Ultrastructure and behavior of actin cytoskeleton during cell wall formation in the fission yeast Schizosaccharomyces pombe

Fluorescence microscopy has shown that F-actin of the fission yeast Schizosaccharomyces pombe forms patch, cable and ring structures. To study the relationship between cell wall formation and the actin cytoskeleton, the process of cell wall regeneration from the protoplast was investigated by transmission electron microscopy (TEM), immunoelectron microscopy (IEM) and three-dimensional reconstruction analysis. During cell wall regeneration from the protoplast, localization of F-actin patches was similar to that of the newly synthesized cell wall materials, as shown by confocal laser scanning microscopy (CLSM). In serial sectioned TEM images, filasomes were spherical, 100-300 nm in diameter and consisted of a single microvesicle (35-70 nm diameter) surrounded by fine filaments. Filasomes were adjacent to the newly formed glucan fibrils in single, cluster or rosary forms. By IEM analysis, we found that colloidal gold particles indicating actin molecules were present in the filamentous area of filasomes. Three-dimensional reconstruction images of serial sections clarified that the distribution of filasomes corresponded to the distribution of F-actin patches revealed by CLSM. Thus, a filasome is one of the F-actin patch structures appearing in the cytoplasm at the site of the initial formation of the cell wall and it may play an important role in this action.     

Scanning and negative-staining electron microscopy of protoplast regeneration of a wild-type and two chitin synthase mutants in the pathogenic yeast Candida glabrata

Protoplast regeneration of a wild-type and two mutant strains of Candida glabrata defective in CHS3 homologues encoding class IV chitin synthase in Saccharomyces cerevisiae was examined by scanning and negative-staining electron microscopy. In the wild-type strain, small particles and short filaments appeared on the protoplast surface at 10?min, filamentous materials covered the entire surface of the protoplast at 1?h, granular materials started filling interspaces of filamentous materials at 2?h and regeneration was completed at 6?h. The filamentous materials consisted of microfibrils of various widths ranging from ≤5 to 40?nm, and composed of β-glucan. Protoplasts of the two chitin synthase mutant strains of Δchs3A and Δchs3B completed regeneration essentially by the same process as wild-type strain, although it took more time. These results suggest that CHS3A and CHS3B genes may have important roles in cell wall formation during protoplast regeneration, but can be compensated by other cell wall enzymes.     

Filament Nucleation Tunes Mechanical Memory in Active Polymer Networks

Incorporating growth into contemporary material functionality presents a grand challenge in materials design. The F‐actin cytoskeleton is an active polymer network that serves as the mechanical scaffolding for eukaryotic cells, growing and remodeling in order to determine changes in cell shape. Nucleated from the membrane, filaments polymerize and grow into a dense network whose dynamics of assembly and disassembly, or “turnover,” coordinates both fluidity and rigidity. Here, the extent of F‐actin nucleation is varied from a membrane surface in a biomimetic model of the cytoskeleton constructed from purified protein. It is found that nucleation of F‐actin mediates the accumulation and dissipation of polymerization‐induced F‐actin bending energy. At high and low nucleation, bending energies are low and easily relaxed yielding an isotropic material. However, at an intermediate critical nucleation, stresses are not relaxed by turnover and the internal energy accumulates 100‐fold. In this case, high filament curvatures template further assembly of F‐actin, driving the formation and stabilization of vortex‐like topological defects. Thus, nucleation coordinates mechanical and chemical timescales to encode shape memory into active materials.     

Cell wall formation in regenerating protoplasts of Schizosaccharomyces pombe: study by high resolution, low voltage scanning electron microscopy

The ultrastructure of regenerating cell wall in Schizosaccharomyces pombe protoplasts was studied with a high resolution, low voltage scanning electron microscope (LVSEM). In contrast to the transmission electron microscopy, the LVSEM images give three-dimensional information on the cell wall regeneration in yeast protoplasts. We found that, after only a few minutes of incubation, the protoplasts began to show protuberances in a unipolar manner, and a fibrilar network was formed asymmetrically which covered the whole surface of the protoplasts after 5 hr. The network consisted of microfibrils about 8 to 10 nm wide, forming flat and wavy bundles of various widths and lengths, up to about 200 nm wide and 1 micron long, mainly made of yeast glucan. Free ends of microfibrils were seldom found. Interfibrillar spaces were progressively filled with granular particles and finally the complete cell wall was formed after 12 hr. The fibrillar network was destroyed by the digestion with beta (1----3)-glucanase. When protoplasts were regenerating in the presence of aculeacin A, the fibrillar networks were not formed, resulting in incomplete cell wall formation. These observations suggest that beta-glucan is the main component of the microfibrils and that it plays an important role in the formation of the cell wall in S. pombe.     

