Differentiation Disorders of Chara vulgaris Spermatids following Treatment with Propyzamide.

Microtubules are cytoskeletal cell elements that also build flagella and cilia. Moreover, these structures participate in spermatogenesis and form a microtubular manchette during spermiogenesis. The present study aims to assess the influence of propyzamide, a microtubule-disrupting agent, on alga Chara vulgaris spermatids during their differentiation by means of immunofluorescent and electron microscopy methods. Propyzamide blocks the functioning of the ß-tubulin microtubule subunit, which results in the creation of a distorted shape of a sperm nucleus at some stages. Present ultrastructural studies confirm these changes. In nuclei, an altered chromatin arrangement and nuclear envelope fragmentation were observed in the research as a result of incorrect nucleus-cytoplasm transport behavior that disturbed the action of proteolytic enzymes and the chromatin remodeling process. In the cytoplasm, large autolytic vacuoles and the dilated endoplasmic reticulum (ER) system, as well as mitochondria, were revealed in the studies. In some spermatids, the arrangement of microtubules present in the manchette was disturbed and the structure was also fragmented. The observations made in the research at present show that, despite some differences in the manchette between Chara and mammals, and probably also in the alga under study, microtubules participate in the intramanchette transport (IMT) process, which is essential during spermatid differentiation. In the present study, the effect of propyzamide on Chara spermiogenesis is also presented for the first time; however, the role of microtubule-associated proteins in this process still needs to be elucidated in the literature.

The Role of Cutinsomes in Plant Cuticle Formation.

The cuticle commonly appears as a continuous lipophilic layer located at the outer epidermal cell walls of land plants. Cutin and waxes are its main components. Two methods for cutin synthesis are considered in plants. One that is based on enzymatic biosynthesis, in which cutin synthase (CUS) is involved, is well-known and commonly accepted. The other assumes the participation of specific nanostructures, cutinsomes, which are formed in physicochemical self-assembly processes from cutin precursors without enzyme involvement. Cutinsomes are formed in ground cytoplasm or, in some species, in specific cytoplasmic domains, lipotubuloid metabolons (LMs), and are most probably translocated via microtubules toward the cuticle-covered cell wall. Cutinsomes may additionally serve as platforms transporting cuticular enzymes. Presumably, cutinsomes enrich the cuticle in branched and cross-linked esterified polyhydroxy fatty acid oligomers, while CUS1 can provide both linear chains and branching cutin oligomers. These two systems of cuticle formation seem to co-operate on the surface of aboveground organs, as well as in the embryo and seed coat epidermis. This review focuses on the role that cutinsomes play in cuticle biosynthesis in S. lycopersicum, O. umbellatum and A. thaliana, which have been studied so far; however, these nanoparticles may be commonly involved in this process in different plants.

Blocking the Bromodomains Function Contributes to Disturbances in Alga Chara vulgaris Spermatids Differentiation.

Bromodomain containing (BRD) proteins play an essential role in many cellular processes. The aim of this study was to estimate activity of bromodomains during alga Chara vulgaris spermatids differentiation. The effect of a bromodomain inhibitor, JQ1 (100 µM), on the distribution of individual stages of spermatids and their ultrastructure was studied. The material was Feulgen stained and analysed in an electron microscope. JQ1 caused shortening of the early stages of spermiogenesis and a reverse reaction at the later stages. Additionally, in the same antheridium, spermatids at distant developmental stages were present. On the ultrastructural level, chromatin fibril system disorders and significantly distended endoplasmic reticulum (ER) cisternae already at the early stages were observed. Many autolytic vacuoles were also visible. The ultrastructural disturbances intensified after prolonged treatment with JQ1. The obtained data show that JQ1 treatment led to changes in the spermatid number and disturbances in chromatin condensation and to cytoplasm reduction. The current studies show some similarities between C. vulgaris and mammals spermiogenesis. Taken together, these results suggest that JQ1 interferes with the spermatid differentiation on many interdependent levels and seems to induce ER stress, which leads to spermatid degeneration. Studies on the role of bromodomains in algae spermiogenesis have not been conducted so far.

