Abnormal formation of the glucan network from regenerating protoplasts in Schizosaccharomyces pombe cps8 actin point mutant

To study the close relationship between the actin cytoskeleton and cell wall formation, the process of cell wall formation in reverting protoplasts of the fission yeast, Schizosaccharomyces pombe, cps8 actin point mutant was investigated by ultra-high-resolution low-voltage scanning electron microscopy (UHR-LVSEM) and transmission electron microscopy (TEM). The protoplast of the cps8 mutant began to form a glucan network in a unipolar manner and to secrete alpha-galactomannan. The site of cell wall formation grew in a cylindrical shape in the wild-type protoplast. The alpha-galactomannan did not fill in the intrafibrillar spaces completely, however, and the fibrils were exposed on the cell surface. UHR-LVSEM images indicated that the glucan fibrils were thin and rope-shaped, forming a looser network than the wild-type. TEM images indicated the finest fibrils were approximately 1.5 nm in diameter, the same diameter as the wild-type. These results suggest that the cps8 mutant was insufficient in developing cross-linkage with the glucan fibrils up to the wide ribbon shape as found in the wild-type [Osumi M et al. (1989) J. Electron Microsc. 38: 457-468; Osumi M (1998) Micron 29: 207-233]. These findings appear to indicate that the actin cytoskeleton controls formation of the glucan network and secretion of beta-1,6-glucan, and confirm the close relationship of the actin cytoskeleton and glucan formation.     

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.     

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.     

The cell cycle, including the mitotic cycle and organelle division cycles, as revealed by cytological observations

It is generally believed that the cell cycle consists essentially of the mitotic cycle, which involves mitosis and cytokinesis. These processes are becoming increasingly well understood at the molecular level. However, successful cell reproduction requires duplication and segregation (inheritance) of all of the cellular contents, including not only the cell-nuclear genome but also intracellular organelles. Eukaryotic cells contain at least three types of double membrane-bounded organelles (cell nucleus, mitochondria and plastids), four types of single membrane-bounded organelles (endoplasmic reticulum, Golgi apparatus, lysosomes and microbodies) and the cytoskeleton, which comprises tubulin-based structures (including microtubules, centrosome and spindle) and actin microfilaments. These membrane-bounded organelles cannot be formed de novo and daughter organelles must be inherited from parent organelles during cell cycle. Regulation of organelle division and its coordination with the progression of the cell cycle involves a sequence of events that are subjected to precise spatio-temporal control. Considering that the cells of higher animals and plants contain many organelles which tend to behave somewhat randomly, there is little information concerning the division and inheritance of these double- and single-membrane-bounded organelles during the cell cycle. Here, we summarize the current cytological and morphological knowledge of the cell cycle, including the division cycles of seven membrane-bounded and some non-membrane-bounded organelles. The underlying mechanisms and the biological relevance of these processes are discussed, particularly with respect to cells of the primitive alga Cyanidioschyzon merolae that have a minimum of organelles. We discuss unsolved problems and future perspectives opened by recent studies.     

Three-dimensional studies of cytoskeletal organizations in cultured thyroid cells by quick-freezing and deep-etching method

Isolated porcine thyroid cells were cultured on collagen gels (control group, TSH-stimulated group, and double-layered culture). They were split or cut to remove cytoplasmic soluble proteins for replica preparations. Some specimens were immunostained with anti-actin antibody or decorated with S1 myosin fragments to identify actin filaments. The basal cell membranes of thyroid cells of monolayer culture were in contact with collagen gels and the apical cell membranes faced the culture medium. Networks of actin filaments were attached to the cytoplasmic sides of the apical cell membranes, while intermediate filaments were localized along the basal ones. The thyroid-stimulating hormone (TSH) treatment induced the formation of microvilli only on the apical cell membranes and the accumulation of actin filaments under the apical cell membranes, indicating the apical-basal polarity of the cells. In double-layered culture, the primitive follicular lumens with microvilli appeared between two adjacent cells. The interaction of cell membranes with collagen gels is a determinant factor in the orientation of apical-basal polarity. Moreover, the TSH treatment and cell-cell contact further intensify the polarization through reorganizing the cytoskeletons.     

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.     

