A light and electron microscopical study on the resting cyst of the tintinnid Schmidingerella (Alveolata, Ciliophora) including a phylogeny-aware comparison.

Resting cysts protect ciliates against adverse environmental conditions. The morphology and ultrastructure of resting cysts has been described in very few Oligotrichea, a group of mainly marine planktonic ciliates. The present study provides the first ultrastructural data for loricate choreotrichids, applying light and electron microscopy on the cysts of the tintinnid Schmidingerella meunieri (Kofoid and Campbell, 1929) Agatha and Strüder-Kypke, 2012. The morphology of live cysts and the wall ultrastructure of cryofixed cysts were morphometrically analysed. The resting cyst is roughly flask-shaped, broadening to a slightly concave, laterally protruding anterior plate. An emergence pore closed by a skull cap-shaped papula is directed to the bottom of the lorica on the opposite side of the cyst. The cyst wall consists of an ectocyst, mesocyst, and endocyst differing in thickness, structure, and nitrogen concentration as revealed by conventional transmission electron microscopy, electron energy loss spectroscopy, and electron spectroscopic imaging. The cysts of S. meunieri belong to the kinetosome-resorbing type, which also occurs in the majority of hypotrich ciliates. Two main features (flask-shape and presence of an emergence pore) are shared with the closely related aloricate choreotrichids and oligotrichids, distinguishing the Oligotrichea from the hypotrich and the more distantly related euplotid ciliates.

Raman imaging of Micrasterias: new insights into shape formation.

The algae Micrasterias with its star-shaped cell pattern is a perfect unicellular model system to study morphogenesis. How the indentations are formed in the primary cell wall at exactly defined areas puzzled scientists for decades, and they searched for chemical differences in the primary wall of the extending tips compared to the resting indents. We now tackled the question by Raman imaging and scanned in situ Micrasterias cells at different stages of development. Thousands of Raman spectra were acquired from the mother cell and the developing semicell to calculate chemical images based on an algorithm finding the most different Raman spectra. Each of those spectra had characteristic Raman bands, which were assigned to molecular vibrations of BaSO4, proteins, lipids, starch, and plant cell wall carbohydrates. Visualizing the cell wall carbohydrates revealed a cell wall thickening at the indentations of the primary cell wall of the growing semicell and uniplanar orientation of the cellulose microfibrils to the cell surface in the secondary cell wall. Crystalline cellulose dominated in the secondary cell wall spectra, while in the primary cell wall spectra, also xyloglucan and pectin were reflected. Spectral differences between the indent and tip region of the primary cell wall were scarce, but a spectral mixing approach pointed to more cellulose fibrils deposited in the indent region. Therefore, we suggest that cell wall thickening together with a denser network of cellulose microfibrils stiffens the cell wall at the indent and induces different cell wall extensibility to shape the lobes.

Winter survival of the unicellular green alga Micrasterias denticulata: insights from field monitoring and simulation experiments.

Peat bog pools around Tamsweg (Lungau, Austria) are typical habitats of the unicellular green alga Micrasterias denticulata. By measurement of water temperature and irradiation throughout a 1-year period (2018/2019), it was intended to assess the natural environmental strain in winter. Freezing resistance of Micrasterias cells and their ability to frost harden and become tolerant to ice encasement were determined after natural hardening and exposure to a cold acclimation treatment that simulated the natural temperature decrease in autumn. Transmission electron microscopy (TEM) was performed in laboratory-cultivated cells, after artificial cold acclimation treatment and in cells collected from field. Throughout winter, the peat bog pools inhabited by Micrasterias remained unfrozen. Despite air temperature minima down to -17.3 °C, the water temperature was mostly close to +0.8 °C. The alga was unable to frost harden, and upon ice encasement, the cells showed successive frost damage. Despite an unchanged freezing stress tolerance, significant ultrastructural changes were observed in field-sampled cells and in response to the artificial cold acclimation treatment: organelles such as the endoplasmic reticulum and thylakoids of the chloroplast showed distinct membrane bloating. Still, in the field samples, the Golgi apparatus appeared in an impeccable condition, and multivesicular bodies were less frequently observed suggesting a lower overall stress strain. The observed ultrastructural changes in winter and after cold acclimation are interpreted as cytological adjustments to winter or a resting state but are not related to frost hardening as Micrasterias cells were unable to improve their freezing stress tolerance.