Ultrastructural disorder of actin mutant suggests uncoupling of actin-dependent pathway from microtubule-dependent pathway in budding yeast

Temperature-sensitive actin mutant of Saccharomyces cerevisiae act1-1 was studied at a permissive temperature of 23°C by light, fluorescent and electron microscopy to elucidate the roles of actin cytoskeleton in the cycling eukaryotic cells. Mutant cells that grew slowly at the permissive temperature showed aberrations in the cytoskeleton and cell cycle. Mutant cells contained aberrant 'faint actin cables,' that failed in directing of mitochondria, vacuoles and secretory vesicles to the bud and the stray vesicles delivered their content to the mother wall instead of the bud. Bud growth was delayed. Spindle pole bodies and cytoplasmic microtubules did not direct to the bud, and nucleus failed to migrate to the bud. Repeated nuclear divisions produced multinucleated cells, indicating continued cycling of actin mutant cells that failed in the morphogenetic checkpoint, the spindle position checkpoint and cytokinesis. Thus, a single actin mutation appears to indicate uncoupling in space and time of the 'actin cytoskeleton-dependent cytoplasmic pathway of bud development and organelle positioning and inheritance' from the 'microtubule-dependent nuclear division pathway' in a budding yeast cell cycle.     

Morphological and functional analysis of Rac1B in Dictyostelium discoideum

Rac1B, a small GTP-binding protein in Dictyostelium discoideum, is involved in regulation of the actin cytoskeleton. Scanning electron microscopy revealed distinctive phenotypes for the wild-type, constitutively active, constitutively inactive and overexpressing cell lines. Immunofluorescence showed constitutively active Rac1B localized to lamellipodia and sites of cell-to-cell contact. In contrast, constitutively inactive Rac1B was homogeneously distributed throughout the cell. Phalloidin staining demonstrated that active Rac1B co-localizes with F-actin. Amoebae expressing mutant Rac1B exhibited defects in endocytosis, cytokinesis and multicellular development. Overexpression of wild-type Rac1B positively affected fluid-phase endocytosis, whereas expression of either constitutively active or inactive forms of Rac1B inhibited endocytic rates. The greatest defects in cytokinesis were observed in amoebae producing constitutively active Rac1B or overexpressing wild-type Rac1B. These cells were severely multinucleated and divided by traction-mediated cytofission when placed onto a solid surface. Cells expressing mutant Rac1B were unable to form viable fruiting bodies. Elucidating the role of Rac1B in filamentous actin dynamics will lead to a better understanding of cell adhesion, development and cell motility.     

In situ localization of cell wall alpha-1,3-glucan in the fission yeast Schizosaccharomyces pombe

The yeast cell walls of the budding yeast Saccharomyces cerevisiae are well studied and the results show the existence of a framework composed of beta-1,3-glucan. It is reported that the cell wall of the fission yeast Schizosaccharomyces pombe has different components and our analysis by 13C-nuclear magnetic resonance (NMR) spectroscopy also showed there is alpha-1,3-glucan in its cell wall. To refine our understanding of the architecture of the yeast cell wall, we re-examined the cell wall glucans of S. pombe by NMR spectroscopy and prepared antibody against alpha-1,3-glucan, which is a characteristic component of this yeast. By the competitive enzyme-linked immunosorbent assay (ELISA) system, specificity of the antibody was restricted to alpha-1,3-glucan, which did not take a highly ordered structure. We analysed the localization of the cell wall glucans by immunoelectron microscopy. Transmission electron microscope (TEM) images showed that most of the alpha-1,3-glucan was along the cell membrane and appeared to enclose the cytoplasm, supporting previous reports that this glucan is synthesized on the cell membrane.     