Modification of physicochemical properties of actin filaments suppresses cell fragmentation in the execution phase of staurosporine-induced apoptotic processes

Effects of jasplakinolide (JSP), a stabilizer of F-actin, and latrunculin A (LTA), a destabilizer of F-actin, on a series of events occurring in the execution phase of staurosporine (STS)-induced apoptotic processes were studied using human osteosarcoma 143B cells. Time-dependent apparent increases of the population of cells with collapsed membrane potential of mitochondria (Delta Psi(m)) caused by STS treatment were not due to actual decreases in the Delta Psi(m) per cell, but due to the fragmentation of cells resulting in decreases in the number of active mitochondria per cell. Decreases in the Delta Psi(m) in fragmented cells occurred late in the execution phase. Both JSP and LAT failed to prevent STS-induced release of cytochrome c from mitochondria followed by the activation of caspases 3 and 9, the cleavage of poly (ADP-ribose) polymerase (PARP) and apoptotic nuclear fragmentation. However, both drugs prevented STS-induced apoptotic cell fragmentation and decreases in the Delta Psi(m). These results indicate that physicochemical states of actin filaments play a certain role in the execution phase of STS-induced apoptotic processes.     

Planar Sodium-Nickel Chloride Batteries with High Areal Capacity for Sustainable Energy Storage

High-temperature sodium-nickel chloride (Na-NiCl₂) batteries are a promising solution for stationary energy storage, but the complex tubular geometry of the solid electrolyte represents a challenge for manufacturing. A planar electrolyte and cell design is more compatible with automated mass production. However, the planar cell design also faces a series of challenges, such as the management of molten phases during cycling. As a result, cycling of planar high-temperature cells until now focused on moderate areal capacities and current densities. In this work, planar cells capable of integrating cost-efficient nickel/iron electrodes at a substantially enhanced areal capacity of 150 mAh cm⁻² is presented. Due to a low cell resistance during operation at 300 °C, these cells deliver a specific discharge energy of 300 Wh kg⁻¹ at high discharge current densities of 80 mA cm⁻² (C/2, 10%–100% state-of-charge). This results represent the first demonstration of planar Na-NiCl₂ cells at a commercially relevant combination of areal capacity, cycling rate, and energy efficiency. It is further identified the secondary molten NaAlCl₄ electrolyte to contribute to the cell capacity during cycling. Mitigating electrochemical decomposition of NaAlCl₄ will play an important role in further enhancing both cycling rates and cycle life of high temperature Na-NiCl₂ batteries.     

Failure study of Sn37Pb PBGA solder joints using temperature cycling,random vibration and combined temperature cycling and random vibration tests

In this paper, the tin-lead (Sn-37wt%Pb) eutectic solder joints of plastic ball grid array (PBGA) assemblies are tested using temperature cycling, random vibrations, and combined temperature cycling and vibration loading conditions. The fatigue lives, failure modes for the solder joints and the typical locations of the failed solder joints for single-variable loading and combined loading conditions are compared and analyzed. The results show much earlier solder joint failure for combined loading than that for either temperature cycling or pure vibration loading at room temperature. The primary failure mode is cracking within the bulk solder under temperature cycling, whereas the crack propagation path is along the intermetallic compound (IMC) layer for vibration loading. The solder joints subjected to combined loading exhibit both types of failure modes observed for temperature cycling and vibration loading; in addition, cracking through the IMC and the bulk solder is observed in the combined test. For temperature cycling and vibration loading, the components in the central region of the printed circuit board (PCB) have more failed solder joints than other components, whereas for combined loading, the number of failed solder joints in the components in different locations of the PCB is approximately the same.     

Reshaping Antitumor Immunity with Chemo-Photothermal Integrated Nanoplatform to Augment Checkpoint Blockade-Based Cancer Therapy

Immune checkpoint therapy promotes cytotoxic T lymphocytes (CTLs) activity to eliminate tumors. Nevertheless, their effectiveness in solid tumors is limited by inadequate infiltration of CTLs and suppressive tumor microenvironment (TME). Herein, an anti-PD-1 antibody coupled chemo-photothermal integrated nanoplatform (A/Au@MSMs-P) is proposed to reshape antitumor immunity against cancer. The matrix metalloproteinase-2 (MMP-2) responsive A/Au@MSMs-P promotes the separation of abemaciclib-loaded gold-silica nanoparticles (A/Au@MSMs) and anti-PD-1 antibody, achieving a triple-coordinated strategy to enhance checkpoint blockade therapy. First, chemo-photothermal therapy of A/Au@MSMs induces cell cycle arrest in G1 phase and promotes tumor cells apoptosis to achieve local ablation. Second, immunogenic death of tumor cells promotes the maturation of dendritic cells and recruits antigen-specific CTLs into tumor tissue to promote immune activation. Third, abemaciclib markedly suppresses the proliferation of regulatory T cells (Tregs) to alleviate the immunosuppression of the TME and potentiates the effectiveness of CTLs. This triple-coordinated strategy not only reshapes the antitumor immunity to enhance checkpoint blockade, but also cooperates with chemo-photothermal therapy, leading to improved antitumor efficiency and prolonged survival rate. Taken together, this study presents a promising strategy for improving checkpoint therapy response and has great potential in future cancer therapy.     