The red alga Tsunamia transpacifica (Stylonematophyceae) from plastic drift shows adaptation to its uncommon habitat in ultrastructure and soluble low molecular weight carbohydrate composition.

The recently described red alga Tsunamia transpacifica (Stylonematophyceae) was previously isolated from plastic drift found at the pacific coast, but the natural habitat remains unknown. Here, we investigate ultrastructural details and the low molecular weight soluble carbohydrate composition to get further insight into the adaptation to this uncommon habitat. By means of high pressure freeze fixation, followed by freeze substitution, we could detect an up to 2-µm-thick cell wall surrounded by a distinct layer of extracellular polymeric substances (EPS), likely responsible for the adhering capacities of Tsunamia. The central position of the nucleus and multilobed parietal chloroplast, already observed by light microscopy, could be confirmed. The ultrastructure revealed large electron-dense bodies (EB) in the central cytoplasm, likely resembling degradation products of the chloroplast. Interestingly, these structures contained phosphorous and cobalt, and iron was found in smaller rounded electron-dense bodies by electron energy loss spectroscopy (EELS). Accumulation of these elements suggests a high biosorption activity of Tsunamia. Liquid chromatography-mass spectrometry (LC-MS) data showed the presence of two heterosides (floridoside and digeneaside) together with the polyol sorbitol, which are known as organic osmolytes and compatible solutes. Taken together, these are the first observations on ultrastructural details, element storage and accumulation of protective compounds are contributing to our understanding of the ultrastructural and osmotic solute basis for the ability of Tsunamia to thrive on plastic surfaces.

Gold nanoparticles (AuNPs) impair LPS-driven immune responses by promoting a tolerogenic-like dendritic cell phenotype with altered endosomal structures.

Dendritic cells (DCs) shape immune responses by influencing T-cell activation. Thus, they are considered both an interesting model for studying nano-immune interactions and a promising target for nano-based biomedical applications. However, the accentuated ability of nanoparticles (NPs) to interact with biomolecules may have an impact on DC function that poses an unexpected risk of unbalanced immune reactions. Here, we investigated the potential effects of gold nanoparticles (AuNPs) on DC function and the consequences for effector and memory T-cell responses in the presence of the microbial inflammatory stimulus lipopolysaccharide (LPS). Overall, we found that, in the absence of LPS, none of the tested NPs induced a DC response. However, whereas 4-, 8-, and 11 nm AuNPs did not modulate LPS-dependent immune responses, 26 nm AuNPs shifted the phenotype of LPS-activated DCs toward a tolerogenic state, characterized by downregulation of CD86, IL-12 and IL-27, upregulation of ILT3, and induction of class E compartments. Moreover, this DC phenotype was less proficient in promoting Th1 activation and central memory T-cell proliferation. Taken together, these findings support the perception that AuNPs are safe under homeostatic conditions; however, particular care should be taken in patients experiencing a current infection or disorders of the immune system.

Fusion of Mitochondria to 3-D Networks, Autophagy and Increased Organelle Contacts are Important Subcellular Hallmarks during Cold Stress in Plants.