Cutinsomes as building-blocks of Arabidopsis thaliana embryo cuticle.

Cutinsomes, spherical nanoparticles containing cutin mono- and oligomers, are engaged in cuticle formation. Earlier they were revealed to participate in cuticle biosynthesis in Solanum lycopersicum fruit and Ornithogalum umbellatum ovary epidermis. Here, transmission electron microscopy (TEM) and immunogold labeling with antibody against the cutinsomes were applied to aerial cotyledon epidermal cells of Arabidopsis thaliana mature embryos. TEM as well as gold particles conjugated with the cutinsome antibody revealed these structures in the cytoplasm, near the plasmalemma, in the cell wall and incorporated into the cuticle. Thus, the cutinsomes most probably are involved in the formation of A. thaliana embryo cuticle and this model plant is another species in which these specific structures participate in the building of cuticle in spite of the lack of the lipotubuloid metabolon. In addition, a mechanism of plant cuticle lipid biosynthesis based on current knowledge is proposed.

Cutinsomes and cuticle enzymes GPAT6 and DGAT2 seem to travel together from a lipotubuloid metabolon (LM) to extracellular matrix of O. umbellatum ovary epidermis.

In the ovary epidermis of O. umbellatum there are lipotubuloid metabolons (LMs), in which synthesis of lipids takes place. This process partly provides nourishment, and partly cuticle building blocks, transformed, among others, with the participation of cutinsomes. The cutinsomes are cutin-building structures, 40-200nm in size, which are formed as a result of self-assembly and self-esterification of hydroxy fatty acids. The cutinsomes, by binding to the cuticle, introduce into it nonlinear, amorphous and cross-linked polymers. Double-immunogold EM observations revealed that enzymes producing elements of cutin (GPAT6) and waxes (WS/DGAT) were found not only as free cytoplasmic molecules but also in many cases they were bound to carboxylate-carboxylic shell of cuntinsomes. Hence, we suppose that these enzymes can move alone or together with the cutinsomes through cytoplasm (pH 6.8-7.0), plasmalemma and the polysaccharide layer of a cell wall to the site of their functioning i.e. to the cuticle (pH 5.0).

Immunocytochemical and immunogold analyses of histone H4 acetylation during Chara vulgaris spermiogenesis.

Histone acetylation is one of the epigenetic modifications which play a significant role in chromatin remodeling during spermiogenesis. Acetylation of the histone H4 makes the exchange of nucleoproteins easy. Research on mouse spermatogenesis showed that H4 histone acetylated at Lys 12 (H4K12ac) was specific only to spermatids. Immunocytochemical studies of Chara vulgaris spermatids with the use of antibodies against the histone H4K12ac revealed positive reactions in spermatid nuclei at stages I-VII. This reaction, connected with nuclear condensation, was much stronger at the early stages of spermiogenesis than later on. Moreover, it showed that at the stages V-VII in spermatid nuclei the presence of the histone H4K12ac corresponded with DNA double-strand breaks. Electron microscopy studies with the use of immunogold technique revealed an almost twofold difference between the mean total numbers of gold grains in the examined chromatin in both stages. This study showed nearly equal distribution of gold grains on condensed and non-condensed chromatin of spermatids at the stage III/IV (48.11% and 51.89%, respectively). In the later stage-VI, when chromatin condensation proceeded, labeling of condensed chromatin reached 57.27%, while in the case of non-condensed chromatin it dropped to 42.73%. The percentage analysis also revealed an increase (above 9%) in condensed chromatin labeling in relation to the stage III/IV. Intensive acetylation of histone H4 at the early stages is correlated with DNA DSBs and transcriptional activity. It facilitates chromatin loosening, which enables the correct course of chromatin remodeling at a later stage. Histone γH2AX also influences chromatin structure in many biological processes in different cell types. Current studies reveal other similarities regarding histone H4 acetylation, not only between Chara and mammals but between invertebrates (molluscs) and vertebrates (bony fishes) as well.

