C. Katsaros

Microtubules and their organizing centres in differentiating guard cells ofAdiantum capillus veneris

SummaryThe cortical cytoplasm of the young guard cells ofAdiantum capillus veneris is locally differentiated. At an early post-telophase stage, numerous microtubules diverge from the cytoplasm occupying the junctions of the midregion of the ventral wall with the periclinal ones, towards the periclinal and ventral wall faces as well as towards the inner cytoplasm. Microtubule-vesicle complexes (MVCs) are detected in these regions. Their appearance is accompanied by the initiation of local wall thickenings in the same areas.Afterwards, more distinct MVCs anchored to the plasmalemma were seen in the cortical cytoplasm of the periclinal walls, close to the growing thickenings, usually at a distance up to 3μm from them. Sometimes, they seemed to contain an electron dense substance in which the microtubules were embedded. Cortical microtubules converging from more than one direction terminate at the MVCs. Besides, the microtubule population lining the periclinal walls radiate from the regions where the above cytoplasmic formations are localized. The overlying cellulose microfibrils exhibit the same orientation. The vesicles localized at the MVCs appear to be of dictyosomal origin, very electron dense and react positively to periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) test. Another population of microtubules fan out from the MVCs, entering deeper into the cytoplasm. They become associated with the nucleus and mitochondria, and traverse the peridictyosomal cytoplasm. In some instances the nucleus formed a protrusion towards an MVC and appeared associated with it via microtubules which radiate from the MVC and flank the nuclear envelope.The observations favour the hypothesis that prominent microtubule organizing centres (MTOCs) function in the cortical cytoplasm of the midregion of the periclinal walls surrounding the ventral one for a relatively long time. The MVCs and/or their adjacent plasmalemma sites may represent MTOCs or at least they specify the cortical cytoplasmic sites where microtubules are nucleated.

Experimental studies on the function of the cortical cytoplasmic zone of the preprophase microtubule band

SummaryCentrifugation of young seedlings ofTriticum durum andTriticum aestivum for 8–10 hours at 1,500–2,000 x g causes a serious disorder of the spatial organelle relationships in the interphase as well as the preprophase and mitotic subsidiary cell mother cells (SMCs). The nucleus, most organelles and cytoplasm are displaced to the centrifugal end of the cell, while the vacuoles lie at the other end. However, after centrifugation, the preprophase microtubule bands (PMBs) are nucleated and remain at the expected position close to the guard cell mother cells (GMCs). In some elongated SMCs the PMBs become completely separated from the nucleus. The mitotic spindle exhibits variable orientation and is usually formed at some distance from the PMB cortical zone.Cytokinesis in SMCs is spatially highly disturbed and the cell plate shows a variety of unpredictable dispositions, which seem to be determined by: 1. the position of the preprophase-prophase nucleus and the orientation of the mitotic spindle as well as their spatial relationships to the PMB cortical zone, and 2. the space available for cell plate growth. Many of the daughter cells exhibit a highly variable shape and size in different planes. Usually one edge of the cell plate partly or totally joins the anticlinal parent wall adjacent to the PMB cortical zone.In some SMCs ofZea mays andTriticum aestivum, the junction regions of the periclinal walls with the anticlinal ones, lined by the PMB cortical zone in normal SMCs, are detectably thickened after the arrest of mitosis and the prevention of interphase microtubule formation by a prolonged colchicine treatment. In a small number of protodermal cells of the same plants, participating in the development of stomatal complexes, irregular wall bodies or incomplete wall sheets were formed at wall regions lined by the PMB cortical zone.The presented observations are in line with the following hypotheses: 1. the PMB cortical zone interacts with the growing edges of the cell plate “attracting” it to fuse with the underlying parent wall when the latter approaches the former at a critical distance, and 2. in SMCs particular regions of the PMB cortical zone and/or the adjacent plasmalemma promote the local wall deposition in the absence of microtubules.