Low temperature stress has a severe impact on the distribution, physiology, and survival of plants in their natural habitats. While numerous studies have focused on the physiological and molecular adjustments to low temperatures, this study provides evidence that cold induced physiological responses coincide with distinct ultrastructural alterations. Three plants from different evolutionary levels and habitats were investigated: The freshwater alga Micrasterias denticulata, the aquatic plant Lemna sp., and the nival plant Ranunculus glacialis. Ultrastructural alterations during low temperature stress were determined by the employment of 2-D transmission electron microscopy and 3-D reconstructions from focused ion beam-scanning electron microscopic series. With decreasing temperatures, increasing numbers of organelle contacts and particularly the fusion of mitochondria to 3-dimensional networks were observed. We assume that the increase or at least maintenance of respiration during low temperature stress is likely to be based on these mitochondrial interconnections. Moreover, it is shown that autophagy and degeneration processes accompany freezing stress in Lemna and R. glacialis. This might be an essential mechanism to recycle damaged cytoplasmic constituents to maintain the cellular metabolism during freezing stress.

Cell Wall Reinforcements Accompany Chilling and Freezing Stress in the Streptophyte Green Alga Klebsormidium crenulatum.

Adaptation strategies in freezing resistance were investigated in Klebsormidium crenulatum, an early branching streptophyte green alga related to higher plants. Klebsormidium grows naturally in unfavorable environments like alpine biological soil crusts, exposed to desiccation, high irradiation and cold stress. Here, chilling and freezing induced alterations of the ultrastructure were investigated. Control samples (kept at 20°C) were compared to chilled (4°C) as well as extracellularly frozen algae (-2 and -4°C). A software-controlled laboratory freezer (AFU, automatic freezing unit) was used for algal exposure to various temperatures and freezing was manually induced. Samples were then high pressure frozen and cryo-substituted for electron microscopy. Control cells had a similar appearance in size and ultrastructure as previously reported. While chilling stressed algae only showed minor ultrastructural alterations, such as small inward facing cell wall plugs and minor alterations of organelles, drastic changes of the cell wall and in organelle distribution were found in extracellularly frozen samples (-2°C and -4°C). In frozen samples, the cytoplasm was not retracted from the cell wall, but extensive three-dimensional cell wall layers were formed, most prominently in the corners of the cells, as determined by FIB-SEM and TEM tomography. Similar alterations/adaptations of the cell wall were not reported or visualized in Klebsormidium before, neither in controls, nor during other stress scenarios. This indicates that the cell wall is reinforced by these additional wall layers during freezing stress. Cells allowed to recover from freezing stress (-2°C) for 5 h at 20°C lost these additional cell wall layers, suggesting their dynamic formation. The composition of these cell wall reinforcement areas was investigated by immuno-TEM. In addition, alterations of structure and distribution of mitochondria, dictyosomes and a drastically increased endoplasmic reticulum were observed in frozen cells by TEM and TEM tomography. Measurements of the photosynthetic oxygen production showed an acclimation of Klebsormidium to chilling stress, which correlates with our findings on ultrastructural alterations of morphology and distribution of organelles. The cell wall reinforcement areas, together with the observed changes in organelle structure and distribution, are likely to contribute to maintenance of an undisturbed cell physiology and to adaptation to chilling and freezing stress.

A new technical approach for preparing frozen biological samples for electron microscopy.