Lipotubuloids in ovary epidermis of Ornithogalum umbellatum act as metabolons: suggestion of the name 'lipotubuloid metabolon'.

A metabolon is a temporary, structural-functional complex formed between sequential metabolic enzymes and cellular elements. Cytoplasmic domains called lipotubuloids are present in Ornithogalum umbellatum ovary epidermis. They consist of numerous lipid bodies entwined with microtubules, polysomes, rough endoplasmic reticulum (RER), and actin filaments connected to microtubules through myosin and kinesin. A few mitochondria, Golgi structures, and microbodies are also observed and also, at later development stages, autolytic vacuoles. Each lipotubuloid is surrounded by a tonoplast as it invaginates into a vacuole. These structures appear in young cells, which grow intensively reaching 30-fold enlargement but do not divide. They also become larger due to an increasing number of lipid bodies formed in the RER by the accumulation of lipids between leaflets of the phospholipid bilayer. When a cell ceases to grow, the lipotubuloids disintegrate into individual structures. Light and electron microscope studies using filming techniques, autoradiography with [(3)H]palmitic acid, immunogold labelling with antibodies against DGAT2, phospholipase D1 and lipase, and double immunogold labelling with antibodies against myosin and kinesin, as well as experiments with propyzamide, a microtubule activity inhibitor, have shown that lipotubuloids are functionally and structurally integrated metabolons [here termed lipotubuloid metabolons (LMs)] occurring temporarily in growing cells. They synthesize lipids in lipid bodies in cooperation with microtubules. Some of these lipids are metabolized and used by the cell as nutrients, and others are transformed into cuticle whose formation is mediated by cutinsomes. The latter were discovered in planta using specific anti-cutinsome antibodies visualized by gold labelling. Moreover, LMs are able to rotate autonomously due to the interaction of microtubules, actin filaments, and motor proteins, which influence microtubules by changing their diameter.

Cutinsomes and lipotubuloids appear to participate in cuticle formation in Ornithogalum umbellatum ovary epidermis: EM-immunogold research.

The outer wall of Ornithogalum umbellatum ovary and the fruit epidermis are covered with a thick cuticle and contain lipotubuloids incorporating (3)H-palmitic acid. This was earlier evidenced by selective autoradiographic labelling of lipotubuloids. After post-incubation in a non-radioactive medium, some marked particles insoluble in organic solvents (similar to cutin matrix) moved to the cuticular layer. Hence, it was hypothesised that lipotubuloids participated in cuticle synthesis. It was previously suggested that cutinsomes, nanoparticles containing polyhydroxy fatty acids, formed the cuticle. Thus, identification of the cutinsomes in O. umbellatum ovary epidermal cells, including lipotubuloids, was undertaken in order to verify the idea of lipotubuloid participation in cuticle synthesis in this species. Electron microscopy and immunogold method with the antibodies recognizing cutinsomes were used to identify these structures. They were mostly found in the outer cell wall, the cuticular layer and the cuticle proper. A lower but still significant degree of labelling was also observed in lipotubuloids, cytoplasm and near plasmalemma of epidermal cells. It seems that cutinsomes are formed in lipotubuloids and then they leave them and move towards the cuticle in epidermal cells of O. umbellatum ovary. Thus, we suggest that (1) cutinsomes could take part in the synthesis of cuticle components also in plant species other than tomato, (2) the lipotubuloids are the cytoplasmic domains connected with cuticle formation and (3) this process proceeds via cutinsomes.

DGAT2 revealed by the immunogold technique in Arabidopsis thaliana lipid bodies associated with microtubules.