系统性淀粉样变性的超微病理诊断

目的:应用光镜(LM)及透射电镜(TEM)观察不同部位淀粉样物质的沉积及超微结构特点,为系统性淀粉样变性提供可靠的病理诊断依据.方法:对21例临床拟诊为系统性淀粉样变性病例的肾脏,腹壁脂肪和直肠粘膜活检组织按常规进行LM及TEM标本制备及病理检查.结果:LM观察显示淀粉样物质在肾脏,腹壁脂肪和直肠粘膜沉积阳性率分别为7...     

基于负染－电镜的Aβ体外聚集表征测试要点分析

本文应用电镜负染色技术,对阿尔茨海默病（ Alzheimer′s disease, AD）中所涉及的β淀粉样肽（β?amyloid peptide,Aβ）的聚集规律进行了分析。通过对样品的浓度、pH值、温度和时间等设置系列梯度,以及调整负染试剂的种类、浓度和染色时间,优化了Aβ寡聚体样品制备以及电镜观察的最佳条件,获得了清晰的Aβ纤维图像并用以估算纤维长度。实验结果表明：经过六氟异丙醇给予单体化处理,并溶于无水二甲基亚砜（ DMSO）的Aβ（100μmol·L－1）,在pH7?2的PBS中4℃孵育24 h,Aβ寡聚体形态清晰,且Aβ42寡聚体比Aβ40寡聚体聚集趋势更明显。在pH 8?0的硼酸缓冲液中,Aβ42（40μmol·L－1）在37℃孵育72 h后,纤维形成明显;而Aβ纤维样品经过煮沸后再制备负染样品,所得电镜图像更为清晰,便于对纤维长度和结构进一步分析。因此电镜负染色技术,可作为一种快捷,直观的Aβ体外聚集形貌表征的质控方法。     

Development of the Fibrillar and Microfibrillar Structure During Biomimetic Mineralization of Wood

Wood is a hierarchical composite, consisting at its lowest hierarchy level of crystalline cellulose elementary fibrils with diameters of 2–4 nm embedded in a matrix of hemicelluloses and lignin. At the micrometer scale, it has a cellular architecture resembling a honeycomb structure. The transformation of the hierarchical wood structure into a silica replica has been reported recently. Its formation process and structural details are studied in this contribution. First, a silica/biopolymer composite is prepared by wood delignification and cell‐wall modification, followed by silica precursor infiltration and condensation. The calcination process is monitored to gain insight into the structure development upon decomposition of the biopolymers. The material changes its architecture gradually from fibrillar structures of 10–20 nm in diameter with homogeneous electron density, into fibrils of 8–10 nm in diameter with inhomogeneous electron density, exhibiting internal sub‐fibrillar structures of about 2 nm in diameter. The steps of the successful replication of the cellulose elementary fibrils into nanopores of similar diameter and orientation in a fibrillar silica matrix are demonstrated. These nanopore replicas of the original cellulose are wound in a steep helix within the macropore walls. These advanced materials may have lightweight structural applications and the nanopores may be advantageous for molecular separation.     

Electron microscopic observation of recombinant Escherichia coli cells overproducing human tumor necrosis factor-alpha mutant as inclusion bodies.

Escherichia coli C600 r-m- carrying plasmid pTNF483 (E. coli [pTNF483]) produces a tumor necrosis factor-alpha (TNF-alpha) mutant protein in an insoluble form. A swollen region was observed in the SEM images to encircle the outside of most of the E. coli [pTNF483] cells just like a bandage. On the other hand, inclusion bodies of the TNF-alpha mutant as large as the short axis of the cell were observed in TEM images. This position was regarded as coinciding with the swollen region of SEM images. The inclusion bodies revealed in the swollen region of the cell envelopes were clearly observed in SEM images of isolated insoluble structures obtained by centrifugal sedimentation of a sonicated cell suspension.     