BACKGROUND: Many methodological approaches have focused so far on physiological and molecular responses of plant tissues to freezing but only little knowledge is available on the consequences of extracellular ice-formation on cellular ultrastructure that underlies physiological reactions. In this context, the preservation of a defined frozen state during the entire fixation procedure is an essential prerequisite. However, current techniques are not able to fix frozen plant tissues for transmission electron microscopy (TEM) without interrupting the cold chain. Chemical fixation by glutaraldehyde and osmium tetroxide is not possible at sub-zero temperatures. Cryo-fixation methods, such as high pressure freeze fixation (HPF) representing the state-of-the-art technique for best structural preservation, are not equipped for freezing frozen samples. In order to overcome this obstacle, a novel technical approach for maintaining the cold chain of already frozen plant samples prior and during HPF is presented. RESULTS: Different algae (Micrasterias denticulata, Klebsormidium crenulatum) and higher plant tissues (Lemna sp., Ranunculus glacialis, Pinus mugo) were successfully frozen and prepared for HPF at freezing temperatures (- 2 °C, - 5 °C, - 6 °C) within a newly developed automatic freezing unit (AFU), that we manufactured from a standard laboratory freezer. Preceding tests on photosynthetic electron transport and ability to plasmolyse show that the temperatures applied did not impair electron transport in PSII nor cell vitality. The transfer of the frozen specimen from the AFU into the HPF-device and subsequently cryo-fixation were performed without intermediate thawing. After cryo-substitution and further processing, the resulting TEM-micrographs showed excellent ultrastructure preservation of the different organisms when compared to specimens fixed at ambient temperature. CONCLUSIONS: The method presented allows preserving the ultrastructure of plant cells in the frozen state during cryo-fixation. The resulting high quality TEM-images represent an important step towards a better understanding of the consequences of extracellular ice formation on cellular ultrastructure. It has the potential to provide new insights into changes of organelle structure, identification of intracellular injuries during ice formation and may help to understand freezing and thawing processes in plant tissues. It may be combined with analytical TEM such as electron energy loss spectroscopy (EELS), X-ray analyses (EDX) and various other electron microscopic techniques.

Biomineralization of strontium and barium contributes to detoxification in the freshwater alga Micrasterias.

The unicellular model alga Micrasterias denticulata inhabits acid peat bogs that are highly endangered by pollutants due to their high humidity. As it was known from earlier studies that algae like Micrasterias are capable of storing barium naturally in form of BaSO4 crystals, it was interesting to experimentally investigate distribution and sequestration of barium and the chemically similar alkaline earth metal strontium. Additionally, we intended to analyze whether biomineralization by crystal formation contributes to diminution of the generally toxic effects of these minerals to physiology and structure of this alga which is closely related to higher plants. The results show that depending on the treatment differently shaped crystals are formed in BaCl2 and Cl2Sr exposed Micrasterias cells. Modern microscopic techniques such as analytical TEM by electron energy loss spectroscopy and Raman microscopy provide evidence for the chemical composition of these crystals. It is shown that barium treatment results in the formation of insoluble BaSO4 crystals that develop within distinct compartments. During strontium exposure long rod-like crystals are formed and are surrounded by membranes. Based on the Raman signature of these crystals their composition is attributed to strontium citrate. These crystals are instable and are dissolved during cell death. During strontium as well as barium treatment cell division rates and photosynthetic oxygen production decreased in dependence of the concentration, whereas cell vitality was reduced only slightly. Together with the fact that TEM analyses revealed only minor ultrastructural alterations as consequence of relatively high concentrated BaCl2 and Cl2Sr exposure, this indicates that biomineralization of Sr and Ba protects the cells from severe damage or cell death at least within a particular concentration range and time period. In the case of Sr treatment where ROS levels were found to be elevated, hallmarks for autophagy of single organelles were observed by TEM, indicating beginning degradation processes.

Ionic stress induces fusion of mitochondria to 3-D networks: An electron tomography study.

Mitochondria are central organelles for energy supply of cells and play an important role in maintenance of ionic balance. Consequently mitochondria are highly sensitive to any kind of stress to which they mainly response by disturbance of respiration, ROS production and release of cytochrome c into the cytoplasm. Many of the physiological and molecular stress reactions of mitochondria are well known, yet there is a lack of information on corresponding stress induced structural changes. 3-D visualization of high-pressure frozen cells by FIB-SEM tomography and TEM tomography as used for the present investigation provide an excellent tool for studying structure related mitochondrial stress reactions. In the present study it is shown that mitochondria in the unicellular fresh-water algal model system Micrasterias as well as in the closely related aquatic higher plant Lemna fuse to local networks as a consequence of exposure to ionic stress induced by addition of KCl, NaCl and CoCl2. In dependence on concentration and duration of the treatment, fusion of mitochondria occurs either by formation of protuberances arising from the outer mitochondrial membrane, or by direct contact of the surface of elongated mitochondria. As our results show that respiration is maintained in both model systems during ionic stress and mitochondrial fusion, as well as formation of protuberances are reversible, we assume that mitochondrial fusion is a ubiquitous process that may help the cells to cope with stress. This may occur by interconnecting the respiratory chains of the individual mitochondria and by enhancing the buffer capacity against stress induced ionic imbalance.