Synchronous organization of two preprophase microtubule bands and final cell plate arrangement in subsidiary cell mother cells of someTriticum species

SummaryIn the primary leaves of threeTriticum species examined, two successive guard cell mother cells (GMCs) are often laterally flanked by one epidermal cell which is induced twice by them and forms two subsidiary cells (SCs). In the case of simultaneous induction of the common subsidiary cell mother cell (SMC) by the GMCs, its nucleus does not migrate towards either of the GMCs, but occupies a position between them. Unexpectedly, in a number of the above “double-polarized” SMCs, two preprophase microtubule bands (PMBs) are organized at the same time, apposed on the GMCs at the expected positions. In some of those cells the daughter wall exhibits aberrant dispositions and either a small lens-shaped cell adjacent to the intervening cell between the GMCs or a large SC alongside both the GMCs and the intervening cell, or an SC flanking one GMC and a part of the intervening cell between the GMCs are separated. Obviously, the sites of fusion of the cell plate with the parent walls do not coincide completely with those of either of the PMB cortical zones. These cell plates appear to intersect those parental walls which are common with the intervening cells at PMB cortical sites belonging to two PMBs. Besides, the one end of the cell plate may pass from the cortical zone of the one PMB to another of the other PMB.The SC nucleus retains the telophase size and chromatin condensation for a longer time period than the other nucleus. This nuclear lag-phase may persist up to the completion of the GMC division, when all the cells of the stomatal complex undergo a coordinated growth and differentiation. The telophase SC nucleus frequently interacts with the phragmoplast microtubules attached on the nuclear envelope. They appear to be pulling the nucleus which forms acute local extensions towards the terminal anticlinal regions of the cell plate. Despite the above-mentioned nuclear behaviour, it does not seem likely that the curved growth of the cell plate is controlled by the SC telophase nucleus. The PMB cortical zone seems to affect the direction of growth of the expanding cell plate, thus controlling its final orientation. In few SMCs in which the prophase nucleus was localized at some distance from the GMC, it formed an acute angular extension towards it. This phenomenon probably indicates the stabilization mechanism of the one pole of the mitotic spindle close to the GMC and implies that the nucleus interacts with the polarized cortical cytoplasm adjacent to the inducing GMC.

Immunofluorescence and electron microscopic studies of microtubule organization during the cell cycle ofDictyota dichotoma (Phaeophyta, Dictyotales)

SummaryInterphase cells ofDictyota dichotoma (Hudson) Lamour. lack cortical microtubules (Mts) but display an impressive network of cytoplasmic microtubules (c-Mts). These are focussed on two opposed perinuclear centriolar sites where centrin or a centrin-homologue is localized. Some of the Mts surround the nucleus, but the majority traverse the cytoplasm as bundles variously directed towards the plasmalemma. In apical cells, and to a lesser extent in the square or slightly elongated meristematic cells, Mts are more or less evenly arranged. In elongated cells they form thick bundles longitudinally traversing the cytoplasm; a pattern maintained in differentiated cells. In early prophase the non-perinuclear Mts disappear but by late prophase a bi-astral arrangement of short Mts is observed. They enter polar nuclear depressions and attach to differentiated regions of the nuclear envelope where polar gaps open. By metaphase the spindle Mts converge on the centrioles at the polar gaps. At anaphase, interzonal Mts are evident and the asters start to reassemble. After telophase disruption of the interzonal Mts, the daughter nuclei approach each other, but move apart again before cytokinesis. The latter movement keeps pace with the development of two interdigitating Mt systems, ensheathing both daughter nuclei. The partition membrane “bisects” this Mt “cage”. Between telophase and cytokinesis the centrosomes separate, finally occupying opposed perinuclear sites. New Mts arise at the new centrosomes, some terminating on the consolidating partition membrane. Our data show thatD. dichotoma vegetative cells display a prominent cytoplasmic Mt cytoskeleton, which undergoes continual, but definite, change in organization during the cell cycle.