Antibody-decorated dystrophin molecule of murine skeletal myofiber as seen by freeze-etching electron microscopy

Dystrophin is the protein product of Duchenne muscular dystrophy gene which is defective in this genetic disorder. Here we identified ultrastructurally the dystrophin molecule from the various cytoskeletons at the cytoplasmic surface of murine myofiber plasma membrane by using quick-freeze, deep-etch, rotary-shadow replica of anti-dystrophin antibody-decorated muscle samples. The molecule was really cytoskeleton and incorporated in the meshwork of the plasma membrane-associated cytoskeletons. The molecule appeared to connect directly and/or through another cytoskeletal molecule with actin filament.     

显微成像技术在氧化石墨烯与A549细胞骨架相互作用研究中的应用

石墨烯及其衍生物纳米材料因具有独特的物理化学性质,被广泛应用于生物医学领域,常被用作抗肿瘤药物的载体。本研究旨在探索氧化石墨烯对非小细胞肺癌细胞A549的生长和迁移的影响及机制。主要实验方法包括live/dead染色、Transwell实验、免疫印迹及原子力显微镜、激光共聚焦显微镜、透射电子显微镜、扫描电子显微镜等显微成像技术。结果显示GO能够穿透细胞膜切入细胞,导致细胞骨架肌动蛋白丝的形态结构发生显著改变;GO能够大量吸附细胞裂解物中F-actin蛋白。这些结果提示GO进入细胞后,可能通过吸附F-actin,破坏细胞骨架的结构,进而影响A549细胞的生长和转移。这些发现为未来以GO为载体的抗肿瘤药物设计提供了新的理论依据。     

Application of a quick-freezing and deep-etching method to pathological diagnosis: a case of elastofibroma

A case of elastofibroma in a middle-aged Japanese woman was examined by the quick-freezing and deep-etching (QF-DE) method, as well as by immunohistochemistry and conventional electron microscopy. The slowly growing tumor developed at the right scapular region and was composed of fibrous connective tissue with unique elastic materials called elastofibroma fibers. A normal elastic fiber consists of a central core and peripheral zone, in which the latter has small aggregates of 10 nm microfibrils. By the QF-DE method, globular structures consisting of numerous fibrils (5-20 nm in width) were observed between the collagen bundles. We could confirm that they were microfibril-rich peripheral zones of elastofibroma fibers by comparing the replica membrane and conventional electron microscopy. One of the characteristics of elastofibroma fibers is that they are assumed to contain numerous microfibrils. Immunohistochemically, spindle tumor cells showed positive immunoreaction for vimentin, whereas alpha-smooth muscle actin, desmin, S-100 protein and CD34 showed negative immunoreaction. By conventional electron microscopy, the tumor cell had thin cytoplasmic processes, pinocytotic vesicles and prominent rough endoplasmic reticulum. Abundant intracytoplasmic filaments were observed in some tumor cells. Thick lamina-like structures along with their inner nuclear membrane were often observed in the tumor cell nuclei. The whole image of the tumor cell was considered to be a periosteal-derived cell, which would produce numerous microfibrils in the peripheral zone of elastofibroma fibers. This study indicated that the QF-DE method could be applied to the pathological diagnosis and analysis of pathomechanism, even for surgical specimens obtained from a patient.     

Manipulating Cell Nanomechanics Using Micropatterns

The nanomechanics of cells have been proven to play important roles in regulating cell behaviors. However, conventional measurement of cell nanomechanics that is processed on uniform surfaces lacks the control of cell morphology, which is reported to significantly influence the cell nanomechanics. This study prepares the micropatterned surfaces using photolithographic micropatterning of photoreactive poly(vinyl alcohol) on cell‐culture polystyrene plates to provide controllable and reproducible cell morphology. The nanomechanics of osteoblasts (NHOst), mesenchymal stem cells (MSCs), and osteosarcoma cell line (MG‐63) are compared on micropatterns. Cell stiffness increases with increase of spreading area due to the ordering of cytoskeleton. Disrupting F‐actin assembly reduces cell stiffness. Meanwhile, cell spreading area influences the expression of phosphoezrin that affects cell surface roughness. Rough membrane is accompanied with high non‐specific adhesion force and migration rate. The influence of spreading area on cancer cell nanomechanics is not as evident as that of normal cells indicating cancer cells behave less dependently on their microenvironment compared to normal cells. The findings of this study suggest that the nanomechanical differences between normal and cancer cells can be used as a biomarker to enhance the diagnosis of cancers. The use of micropatterns should be very useful to compare the nanomechanics of cells.     