The immunogold technique with anti-diacylglycerol acyltransferase 2 (DGAT2) antibody revealed in A. thaliana embryo and root meristematic cells gold particles manifesting the presence of DGAT2 in ER as well as in lipid bodies. This being so, lipid synthesis could take place both in ER and in the lipid bodies. The presence of microtubules around the lipid bodies was evidenced under transmission EM. Detection of tubulin around the lipid bodies using the immunogold technique with anti-a-tubulin is in agreement with the above observations. Connection of lipid bodies with microtubules was also detected by us in other plants where they probably participated in lipid synthesis. A similar phenomenon may take place in A. thaliana.

Lipid body biogenesis and the role of microtubules in lipid synthesis in Ornithogalum umbellatum lipotubuloids.

Lipid bodies present in lipotubuloids of Ornithogalum umbellatum ovary epidermis take the form of a lens between leaflets of ER (endoplasmic reticulum) membrane filled with a highly osmiophilic substance. The two enzymes, DGAT1 [DAG (diacylglycerol) acyltransferase 1] and DGAT2 (DAG acyltransferase 2), involved in this process are synthesized on rough ER and localized in the ER near a monolayer surrounding entities like lipid bodies. After reaching the appropriate size, newly formed lipid bodies transform into mature spherical lipid bodies filled with less osmiophilic content. They appear to be surrounded by a half-unit membrane, with numerous microtubules running adjacently in different directions. The ER, no longer continuous with lipid bodies, makes contact with them through microtubules. At this stage, lipid synthesis takes place at the periphery of lipid bodies. This presumption, and a hypothesis that microtubules are involved in lipid synthesis delivering necessary components to lipid bodies, is based on strong arguments: (i) silver grains first appear over microtubules after a short [3H]palmitic acid incubation and before they are observed over lipid bodies; (ii) blockade of [3H]palmitic acid incorporation into lipotubuloids by propyzamide, an inhibitor of microtubule function; and (iii) the presence of gold grains above the microtubules after DGAT1 and DGAT2 reactions, as also near microtubules after an immunogold method that identifies phospholipase D1.

Microtubule heterogeneity of Ornithogalum umbellatum ovary epidermal cells: non-stable cortical microtubules and stable lipotubuloid microtubules.

Lipotubuloids, structures containing lipid bodies and microtubules, are described in ovary epidermal cells of Ornithogalum umbellatum. Microtubules of lipotubuloids can be fixed in electron microscope fixative containing only buffered OsO(4) or in glutaraldehyde with OsO(4) post-fixation, or in a mixture of OsO(4) and glutaraldehyde. None of these substances fixes cortical microtubules of ovary epidermis of this plant which is characterized by dynamic longitudinal growth. However, cortical microtubules can be fixed with cold methanol according immunocytological methods with the use of ß-tubulin antibodies and fluorescein. The existence of cortical microtubules has also been evidenced by EM observations solely after the use of taxol, microtubule stabilizer, and fixation in a glutaraldehyde/OsO(4) mixture. These microtubules mostly lie transversely, sometimes obliquely, and rarely parallel to the cell axis. Staining, using Ruthenium Red and silver hexamine, has revealed that lipotubuloid microtubules surface is covered with polysaccharides. The presumption has been made that the presence of a polysaccharide layer enhances the stability of lipotubuloid microtubules.

Immunogold evidence suggests that endoplasmic reticulum is the site of protamine-type protein synthesis and participates in translocation of these proteins into the nucleus during Chara vulgaris spermiogenesis.

