Presence and absence of the preprophase band of microtubules in moss protonemata: a clue to understanding its function? (original) (raw)
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In the presence of cytokinin, undetermined side branch initials of the moss, Physcomitrella patens, are induced to form buds and then leafy shoots rather than to develop as tip-growing filaments. This represents a transition between the two modes of plant cell expansion-tip growth and uniform intercalary growth. The organization of microtubules in filaments is different from that in leafy shoots and can be traced back to the influence of phytohormones on side branch initials. Microtubules either focus at a particular region (as in tip-growing cells) or in the presence of high levels of cytokinin form swollen bud initials in which microtubules are more diffusely organized. Higher levels of cytokinin are capable of destabilizing tip microtubules in caulonemal filaments. Although caulonemata are not normally target cells, this implies that cytokinin may exert its morphogenetic effects by altering microtubule organization. IR tip-growing filaments, interphase microtubules trace a meandering course through the cytoplasm towards the tip and are not for the main part associated with the plasma membrane as are cortical arrays. There is no pre-prophase band of microtubules to indicate the future division plane, even though the oblique division plane is known to be precisely controlled relative to environmental factors. This microtubule cycle contrasts with cells of the leafy shoots that develop from buds: in these, the interphase array is cortical, consisting of flat-pitched microtubular helices that do not focus upon a growing tip. It is now shown that pre-prophase bands occur at this stage. The absence of bands does not readily correlate with imprecise control of the division plane. Instead, it is proposed that the ability to form pre-prophase bands depends upon the arrangement of microtubules in the preceding interphase array. Ways in which bands might be formed are discussed and the generality of these ideas is tested by observations on higher plant cells.
1985
SUMMARY Monoclonal antibodies to yeast tubulin have been used to visualize the distribution of microtubules in the intact filamentous protonemata of the moss Physcomitrella patens. Protonemata were prepared for immunofluorescence by fixation in formaldehyde and cells were made permeable with Driselase. Extensive cell files were preserved by 'blotting' the moss onto glutaraldehydederivatized coverslips. Problems due to fluorescence from chloroplasts were obviated by extraction with dimethyl sulphoxide and the non-ionic detergent, Nonidet NP40. These improvements allowed us to determine that microtubules were present throughout the cell cycle in the apical dome of caulonemal tip cells, that there was a pronounced association of microtubules with the nucleus, that 'astral' microtubules were associated with the mitotic spindle and during anaphase may be involved in reorientation of the spindle before an oblique cytokinesis in caulonemata and that the cytokinetic phragmoplast appeared identical to the structure described for higher plants. Microtubules appeared to converge at the very tip of apical caulonemal cells and this was studied further by treating cells with CIPC — a drug that is known to produce multiple microtubuleorganizing centres — and which here produces multiple foci for microtubules at the tip. These observations emphasize the involvement of microtubules in tip growth, alignment of the cell plate and nuclear migration - processes that are fundamental to the morphogenesis of filamentous organisms
In this study we compare the contributions of Factin and microtubules to tip growth in filamentous cells of the moss Physcomitrella patens. In tip growth, expansion seems to be restricted to the hemispherical apical dome. Cytoskeletal elements have been suspected, from drug studies, to be involved in this but electron microscopy has generally not confirmed the presence of an apical cytoskeleton. However, in a previous immunofluorescence study we reported that microtubules could be seen to focus upon the apical dome in tip cells of the moss P. patens. In the present investigation F-actin has also been detected at the apices of these cells. Anti-cytoskeletal drugs were therefore used to differentiate between the roles of actin filaments and microtubules in tip growth. At high concentrations (30//M), the herbicide cremart de-polymerized microtubules and caused tip swelling. F-actin was still present under such conditions but its fragmentation by cytochalasin D suppressed this herbicide-induced swelling. On its own, cytochalasin D arrested tip growth without causing swollen tips. At lower concentrations, cremart disorganized microtubules rather than causing their complete depolymerization. Under these conditions, new but swollen growing points were initiated along the filament. The addition of taxol to cremarttreated filaments tended to reduce swelling and to re-polarize outgrowth. With particular combinations of these drugs, multiple lateral outgrowths were initiated in the vicinity of the nucleus. It is concluded: (1) that F-actin is present at the tips of Physcomitrella caulonemal apical cells; (2) that unfragmented F-actin is necessary for outgrowth; (3) that even disorganized microtubules permit some degree of outgrowth but that an unperturbed distribution of axial microtubules, focussing upon an apex, is essential in order to impose tubular shape and directionality upon expansion.
Journal of Cell Science, 1983
Antibody against tubulin from porcine brain was used to examine the distribution of tubulin in developing spermatids of Polytrichum and mature spermatozoids of Sphagnum. Cells were prepared for indirect immunofluorescence microscopy after fixation in buffered paraformaldehyde and brief incubation in cellulase. Pretreatment with cold methanol resulted in considerably enhanced immunofluorescence but exposure to Triton X-100, with or without sonication, had no effect. The antibody showed similar immunological cross-reactivity with the flagella (both basal bodies and axonemes) and the spline microtubules of the multilayered structure. This is the first direct evidence that this rigid array of stable cytoskeletal microtubules consists of tubulin. Particularly intense fluorescence from the lamellar strata of the MLS in developing spermatids provides strong support for the notion that the lamellae comprise a highly structured microtubule organizing centre (MTOC), responsible for the ordere...