毛竹茎秆纤维发育过程中Ca2+分布的时空变化

利用焦锑酸盐沉淀技术，对毛竹茎秆纤维发育过程中Ca^2＋的时空变化进行了研究。在纤维原始细胞发育期．Ca^2＋主要分布在细胞壁和细胞核染色质中；随着初生壁的形成，大量的Ca^2＋在液泡膜内侧成稀疏点状分布，在吞噬泡和降解的细胞质中出现Ca^2＋反应颗粒的沉积，液泡内逐渐出现大量的絮状Ca^2＋沉积，在细胞核和细胞壁上没有Ca^2＋分布；随后细胞质中Ca^2＋大量聚集，少量Ca^2＋沿液泡膜内侧分布，而液泡中Ca^2＋含量变得极少，在细胞壁、胞间连丝和细胞核中几乎没有Ca^2＋分布；随着次生壁的形成，胞质内的Ca^2＋浓度增加，并随着次生壁的逐渐加厚而聚集成块状；在次生壁形成的整个过程中，在降解的细胞质、凝聚的染色质、纹孔和运输小泡膜上一直存在Ca^2＋。结果表明：Ca^2＋参与了毛竹茎秆纤维细胞的增殖过程和细胞壁的整个形成过程；胞质Ca^2＋的内流是引起纤维细胞PCD的重要原因之一，并参与了PCD过程中原生质体的降解。     

A comparative atomic force microscopy study on living skin fibroblasts and liver endothelial cells

Atomic force microscopy (AFM) has been used to image a wide variety of cells and has proven to be successful in cellular imaging, by comparing results obtained by AFM with SEM or TEM. The aim of the present study was to investigate further the conditions for AFM imaging of living cells and compare the results with those obtained by SEM. We chose to image skin fibroblast and liver sinusoidal endothelial cells of two different sources, because these cells have been well described and characterized in earlier studies. AFM imaging of living cells mainly reveals submembranous structures, which could not be observed by SEM. This concerns the visualization of the overall cytoskeletal architecture and organelles, without the necessity of any preparative steps. The AFM study of living cells allows a time lapse study of dynamic changes of the actin cytoskeleton under the influence of the cytoskeleton-disturbing drug cytochalasin B in cells that can be followed individually during the process. However, softer samples, such as the fenestrated parts of living rat liver sinusoidal endothelial cells in culture could not be visualized. Apparently, these cell parts are disrupted due to tip-sample interaction in contact mode. To avoid the lateral forces and smearing artefacts of contact mode AFM, non-contact imaging was applied, resulting in images of higher quality. Still, endothelial fenestrae could not be visualized. In contrast, contact imaging of immortomouse liver sinusoidal endothelial cells, which are devoid of fenestrae, could easily be performed and revealed a detailed filamentous cytoskeleton.     

The ruffled border and attachment regions of the apposing membrane of resorbing osteoclasts as visualized from the cytoplasmic face of the membrane

The aim of our present research was to visualize how the plasma membrane is modified and how the cytoskeleton interacts with the attachment and ruffled border regions of resorbing osteoclasts. In order to view the surface modification of membranes and associated cytoskeleton, we employed the method of cell-shearing combined with quick-freezing and rotary replication to expose and replicate an extensive area of the cytoplasmic face of the surface membrane of osteoclasts in contact with synthetic apatite as a substratum. The membrane apposed to the apatite was composed of three different domains: the attachment zone, ruffled border and the remainder. In the attachment zone, a highly organized actin filament network formed dot-shaped, F-actin rich adhesion sites, so-called podosomes, and the actin ring. The cytoskeletal filament of podosomes and actin ring appeared to be in direct contact with the cytoplasmic surface of the underlying membrane. Within the actin ring, individually recognizable podosomes were well preserved, which indicates that the actin ring was probably derived from the fusion of podosomes. After shearing at the ruffled border region, the ruffled border projections and membrane regions among the projections were left behind. These ruffled border projections contained the cytoskeletal network. These actin networks also appeared to be in direct contact with the inner side of the ruffled border membrane or in contact with it via membrane-associated particles. At the basal portion of the ruffled border, numerous clathrin-coated patches or pits were well preserved. Deeper clathrin-coated pits and vesicles were also found, which indicates an active site for receptor-mediated endocytotic events. Clathrin sheets were also observed in the cell periphery outside of the actin ring. This type of clathrin sheets adhered to the apatite substrate, but was not anchored to the actin microfilaments. Our study thus clearly visualized the interaction between the cytoskeletal filaments and the underlying membrane at the ruffled border, attachment zone and podosome in osteoclasts cultured on apatitepellets.     

