Centrosome and microtubule dynamics in apical cells of Sphacelaria rigidula (Phaeophyceae) treated with nocodazole

Treatment of young thalli of Sphacelaria rigidula with 0.04 μg of nocodazole (Nz) per ml for up to 36 h affects microtubules (Mts) only slightly, but blocks a large number of mitotic cells in metaphase, without disruption of the metaphase plate. Higher concentrations of Nz (0.1 μg/ml) depolymerize interphase Mts. Only a few perinuclear and some short Mrs resist and remain associated with the centrosomes. Fragmented Mrs or groups of Mts sometimes remain in the apical dome. After treatment with 0.1 μg of Nz per ml, prometaphase cells are blocked at metaphase, while post-metaphase cells become binuclear, due to the failure of cytokinesis. With anticentrin immunofluorescence, a positive centrin signal is always observed in the centrosome area. Centrosome duplication is not affected by Nz, but separation is disturbed. After recovering for 2-4 h, most of the blocked metaphases proceed normally. In such cells duplicated centrosomes are seen in different stages of separation. In some cells independent aster-like microtubule configurations appear in the apical dome, occasionally displaying centrin at their centre. During recovery various configurations of bimitosis or multipolar mitosis were found. The multipolar spindles may share common centrosomes. Up to four centrosomes may accompany each nucleus. In some 24 h treated cells, as well as in cells recovering for 2 h, the centrin-positive structure is rod-like, extending in opposite directions from the usual position to the poles. Electron microscopical examination of thin sections revealed that the growth pattern of the apical cells is disrupted after Nz treatment. The observations show that: (a) the Mt cytoskeleton is involved in maintaining the polarity and growth pattern of apical cells, (b) mitosis is blocked by low concentrations of Nz without significant depolymerization of Mrs, (c) the centrosome cycle is independent of the nuclear cycle, (d) centrosome separation and differentiation are disturbed by Nz treatment, (e) during recovery from Nz treatment, centrosomal material that may have separated from the centrosomes, as well as Mt fragments that resisted depolymerization, may operate as Mt nucleation centres

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. © 2000 Taylor & Francis Group, LLC

Cytoskeleton and morphogenesis in brown algae

• Background: Morphogenesis on a cellular level includes processes in which cytoskeleton and cell wall expansion are strongly involved. In brown algal zygotes, microtubules (MTs) and actin filaments (AFs) participate in polarity axis fixation, cell division and tip growth. Brown algal vegetative cells lack a cortical MT cytoskeleton, and are characterized by centriole-bearing centrosomes, which function as microtubule organizing centres. • Scope: Extensive electron microscope and immunofluorescence studies of MT organization in different types of brown algal cells have shown that MTs constitute a major cytoskeletal component, indispensable for cell morphogenesis. Apart from participating in mitosis and cytokinesis, they are also involved in the expression and maintenance of polarity of particular cell types. Disruption of MTs after Nocodazole treatment inhibits cell growth, causing bulging and/or bending of apical cells, thickening of the tip cell wall, and affecting the nuclear positioning. Staining of F-actin using Rhodamine-Phalloidin, revealed a rich network consisting of perinuclear, endoplasmic and cortical AFs. AFs participate in mitosis by the organization of an F-actin spindle and in cytokinesis by an F-actin disc. They are also involved in the maintenance of polarity of apical cells, as well as in lateral branch initiation. The cortical system of AFs was found related to the orientation of cellulose microfibrils (MFs), and therefore to cell wall morphogenesis. This is expressed by the coincidence in the orientation between cortical AFs and the depositing MFs. Treatment with cytochalasin B inhibits mitosis and cytokinesis, as well as tip growth of apical cells, and causes abnormal deposition of MFs. • Conclusions: Both the cytoskeletal elements studied so far, i.e. MTs and AFs are implicated in brown algal cell morphogenesis, expressed in their relationship with cell wall morphogenesis, polarization, spindle organization and cytokinetic mechanism. The novelty is the role of AFs and their possible co-operation with MTs. © The Author 2006. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved

Centrosome and microtubule dynamics in apical cells of Sphacelaria rigidula (Phaeophyceae) treated with nocodazole

Treatment of young thalli of Sphacelaria rigidula with 0.04 μg of nocodazole (Nz) per ml for up to 36 h affects microtubules (Mts) only slightly, but blocks a large number of mitotic cells in metaphase, without disruption of the metaphase plate. Higher concentrations of Nz (0.1 μg/ml) depolymerize interphase Mts. Only a few perinuclear and some short Mrs resist and remain associated with the centrosomes. Fragmented Mrs or groups of Mts sometimes remain in the apical dome. After treatment with 0.1 μg of Nz per ml, prometaphase cells are blocked at metaphase, while post-metaphase cells become binuclear, due to the failure of cytokinesis. With anticentrin immunofluorescence, a positive centrin signal is always observed in the centrosome area. Centrosome duplication is not affected by Nz, but separation is disturbed. After recovering for 2-4 h, most of the blocked metaphases proceed normally. In such cells duplicated centrosomes are seen in different stages of separation. In some cells independent aster-like microtubule configurations appear in the apical dome, occasionally displaying centrin at their centre. During recovery various configurations of bimitosis or multipolar mitosis were found. The multipolar spindles may share common centrosomes. Up to four centrosomes may accompany each nucleus. In some 24 h treated cells, as well as in cells recovering for 2 h, the centrin-positive structure is rod-like, extending in opposite directions from the usual position to the poles. Electron microscopical examination of thin sections revealed that the growth pattern of the apical cells is disrupted after Nz treatment. The observations show that: (a) the Mt cytoskeleton is involved in maintaining the polarity and growth pattern of apical cells, (b) mitosis is blocked by low concentrations of Nz without significant depolymerization of Mrs, (c) the centrosome cycle is independent of the nuclear cycle, (d) centrosome separation and differentiation are disturbed by Nz treatment, (e) during recovery from Nz treatment, centrosomal material that may have separated from the centrosomes, as well as Mt fragments that resisted depolymerization, may operate as Mt nucleation centres

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. © 2000 Taylor & Francis Group, LLC

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. © 2000 Taylor & Francis Group, LLC

Changes in Cell Wall Structure During Rhizoid Formation of Silvetia babingtonii

We examined the ultrastructure of the cell wall and immunolocalization of alginates using specific antibodies against M-rich alginates and MG blocks during rhizoid formation in fucoid zygotes, Silvetia babingtonii. The thallus region of 24-h-old zygotes had a cell wall made of three layers with different fiber distribution. In the 12-h-old zygotes, three layers in the thallus were observed before rhizoid formation, namely the inner, middle, and outer layers. During rhizoid elongation, only the inner layer was apparent close to the rhizoid tip area. Immunoelectron microscopy detected M-rich blocks of alginate on the inner half of the cell wall, irrespective of the number of layers in the thallus and rhizoid regions. The MG blocks were seen to cover a slightly wider area than M-rich alginate blocks. It was suggested that parts of M in mannuronan would be rapidly converted to G, and MG-blocks are generated. Transcriptome analysis was performed using 3 -, 10 -, and 24-h-old zygotes after fertilization to examine the relationship between gene expression and alginate synthesis over time. The expression of two mannuronan C5-epimerase homologs that convert mannuronic acid into guluronic acid in alginates was upregulated or downregulated over the course of the examination