The Recombinant Inhibitor of DNA Binding Id2 Forms Multimeric Structures via the Helix-Loop-Helix Domain and the Nuclear Export Signal.

The inhibitor of DNA binding and cell differentiation 2 (Id2) is a helix-loop-helix (HLH) protein that acts as negative dominant regulator of basic-HLH transcription factors during development and in cancer. The structural properties of Id2 have been investigated so far by using synthetic or recombinant fragments reproducing single domains (N-terminus, HLH, C-terminus): the HLH domain tends to dimerize into a four-helix bundle, whereas the flanking regions are flexible. In this work, the intact protein was expressed in E. coli, solubilized from inclusion bodies with urea, purified and dissolved in water at pH~4. Under these conditions, Id2 was obtained with both cysteine residues disulfide-bonded to ß-mercaptoethanol that was present during the solubilization process. Moreover, it existed in a self-assembled state, in which the N-terminus remained highly flexible, while the HLH domain and, surprisingly, part of the C-terminus, which corresponds to the nuclear export signal (NES), both were involved in slowly tumbling, rigid structures. The protein oligomers also formed twisted fibrils that were several micrometers long and up to 80 nm thick. These results show that self-assembly decreases the backbone flexibility of those two protein regions (HLH and NES) that are important for interaction with basic-HLH transcription factors or for nucleocytoplasmic shuttling.

Carbon starvation induces lipid degradation via autophagy in the model alga Micrasterias.

Autophagy is regarded as crucial intracellular process in plant development but also in intracellular stress response. It is known to be controlled by the energy level of the cell and consequently can be triggered by energy deprivation. In this study carbon starvation evoked in different ways was investigated in the freshwater algae model system Micrasterias denticulata (Streptophyta) which is closely related to higher plants. Cells exposed to the photosynthesis inhibiting herbicide DCMU, to the glycolysis inhibitor 2-Deoxy-d-glucose and to complete darkness over up to 9 weeks for preventing metabolism downstream of glucose supply, were investigated by means of Nile red staining and analyses in CLSM, and TEM after cryo-preparation. Our results show that lipid bodies containing both neutral and polar lipids are evenly distributed inside the chloroplast in control cells. During carbon starvation they are displaced into the cytoplasm and are either degraded via autophagy and/or excreted from the cell. Upon discharge from the chloroplast lipid bodies become engulfed by double membranes probably deriving from the ER, thus forming autophagosomes which later fuse with vacuoles. Coincidently indications for autophagy of other organelles and cytoplasmic portions were found during starvation and particularly in DCMU treated cells the number of starch grains decreased and pyrenoids disintegrated. Additionally our molecular data provide first evidence for the existence of a single ATG8 isoform in Micrasterias. ATG8 is known as main regulator of both bulk and selective autophagy in eucaryotes. Our study indicates that lipid degradation during carbon starvation is achieved via "classical" autophagy in the alga Micrasterias. This process has so far only been very rarely observed in plant cells and seems to allow recruitment of lipids for energy supply on the one hand and elimination of unusable or toxicated lipids on the other hand.

Micrasterias as a Model System in Plant Cell Biology.