F-Actin organization during the cell cycle of Sphacelaria rigidula (Phaeophyceae)

The organization of F-actin in vegetative cells of the brown alga Sphacelaria rigidula was studied after staining with a modified rhodaminephalloidin (Rh-Ph) protocol. The interphase vegetative cells display a well-organized F-actin cytoskeleton, consisting of cortical, endoplasmic and perinuclear arrays of actin filaments (AFs). The organization of these AFs changes slightly during mitosis, while they almost disappear at cytokinesis. The perinuclear AF population becomes more obvious during prophase, especially at the poles. At metaphase, an actin spindle is organized, co-localized with the microtubule spindle, while at anaphase an interzonal AF population appears, which persists at early telophase. At advanced telophase the image changes to a rather diffuse actin meshwork in the mid-area between the daughter nuclei. During post-telophase-early cytokinesis this AF system becomes gradually disassembled and a conspicuous actin plate is formed on the cytokinetic plane. This plate becomes more compact with the progress of cytokinesis, and seems to overcoat the patches of the forming diaphragm, and finally the daughter plasmalemmata. Treatment with cytochalasin B disturbs mitosis, not allowing the cells to proceed to anaphase, and prevents cytokinesis. These observations show that AFs are ubiquitous cytoskeletal elements of the vegetative cells of brown algae. The probable role of AFs during the cell cycle and particularly the unique actin configuration involved in cytokinesis are discussed.

Tubulin conformation in microtubule-free cells ofVigna sinensis

SummaryThe primary leaf, epicotyl, and root cells ofVigna sinensis seedlings grown continuously in a 0.08% colchicine solution, become microtubule-free and polyploid. In meristematic root cells a tubulin transformation is detected 1–3 h after the treatment had begun. Tubulin strands are organized at the positions of the pre-existing microtubules. Frequently, the strands converge on or are organized in the cortical cytoplasmic zone where in normal cells the preprophase microtubule band (PMB) is assembled. In meristematic root cells subjected to a 6–12 h colchicine treatment, the tubulin strands become perinuclear, entering the cortical cytoplasm at regions close to the nucleus. One day after the onset of the treatment, tubulin generally forms a continuous reticulum of interconnected strands in all the organs examined. In most cells this reticulum surrounds the nucleus partly or totally or lies close to it, exhibiting variable configurations in different cells. After prolonged treatments, the organization of the tubulin reticulum changes further. Now this consists of crystal-like structures interconnected by thin strands.On thin sections of fixed tissue the tubulin strands consist of paracrystalline material. The distribution of this material in the affected cells coincides with that of tubulin reticulum visualized by immunofluorescence. In transverse planes each strand exhibits circular subunits arranged close to one another in a hexagonal pattern but in longitudinal ones variable images were observed. The paracrystalline material persists in root cells subjected to an 8-day continuous colchicine treatment. The immunolabeled strands seem to be composed of tubulin-colchicine complexes and not pure tubulin.

Thallus development in Dictyopteris membranacea (Phaeophyta, Dictyotales)

The thallus ontogeny of Dictyopteris membranacea is the outcome of a highly co-ordinated series of cell divisions, which is completed at a long distance from the apex. Three types of “meristems” function: (a) the central apical initials, which give rise to the other meristems and to initial cells of the midrib; (b) the marginal apical initials, which contribute mainly to wing formation; and (c) a superficial meristem which is a true meristoderm and contributes to midrib formation. The central apical initials divide symmetrically in a longitudinal plane and asymmetrically in a transverse one. The initial cells of the midrib are derived by asymmetrical divisions of these central apical initials. The symmetrical divisions of the latter cells form new central apical initials and other apical ones of a determinate function. The meristodermal cells are separated by asymmetrical divisions of the initial cells of the midrib, which have been previously divided symmetrically on a periclinal plane. The meristoderm a...

