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.

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.

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.

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.

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.

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.

Down-regulation of UDP-glucuronic acid biosynthesis leads to swollen plant cell walls and severe developmental defects associated with changes in pectic polysaccharides.

UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, as its product UDP-glucuronic acid is the common precursor for arabinose, xylose, galacturonic acid, and apiose residues found in the cell wall. In this study we characterize an Arabidopsis thaliana double mutant ugd2,3 that lacks two of the four UGD isoforms. This mutant was obtained from a cross of ugd2 and ugd3 single mutants, which do not show phenotypical differences compared with the WT. In contrast, ugd2,3 has a strong dwarfed phenotype and often develops seedlings with severe root defects suggesting that the UGD2 and UGD3 isoforms act in concert. Differences in its cell wall composition in comparison to the WT were determined using biochemical methods indicating a significant reduction in arabinose, xylose, apiose, and galacturonic acid residues. Xyloglucan is less substituted with xylose, and pectins have a reduced amount of arabinan side chains. In particular, the amount of the apiose containing side chains A and B of rhamnogalacturonan II is strongly reduced, resulting in a swollen cell wall. The alternative pathway to UDP-glucuronic acid with the key enzyme myo-inositol oxygenase is not up-regulated in ugd2,3. The pathway also does not complement the ugd2,3 mutation, likely because the supply of myo-inositol is limited. Taken together, the presented data underline the importance of UDP GlcA for plant primary cell wall formation.

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.

Perturbation of intracellular K(+) homeostasis with valinomycin promotes cell death by mitochondrial swelling and autophagic processes.

Perturbation of cellular K(+) homeostasis is a common motif in apoptosis but it is unknown whether a decrease in intracellular K(+) alone is sufficient to replicate apoptotic hallmarks. We investigated, which mode of cell death is induced by decreasing the intracellular K(+) concentration using valinomycin, a highly K(+)-selective ionophore. Valinomycin treatment induced mitochondrial swelling and minor nuclear changes in cell lines (BV-2, C6, HEK 293), and in primary mouse microglia and astrocytes. In the microglial cell line BV-2, we identified and quantified three phenotypes in valinomycin-exposed cells. The first and most prevalent phenotype (62 ± 2%) was characterized by swollen mitochondria and no chromatin condensation, and the second (25 ± 3%) by swollen mitochondria and slight chromatin condensation. Only the third phenotype (11 ± 4%) fulfilled criteria of apoptosis by having normal-sized mitochondria and strongly condensed chromatin. Valinomycin-induced swelling of mitochondria was not altered by the adenine nucleotide translocase inhibitor bongkrekic acid (BA), the pan caspase inhibitor Z-VAD-FMK, changing extracellular K(+) or Cl(-) concentrations, or the membrane-permeable Ca(2+) chelator BAPTA-AM. Only co-exposure of cells to valinomycin and the Ca(2+) ionophore ionomycin in high K(+) Cl(-)-free extracellular solution suppressed mitochondrial swelling. Ionomycin alone caused shrinkage of mitochondria. Additionally, valinomycin promoted autophagic processes, which were further enhanced by preincubation with BA or with Z-VAD-FMK. Valinomycin-dependent chromatin condensation was inhibited by BA, Z-VAD-FMK, BAPTA-AM, and ionomycin. Our findings demonstrate that mitochondrial swelling and autophagy are common features of valinomycin-exposed cells. Accordingly, valinomycin promotes an autophagic cell death mode, but not apoptosis.

Transcriptional analysis of cell growth and morphogenesis in the unicellular green alga Micrasterias (Streptophyta), with emphasis on the role of expansin.