During spermiogenesis of an alga Chara vulgaris, which in many aspects resembles that of animals, histones are replaced by protamine-type proteins. Our earlier immunocytochemical studies showed that this replacement started during the short stage V of spermiogenesis, when electronograms revealed an extensive system of cisternae and vesicles of endoplasmic reticulum (ER). The present studies revealed at stage V intensive incorporation of labeled (3)H-arginine and (3)H-lysine quickly translocating into a nucleus visualized with pulse-chase autoradiography of semithin sections. The immunogold technique with the use of the antibodies to protamine-type proteins isolated from Chara tomentosa show that both ER cisternae and vesicles are labeled with gold grains, which are absent from the spermatids not treated with the antibodies; thus, the ER is probably the site of the protamine-type protein synthesis. These proteins then are translocated to a nucleus through ER channels connected with the nuclear envelope, as suggested by gold labeling of an inner membrane of the nuclear envelope adjacent to condensed chromatin. The above results correspond with those of other authors showing that in animals, protamines bind with lamin B receptors localized in the inner membrane of the nuclear envelope. A hypothesis has been put forward that during Chara spermiogenesis the inner membrane of the nuclear envelope invaginates into a nucleus together with protamine-type proteins, which become separated from the membrane and penetrate into chromatin.

Immunocytochemical and ultrastructural analyses of the function of the ubiquitin-proteasome system during spermiogenesis with the use of the inhibitors of proteasome proteolytic activity in the alga, Chara vulgaris.

Spermiogenesis in Chara vulgaris and in animals share many common features, including exchange of nucleohistones into nucleoprotamines, remodeling and extreme condensation of chromatin, formation of flagellae and of microtubule manchette, and decrease in cytoplasm volume. In C. vulgaris, spermiogenesis is not preceded by meiosis since this alga is a haplobiont. In the present work we showed that in early spermiogenesis characterized by a significant metabolic activity of spermatids, the inhibitors of proteasomes did not visibly change their ultrastructure but significantly prolonged this process. At late stages of spermiogenesis, MG-132 and epoxomicin dramatically changed the structure of nuclei: regular fibrillar and lamellar structure of chromatin was disturbed and clusters of grains corresponding to aggresomes appeared, but the nucleus shape and cytoplasm structure were the same as in the controls. Immunocytochemical studies revealed that these inhibitors blocked disappearance of histones from nuclei while the structures corresponding to aggresomes were clusters of undegraded ubiquitinated histones, since they gave positive immunosignals indicating the presence of ubiquitin and histones.

Cytochemical and immunocytochemical studies of the localization of histones and protamine-type proteins in spermatids of Chara vulgaris and Chara tomentosa.

Spermiogenesis in Chara algae, which has been divided into 10 phases (sp I-X), is similar to spermiogenesis in animals. The most important process during spermiogenesis in animals is remodeling of chromatin leading to "sleeping genome", being the result the exchange of histone proteins into protamine-like proteins. Cytochemical studies showed in both Chara species (C. vulgaris, C. tomentosa) that at spI-IV phases only histones were present, at spV-VIII phases--the amount of nuclear protamine-type proteins progressively increased and that of histones decreased while at spIX-X only pro-tamine-type proteins were present. This was also confirmed with capillar electrophoresis. In order to localize more precisely both histones and protamines the immunocytochemical studies with the use of anti-protamine antibodies (protamine-type proteins were obtained from C. tomentosa antheridia) and anti-histone H3 antibodies, have been carried out. More specific immunocytochemical studies confirmed cytochemical results including the exchange of histones into protamine-type during spermiogenesis (spV-VIII) in both Chara species. At phase V spermiogenesis these strong strand-like anti-protamine signals were observed in cytoplasm which might suggest that protamine synthesis took place in ER.

The influence of epoxomicin, inhibitor of proteasomal proteolytic activity, on spermiogenesis in Chara vulgaris.

The influence of 48-h treatment with epoxomicin, an inhibitor of proteolytic activity of proteasomes, at the concentration 10 microM, on spermiogenesis in algae Chara vulgaris was examined. In the presence of the inhibitor, the frequency of early spermiogenesis phases significantly increased, the number of spermatids in mid-phases decreased and disappearance of late phases was observed. A hypothesis has been put forward that epoxomicin stops spermiogenesis during the period of preparation to further deep reorganisation of spermatids by blocking proteolysis of short-lived regulatory proteins which are responsible among others for triggering the exchange of nucleohistones into nucleoprotamines.