The unicellular freshwater alga Micrasterias denticulata is an exceptional organism due to its complex star-shaped, highly symmetric morphology and has thus attracted the interest of researchers for many decades. As a member of the Streptophyta, Micrasterias is not only genetically closely related to higher land plants but shares common features with them in many physiological and cell biological aspects. These facts, together with its considerable cell size of about 200 µm, its modest cultivation conditions and the uncomplicated accessibility particularly to any microscopic techniques, make Micrasterias a very well suited cell biological plant model system. The review focuses particularly on cell wall formation and composition, dictyosomal structure and function, cytoskeleton control of growth and morphogenesis as well as on ionic regulation and signal transduction. It has been also shown in the recent years that Micrasterias is a highly sensitive indicator for environmental stress impact such as heavy metals, high salinity, oxidative stress or starvation. Stress induced organelle degradation, autophagy, adaption and detoxification mechanisms have moved in the center of interest and have been investigated with modern microscopic techniques such as 3-D- and analytical electron microscopy as well as with biochemical, physiological and molecular approaches. This review is intended to summarize and discuss the most important results obtained in Micrasterias in the last 20 years and to compare the results to similar processes in higher plant cells.

Chitosan functionalisation of gold nanoparticles encourages particle uptake and induces cytotoxicity and pro-inflammatory conditions in phagocytic cells, as well as enhancing particle interactions with serum components.

BACKGROUND: Gold nanoparticles (AuNPs) are a popular choice for use in medical and biomedical research applications. With suitable functionalisation AuNPs can be applied in drug delivery systems, or can aid in disease diagnosis. One such functionalisation is with chitosan, which enables efficient interaction and permeation of cellular membranes, providing an effective adjuvant. As both AuNPs and chitosan have been shown to have low toxicity and high biocompatibility their proposed use in nanomedicine, either individually or combined, is expanding. However, further toxicological and immunological assessments of AuNP-chitosan conjugates are still needed. Therefore, we have evaluated how AuNP functionalisation with chitosan can affect uptake, cytotoxicity, and immunological responses within mononuclear cells, and influence the interaction of AuNPs with biomolecules within a complex biofluid. The AuNPs used were negatively charged through citrate-coating, or presented either low or high positive charge through chitosan-functionalisation. Uptake by THP-1 cells was assessed via transmission electron microscopy and electron energy loss spectroscopy, pro-inflammatory responses by ELISA and qRT-PCR, and cell death and viability via lactate dehydrogenase release and mitochondrial activity, respectively. Interactions of AuNPs with protein components of a frequently used in vitro cell culture medium supplement, foetal calf serum, were investigated using mass spectrometry. RESULTS: Although cells internalised all AuNPs, uptake rates and specific routes of intracellular trafficking were dependent upon chitosan-functionalisation. Accordingly, an enhanced immune response was found to be chitosan-functionalisation-dependent, in the form of CCL2, IL-1ß, TNF-α and IL-6 secretion, and expression of IL-1ß and NLRP3 mRNA. A corresponding increase in cytotoxicity was found in response to chitosan-coated AuNPs. Furthermore, chitosan-functionalisation was shown to induce an increase in unique proteins associating with these highly charged AuNPs. CONCLUSIONS: It can be concluded that functionalisation of AuNPs with the perceived non-toxic biocompatible molecule chitosan at a high density can elicit functionalisation-dependent intracellular trafficking mechanisms and provoke strong pro-inflammatory conditions, and that a high affinity of these NP-conjugates for biomolecules may be implicit in these cellular responses.

Enhanced Deposition by Electrostatic Field-Assistance Aggravating Diesel Exhaust Aerosol Toxicity for Human Lung Cells.