Ultrastructural studies on thallus development in Dictyota dichotoma (Phaeophyta, Dictyotales)

Three successive asymmetrical divisions, one occurring in the apical cell of Dictyota dichotoma and two in its first discoid segment, lead to the formation of two different cell types, the epidermal and medullary cells. The change in cell length with distance from the thallus apex is the same for the four series of nascent epidermal cells as for the medullary cells. Thereafter, and for a distance of 2 mm from the apex, the elongation of the epidermal cells differs and reflects a different pattern of cell growth. The derivatives of the initial epidermal cells exhibit greater meristematic activity than those of the initial medullary cell and grow mainly by the synthesis of protoplasm. During differentiation they retain a highly secretory ribosomal cytoplasm and develop well-differentiated chloroplasts and microbodies. Besides, many mitochondria underlie the longitudinal anticlinal walls of the differentiated epidermal cells, forming an intimate relationship with the plasmalemma, and the anticlinal walls bec...

F-actin involvement in apical cell morphogenesis of Sphacelaria rigidula (Phaeophyceae): mutual alignment between cortical actin filaments and cellulose microfibrils

The polarized apical cells of Sphacelaria rigidula display a well-organized cortical F-actin cytoskeleton. This consists of bundles of actin filaments (AFs), assuming definite patterns of organization in different regions of the cell cortex. At the tip region of the apical dome the AFs appear randomly oriented, showing a diffuse fluorescence. Immediately below, at the base of the apical hemisphere, the AFs form a ring-like band around the plasmalemma transverse to the polar cell axis. The rest of the cell cortex is traversed by AFs showing an axial or slightly inclined or helical orientation. Examination of the apical cells of S. rigidula in appropriate thin sections revealed that the wall has a multi-layered structure. In the tip region of the apical dome the cell wall bears randomly oriented cellulose microfibrils (MFs), while in the basal part of the apical dome it is reinforced by a layer of densely arranged transverse MFs. As the cell grows at the apex, the transverse MFs are continuously displaced towards the cell base. Below the transverse MF layer, an additional layer with axial or slightly oblique MFs starts being depositing internally, on the tubular part of the cell. Externally to them, the layer of transversely oriented MFs remains visible. The above observations were confirmed in apical cells of S. tribuloides. MF orientation in the innermost wall layer of the apical cells coincides with that of the cortical AFs observed by fluorescence. This mutual alignment between AFs and MFs in a cell that lacks cortical microtubules (MTs) suggests that the AFs are involved in the oriented deposition of MFs. Experimental disruption of AFs with cytochalasin B caused abnormal MF deposition, a fact strongly supporting the above hypothesis. The transverse MFs forming at the base of the apical dome define the diameter and consequently the cylindrical shape of the apical cells. It is suggested that in the brown algal cells examined the AFs play a morphogenetic role similar to that of cortical microtubules in higher plant cells.

Virus assembly in Hincksia hincksiae (Ectocarpales, Phaeophyceae) an electron and fluorescence microscopic study

SummaryThe filamentous brown algaHincksia hincksiae can be infected by a large icosahedral double-stranded DNA virus (HincV-1). The virus shows extended latency and is replicated only in cells homologous to sporangia. Virus formation was studied by transmission electron microscopy, DAPI staining, and β-tubulin immunofluorescence. Inhibition of cytokineses results in multinucleate cells, which are the first indication of virus replication in productive cells; the microtubular cytoskeleton does not seem to be affected by the virus. Replication of viral DNA begins in the nuclei, which increase in size and eventually disintegrate. Virus assembly takes place in a mixed nucleo-/cytoplasm. Capsids bud from cisternae, which are interpreted as modified endoplasmic reticulum aggregated to virus assembly centres. The internal membranous component of the virus is thus derived from the endoplasmic reticulum. The particles are empty (electron translucent) when assembled, and the nucleoprotein core seems to be packaged subsequently through an opening in the capsid. A number of fine structural features not previously reported from brown algae and related to virus formation are described. Our results on Hincksia hincksiae virus are compared with observations made on various other icosahedral DNA viruses infecting eukaryotic algae and animals.