In situ high-resolution transmission electron microscopy of photocatalytic reactions by excited electrons in ionic liquid

A transmission electron microscope (TEM) sample for observing photocatalysis in a liquid was prepared by using N,N,N-trimetyl-N-propylammonium-bis(trifluoromethanesulfonyl)imide. The ionic liquid (IL) was used as a reaction solvent. Tetrachloroauric acid was dissolved in the IL as gold ion species. Rutile particles were added in the solution as a photocatalyst. The low vapor pressure of the IL enables a diffusing system in high vacuum of TEM. Rutile particles were UV irradiated in that liquid phase. After 3 h UV irradiation, a gold particle of 8 nm diameter was grown on the TiO2 surface. Photonucleation of Au/TiO2 system was discussed from the high-resolution TEM images.     

The role of oxidizing agents in the structural and morphological properties of CeO2 nanoparticles

Cerium oxide (CeO₂) nanoparticles with good crystallinity and smooth surface are prepared by chemical precipitation method with different bases (NH₃, NaOH and KOH) using cerium nitrate as a source material. The effect of precipitating agents on the growth of cerium oxide nanoparticles are investigated by Photoluminescence (PL), X-ray diffraction (XRD), Fourier transform-infra red spectroscopy (FTIR), thermo gravimetric–differential thermal analysis (TG-DTA), Scanning electron microscope (SEM), Transmission electron microscope (TEM), and X-ray Photoelectron Spectroscopy (XPS). Cubic fluorite crystallites are detected by XRD pattern with preferred orientation along (1 1 1) direction. PL spectra revealed the presence of a strong and broad emission band at425 nm due to the blue shift in the visible region. The broad band below 700 cm⁻¹ is due to the envelope of the phonon band of metal oxide (Ce–O) network as revealed by the IR spectra. The TG-DTA curves revealed that the total weight loss of the samples is 19.67% when the samples are heated upto 800 °C. SEM images exhibits weakly agglomerated spheroid crystallites are obtained with the typical size in the range 10–50 nm. TEM images display that the particles are nearly spherical and square in shape with diameter 8–12 nm. XPS spectrum confirms the existence of Ce⁴⁺ oxidation states in CeO₂samples.     

Calnexin associates with Shaker K+ channel protein but is not involved in quality control of subunit folding or assembly.

Calnexin is part of an ER chaperone system that monitors and promotes the proper folding and assembly of glycosylated membrane proteins. To investigate the role of calnexin in the biogenesis of the voltage-dependent Shaker K+ channel, wild-type and mutant Shaker proteins were expressed in mammalian cells. Association with calnexin was assayed by coimmunoprecipitation. Calnexin interacted transiently with wild-type Shaker protein in the ER. In contrast, calnexin failed to associate with an unglycosylated Shaker mutant that makes active, cell surface channels. Therefore, glycosylation of Shaker protein is required for association with calnexin, but calnexin is not required for the proper folding and assembly of Shaker channels. We also investigated whether calnexin is involved in the ER retention of mutant Shaker proteins defective in subunit folding, assembly, or pore formation. Each of the mutant proteins associated transiently with calnexin during biogenesis. Calnexin dissociated from wild-type and mutant proteins with similar time courses. Thus, non-native Shaker proteins escape the folding sensor of the calnexin chaperone system. Furthermore, stable association with calnexin is not the mechanism by which these mutant proteins are retained in the ER. Our results indicate that calnexin is not involved in the quality control of subunit folding, assembly, or pore formation in Shaker K+ channels.     

Detecting left ventricular endocardial and epicardial boundaries by digital two-dimensional echocardiography

Cardiac function is evaluated using echocardiographic analysis of shape attributes, such as the heart wall thickness or the shape change of the heart wall boundaries. This requires that the complete boundaries of the heart wall be detected from a sequence of two-dimensional ultrasonic images of the heart. The image segmentation process is made difficult since these images are plagued by poor intensity contrast and dropouts caused by the intrinsic limitations of the image formation process. Current studies often require having trained operators manually trace the heart walls. A review of previous work is presented, along with how this problem can be viewed in the context of the computer vision area. A novel algorithm is presented for detecting the boundaries. This algorithm first detects spatially significant features based on the measurement of image intensity variations. Since the detection step suffers from false alarms and missing boundary points, further processing uses high-level knowledge about the heart wall to label the detected features for noise rejection and to fill in the missing points by interpolation.