BACKGROUND: Streptophyte green algae share several characteristics of cell growth and cell wall formation with their relatives, the embryophytic land plants. The multilobed cell wall of Micrasterias denticulata that rebuilds symmetrically after cell division and consists of pectin and cellulose, makes this unicellular streptophyte alga an interesting model system to study the molecular controls on cell shape and cell wall formation in green plants. RESULTS: Genome-wide transcript expression profiling of synchronously growing cells identified 107 genes of which the expression correlated with the growth phase. Four transcripts showed high similarity to expansins that had not been examined previously in green algae. Phylogenetic analysis suggests that these genes are most closely related to the plant EXPANSIN A family, although their domain organization is very divergent. A GFP-tagged version of the expansin-resembling protein MdEXP2 localized to the cell wall and in Golgi-derived vesicles. Overexpression phenotypes ranged from lobe elongation to loss of growth polarity and planarity. These results indicate that MdEXP2 can alter the cell wall structure and, thus, might have a function related to that of land plant expansins during cell morphogenesis. CONCLUSIONS: Our study demonstrates the potential of M. denticulata as a unicellular model system, in which cell growth mechanisms have been discovered similar to those in land plants. Additionally, evidence is provided that the evolutionary origins of many cell wall components and regulatory genes in embryophytes precede the colonization of land.

INTRACELLULAR METAL COMPARTMENTALIZATION IN THE GREEN ALGAL MODEL SYSTEM MICRASTERIAS DENTICULATA (STREPTOPHYTA) MEASURED BY TRANSMISSION ELECTRON MICROSCOPY-COUPLED ELECTRON ENERGY LOSS SPECTROSCOPY1.

Entry of metals in form of aerosols into areas of high air humidity such as peat bogs represents a serious danger for inhabiting organisms such as the unicellular desmid Micrasterias denticulata Bréb. ex Ralfs (Desmidiaceae, Zynematophyceae, Streptophyta). To understand cellular detoxification and tolerance mechanisms, detailed intracellular localization of metal pollutants is required. This study localizes the metals aluminum (Al), zinc (Zn), copper (Cu), and cadmium (Cd) in the green algal model system Micrasterias after experimental exposure to sulfate solutions by highly sensitive TEM-coupled electron energy loss spectroscopy (EELS). Concentrations of the metals shown to induce inhibiting effects on cell development and cytomorphogenesis were chosen for these experiments. Long-term exposure to these metal concentrations led to a pronounced impact on cell physiology expressed by a general decrease in apparent photosynthesis. After long-term treatment, Zn, Al, and Cu were detected in the cell walls by EELS. Zn was additionally found in vacuoles and mucilage vesicles, and Cu in starch grains and also in mucilage vesicles. Elevated amounts of oxygen in areas where Zn, Al, and Cu were localized suggest sequestration of these metals as oxides. The study demonstrated that Micrasterias can cope differently with metal pollutants. In low doses and during a limited time period, the cells were able to compartmentalize Cu the best, followed by Zn and Al. Cu and Zn were taken up into intracellular compartments, whereas Al was only bound to the cell wall. Cd was not compartmentalized at all, which explains its strongest impact on growth, cell division rate, and photosynthesis in Micrasterias.

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.

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.

Salt stress-induced cell death in the unicellular green alga Micrasterias denticulata.

Programmed cell death (PCD) is a key element in normal plant growth and development which may also be induced by various abiotic and biotic stress factors including salt stress. In the present study, morphological, biochemical, and physiological responses of the theoretically immortal unicellular freshwater green alga Micrasterias denticulata were examined after salt (200 mM NaCl or 200 mM KCl) and osmotic stress induced by iso-osmotic sorbitol. KCl caused morphological changes such as cytoplasmic vacuolization, extreme deformation of mitochondria, and ultrastructural changes of Golgi and ER. However, prolonged salt stress (24 h) led to the degradation of organelles by autophagy, a special form of PCD, both in NaCl- and KCl-treated cells. This was indicated by the enclosure of organelles by ER-derived double membranes. DNA of NaCl- and KCl-stressed cells but not of sorbitol-treated cells showed a ladder-like pattern on agarose gel, which means that the ionic rather than the osmotic component of salt stress leads to the activation of the responsible endonuclease. DNA laddering during salt stress could be abrogated by addition of Zn(2+). Neither cytochrome c release from mitochondria nor increase in caspase-3-like activity occurred after salt stress. Reactive oxygen species could be detected within 5 min after the onset of salt and osmotic stress. Respiration, photosynthetic activity, and pigment composition indicated an active metabolism which supports programmed rather than necrotic cell death in Micrasterias after salt stress.