surface of the postcytokinetic nuclear envelope, before becoming organized at the cortex. On the other hand, results of regrowth experiments, following MT depolymerization by drugs, suggest that the array may assemble directly upon the cortex. In this study, we have taken advantage of the significant separation between nucleus and cortex, in large, vacuolated epidermal cells, to determine which of these two potential sites supports the earliest stages of regrowth in the undrugged, native state. MTs in stem epidermis of Datura stramonium L. were stained by indirect immunofluorescence. This was performed on hand-cut sections of tissue in which the cells were not separated by enzymes or distorted by airdrying. Epidermal cells with these sheets were optically sectioned by confocal laser scanning microscopy and
three-dimensional images reconstructed, rotated and viewed stereoscopically using computer methods. During metaphase, no MTs can be detected at the cortex but MTs begin to re-colonize the cell surface during early cytokinesis. Thick cables of MTs radiate from the nucleus parallel to the cell plate as well as in other directions, along transvacuolar strands, out to the cortex. Microtubules grow out over the cortex where the thick bundles make contact, as well as from the edges of the fully developed phragmoplast. These early cortical MTs do not form regular transverse arrays: they either appear to be random or to grow in branching V-shaped patterns. The cortical array is therefore not organized immediately but at a later stage. It is concluded that MT bundles, radiating from the nucleus, are involved in the earliest stages of cortical array formation.
Introduction
root tip cells appear during cytokinesis (Wick, 1985; Clayton et al. 1985), emanating from the nuclear envelope. This is a transient stage, for relatively few are found in populations of squashed root tip cells, and these radiating MTs disappear once the mature cortical array has established itself. Studies in which MT regrowth is observed following deploymerization by drugs or cold, present a different picture. In algae, recovering from chilling (Hogetsu, 1986), or amiprophos-methyl (APM) treatment (Hogetsu, 1987), or oryzalin (Wasteneys and Williamson, 1989), MTs reappear at the cell surface in random configurations. In drug-treated root tip cells (Cleary and Hardham, 1988) and cell suspensions (Falconer et al. 1988), MTs also reappear over the cell cortex once the depolymerizing drug is washed out. . Interpretation of MT-recovery experiments is, however, clouded by various problems. The high concentration of soluble tubulin may force regrowth from physiologically irrelevant sites, such as incompletely
The origin of the cortical microtubule array, following its disappearance prior to mitosis, can be accounted for in two different ways. Either, MTs might be polymerized from sites around the nucleus, or from cortical nucleation sites. Nucleation sites in higher plants have no agreed distinguishing features (see Clayton et al. 1985) and so their location during early interphase has to be implied either from observing the postcytokinetic phases of normal cells or from studying the pattern of regrowth following drug-induced depolymerization. Observations from cells untreated by drugs support the idea of nuclear regrowth. Lilium microspores during their early development (Dickinson and Sheldon, 1984), and Haemanthus endosperm (de Mey et al. 1982) have no regular cortical array and show that MTs radiate from the surface of the interphase nucleus. In cells with a normal cellulosic wall, MTs also radiate from the nucleus. The earliest interphase MTs in onion
Key words: microtubules, interphase microtubule array, 3D imaging, confocal scanning laser microscopy.
898
D. J. Flanders and others
depolymerized MT fragments and MT-linking proteins. This would be of particular concern if native, interphase nucleation sites are active for only a brief postcytokinetic period. In recent studies (Flanders etal. 1989,1990), we have used the large, vacuolated stem epidermal cells of Datura stramonium L. for immunofluorescence. Their advantage over the smaller, densely cytoplasmic, meristematic cells is that the premitotic nucleus makes a pronounced move from the cortex to the centre of the cell. The gap between nucleus and cortex (hypothesized locations of different MT nucleation sites) is a minimum of lOjUm, and such large separation has allowed it to be seen that bundles of MTs emanate from the premitotic nucleus and contribute to the formation of the preprophase band (Flanders et al. 1990). The present study concentrates on the postmitotic phase. It is found that MTs reappear from the nuclear surface during cytokinesis, before the cortical array is established. Where bundles of nucleus-associated MTs contact the cortex, MTs radiate out over the cell surface as well as from the edge of the phragmoplast. The early MT array is disorganized. Organization and interpolation of MTs during subsequent cell expansion are therefore separate subsequent processes, but at this early stage nucleusassociated MTs are involved in establishing the interphase array. This indicates a role for the nuclear surface in initiating the new cortical MT array. Materials and methods Plants of Datura stramonium L. were grown in a glasshouse as described by Flanders et al. (1989, 1990). Split sections of internode, approximately lcm long, were fixed for 4h, initially aided by vacuum infiltration, in freshly prepared 4% (w/v) formaldehyde in extraction buffer (50ITIM Pipes, 5 HIM EGTA, 5 mM MgSO4, 1 % (v/v) DMSO, 0.1 % (v/v) Nonidet NP-40, pH6.9). After several rinses, strips of outer tissue were removed with aflexiblerazor blade. The wet strips were then scored with a razor blade from the internal face to the outer epidermal wall. Only cut cells stain, but this method avoids the distortions of air-drying and separation by enzymes. The strips were then gently placed into chamber slides and stained with the anti-tubulin YOL 1/34 (Kilmartin et al. 1982) and second antibody, as described fully by Flanders et al. (1989, 1990). Both cortical and endoplasmic staining patterns were specific to anti-tubulin and were not observed when primary antibody was omitted.
Data collecting and image-processing The equipment and methods used for 3-dimensional image data collection have been described in detail in Rawlins and Shaw (1990) but the present differs from our previous studies (Flanders et al. 1989, 1990) in the use of confocal scanning laser microscopy coupled to a more advanced imageprocessing system. Slides were examined with a Zeiss Universal microscope linked to a confocal laser scanning head under microcomputer control (Biorad Lasersharp MRC 500, Biorad Lasersharp Ltd, Hemel Hempstead, UK; White et al. 1987). Images were transferred to a Stardent P1500 image-processing computer (Stardent Computers Ltd, Guildford UK), for further processing and display. Optical sections, taken at 1 or 2 pan
intervals, were collected under computer control, the fine focus of the microscope being stepped automatically by a microstepping motor. Frame averaging was used to decrease noise in the images. Stacks of optical sections were projected at several different angles about the y axis (Agard et al. 1989) and the projections either viewed in quick succession to give the effect of rotation, or in pairs to give stereo pairs, or as rotating stereo pairs. Images were recorded onto Kodak Panatomic-X film by photographing the high resolution monitor of the Stardent computer, directly with a 35 mm camera.
Results
In this study, we investigate the earliest appearance of the new interphase microtubule array in cells undergoing cytokinesis. A succession of images is presented which we interpret to represent a progression although, of course, this can only be inferred from fixed cells. However, staging is aided by noting the development of the phragmoplast which expands outwards until it contacts the cortex. Over the course of this study using sheets of epidermal cells several thousand labelled cells were examined by conventional immunofluorescence in order to identify the small proportion of cells (