Air pollution is associated with increased risk of cardiovascular and pulmonary diseases, but conventional air quality monitoring gives no information about biological consequences. Exposing human lung cells at the air-liquid interface (ALI) to ambient aerosol could help identify acute biological responses. This study investigated electrode-assisted deposition of diesel exhaust aerosol (DEA) on human lung epithelial cells (A549) in a prototype exposure chamber. A549 cells were exposed to DEA at the ALI and under submerged conditions in different electrostatic fields (EFs) and were assessed for cell viability, membrane integrity, and IL-8 secretion. Qualitative differences of the DEA and its deposition under different EFs were characterized using scanning mobility particle sizer (SMPS) measurements, transmission electron microscopy (TEM), and electron energy loss spectroscopy (EELS). Upon exposure to DEA only, cell viability decreased and membrane impairment increased for cells at the ALI; submerged cells were unaffected. These responses were enhanced upon application of an EF, as was DEA deposition. No adverse effects were observed for filtered DEA or air only, confirming particle-induced responses. The prototype exposure chamber proved suitable for testing DEA-induced biological responses of cells at the ALI using electrode-assisted deposition and may be useful for analysis of other air pollutants.

Subcellular Sequestration and Impact of Heavy Metals on the Ultrastructure and Physiology of the Multicellular Freshwater Alga Desmidium swartzii.

Due to modern life with increasing traffic, industrial production and agricultural practices, high amounts of heavy metals enter ecosystems and pollute soil and water. As a result, metals can be accumulated in plants and particularly in algae inhabiting peat bogs of low pH and high air humidity. In the present study, we investigated the impact and intracellular targets of aluminum, copper, cadmium, chromium VI and zinc on the filamentous green alga Desmidium swartzii, which is an important biomass producer in acid peat bogs. By means of transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) it is shown that all metals examined are taken up into Desmidium readily, where they are sequestered in cell walls and/or intracellular compartments. They cause effects on cell ultrastructure to different degrees and additionally disturb photosynthetic activity and biomass production. Our study shows a clear correlation between toxicity of a metal and the ability of the algae to compartmentalize it intracellularly. Cadmium and chromium, which are not compartmentalized, exert the most toxic effects. In addition, this study shows that the filamentous alga Desmidium reacts more sensitively to aluminum and zinc when compared to its unicellular relative Micrasterias, indicating a severe threat to the ecosystem.

From the nucleus to the plasma membrane: translocation of the nuclear proteins histone H3 and lamin B1 in apoptotic microglia.

Nuclear autoantibodies have been found in patients with autoimmune diseases. One possible source for nuclear antigens are apoptotic cells. However, the mechanism of how apoptotic cells make nuclear factors accessible to the immune system is still elusive. In the present study, we investigated the redistribution of nuclear components after UV irradiation in the microglial cell line BV-2 and in primary mouse microglia at the ultrastructural level. We used transmission electron microscopy-coupled electron energy loss spectroscopy (EELS) to measure phosphorus as an indicator for nucleic acids and immunogold labeling to detect histone H3 and lamin B1 in apoptotic cells. EELS revealed elevated concentrations of phosphorus in nuclear and cytoplasmic condensed chromatin compared to the remaining cytoplasm. Furthermore, immunolabeling of lamin B1 and histone H3 was detected in apoptotic microglia not only in the nucleus, but also in the cytoplasm, and even at the plasma membrane. Confocal images of apoptotic microglia, which were not previously permeabilized, showed patches of histone H3 and lamin B1 labeling at the cell surface. The pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) prevented the occurrence of cytoplasmic condensed chromatin in apoptotic microglia. Our findings indicate that nuclear components leak from the nucleus into the cytoplasm in apoptotic microglia. At least histone H3 and lamin B1 reach the cell surface, this may promote autoreactive processes.

Rescue of heavy metal effects on cell physiology of the algal model system Micrasterias by divalent ions.

Recent studies have shown that metals such as copper, zinc, aluminum, cadmium, chromium, iron and lead cause severe dose-dependent disturbances in growth, morphogenesis, photosynthetic and respiratory activity as well as on ultrastructure and function of organelles in the algal model system Micrasterias denticulata (Volland et al., 2011, 2012; Andosch et al., 2012). In the present investigation we focus on amelioration of these adverse effects of cadmium, chromium and lead by supplying the cells with different antioxidants and essential micronutrients to obtain insight into metal uptake mechanisms and subcellular metal targets. This seems particularly interesting as Micrasterias is adapted to extremely low-concentrated, oligotrophic conditions in its natural bog environment. The divalent ions of iron, zinc and calcium were able to diminish the effects of the metals cadmium, chromium and lead on Micrasterias. Iron showed most ameliorating effects on cadmium and chromium in short- and long-term treatments and improved cell morphogenesis, ultrastructure, cell division rates and photosynthesis. Analytical transmission electron microscopic (TEM) methods (electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI)) revealed that chromium uptake was decreased when Micrasterias cells were pre-treated with iron, which resulted in no longer detectable intracellular chromium accumulations. Zinc rescued the detrimental effects of chromium on net-photosynthesis, respiration rates and electron transport in PS II. Calcium and gadolinium were able to almost completely compensate the inhibiting effects of lead and cadmium on cell morphogenesis after mitosis, respectively. These results indicate that cadmium is taken up by calcium and iron transporters, whereas chromium appears to enter the algae cells via iron and zinc carriers. It was shown that lead is not taken up into Micrasterias at all but exerts its adverse effects on cell growth by substituting cell wall bound calcium. The antioxidants salicylic acid, ascorbic acid and glutathione were not able to ameliorate any of the investigated metal effects on the green alga Micrasterias when added to the culture medium.

3-D analysis of dictyosomes and multivesicular bodies in the green alga Micrasterias denticulata by FIB/SEM tomography.

In the present study we employ FIB/SEM tomography for analyzing 3-D architecture of dictyosomes and formation of multivesicular bodies (MVB) in high pressure frozen and cryo-substituted interphase cells of the green algal model system Micrasterias denticulata. The ability of FIB/SEM of milling very thin 'slices' (5-10 nm), viewing the block face and of capturing cytoplasmic volumes of several hundred µm(3) provides new insight into the close spatial connection of the ER-Golgi machinery in an algal cell particularly in z-direction, complementary to informations obtained by TEM serial sectioning or electron tomography. Our FIB/SEM series and 3-D reconstructions show that interphase dictyosomes of Micrasterias are not only closely associated to an ER system at their cis-side which is common in various plant cells, but are surrounded by a huge "trans-ER" sheath leading to an almost complete enwrapping of dictyosomes by the ER. This is particularly interesting as the presence of a trans-dictyosomal ER system is well known from mammalian secretory cells but not from cells of higher plants to which the alga Micrasterias is closely related. In contrast to findings in plant storage tissue indicating that MVBs originate from the trans-Golgi network or its derivatives our investigations show that MVBs in Micrasterias are in direct spatial contact with both, trans-Golgi cisternae and the trans-ER sheath which provides evidence that both endomembrane compartments are involved in their formation.

Identification of phytochelatins in the cadmium-stressed conjugating green alga Micrasterias denticulata.

Aquatic environments like peat bogs are affected by anthropogenic metal input into the environment. These ecosystems are inhabited by unicellular green algae of the class Zygnematophyceae. In this study the desmid Micrasterias denticulata was stressed with 600 nM Cd, 10 µM Cr and 300 nM Cu for 3 weeks. GSH levels were measured with HPLC and did not differ between the different treatments or the control. According to the metallo-thiolomics concept, mass spectrometry was used as a method for unambiguous thiol peptide identification. PC2, PC3 and PC4 were clearly identified in the Cd stressed sample with UPLC-MS by their MS spectrum and molecular masses. PC2 and PC3 were determined to be the main thiol compounds, while PC4 was only abundant in traces in Micrasterias. In addition, the identity of PC2 and PC3 was confirmed by MS/MS. No PCs were detected in the Cu stressed algae sample. However, in the Cr stressed sample traces of PC2 were indicated by a peak in UPLC-MS at the retention time of the PC2 standard, but the intensity was too low to acquire reliable MS and MS/MS spectra. In this study PCs have been detected for the first time in a green alga of the division Streptophyta, a close relative to higher plants.