Electron microscopy of pathogenic yeasts Cryptococcus neoformans and Exophiala dermatitidis by high-pressure freezing

A high-pressure freezing method was used to observe the ultrastructure of pathogenic yeasts, Cryptococcus neoformans and Exophiala dermatitidis, after freeze-substitution and ultrathin sectioning. The method well preserved the cell structure in its natural state, since the capsule, cell wall, plasma membrane, nucleus, outer and inner nuclear membranes, nuclear pores, nucleolus, mitochondria, mitochondrial membrane and cristae, vacuoles, endoplasmic reticulum, Golgi apparatus, spindle pole body, ribosomes, lipid droplets, microtubules, actin filaments, and glycogen granules were clearly visible. The method was shown to freeze cells as deep as 0.1 mm by sectioning the sample perpendicular to specimen surface. The quality of the cell image was similar to that obtained by a rapid freezing method when compared using the same materials. Thus, high-pressure freezing would be useful for making serial ultrathin sections for three-dimensional analysis of cells, which should give basic information of structure and function of pathogenic yeast cells necessary for finding an effective therapy for mycoses.     

Reversible Conversion Reactions and Small First Cycle Irreversible Capacity Loss in Metal Sulfide‐Based Electrodes Enabled by Solid Electrolytes

Solid‐state batteries can potentially enable new classes of electrode materials which are unstable against liquid electrolytes. Here, SnS nanocrystals, synthesized by a wet chemical method, are used to fabricate a Li‐ion electrode, and the electrochemical properties of this electrode are examined in both solid and liquid electrolyte designs. The SnS‐based solid‐state cell delivers a capacity of 629 mAh g^?1 after 100 cycles and exhibits an unprecedentedly small irreversible capacity in the first cycle (8.2%), while the SnS‐based liquid cell shows a rapid capacity decay and large first cycle irreversible capacity (44.6%). Cyclic voltammetry (CV) experiments show significant solid electrolyte interphase (SEI) formation in the liquid cell during the first discharge while SEI formation by electrolyte reduction in the solid‐state cell appears negligible. Along with CV, X‐ray photoelectron spectroscopy and energy dispersive spectroscopy are used to investigate the differences between the solid‐state and liquid cells. The reaction chemistry of SnS in solid‐state cells is also studied in detail by ex situ X‐ray diffraction and X‐ray absorption spectroscopy. The overarching findings are that use of a solid electrolyte suppresses materials degradation and electrolyte reduction which leads to a small first cycle irreversible capacity and stable cycling.     

Effect of temperature cycling on joint strength of PbSn and AuSnsolders in laser packages

The effect of temperature cycle testing on the joint strength of PbSn and AuSn solders in laser diode packages has been studied experimentally and numerically. Experimental results showed that the joint strength increased as the temperature cycle number increased initially, and then became steady after 400 cycles. The joint strengths of PbSn and AuSn solders increased about 40% to 20% after undergoing 500 temperature cycles, respectively. A finite-element method (FEM) analysis was performed on the calculation of joint strength variation of PbSn and AuSn solders in temperature cycling tests. The coupled thermal-elasticity-plasticity model was employed in the solidification and residual stresses calculation. Simulation results were in good agreement with the experimental measurements that the solder joint strength increased as the temperature cycle increased. Numerical results indicate that the increasing solder joint strength comes from the redistribution of the residual stresses within the solder during temperature cycling tests. The local yielding and the creep effects on the low melting temperature solders will make uniform the residual stresses distribution introduced in the solidification process and increasing the solder joint strength as the temperature cycle number increased. The result suggests that the FEM is an effective method for analyzing and predicting the solder joint strength in laser diode packages     

Impact of temperature cycle profile on fatigue life of solder joints

In this paper the influence of the temperature cycle time history profile on the fatigue life of ball grid array (BGA) solder joints is studied. Temperature time history in a Pentium processor laptop computer was measured for a three-month period by means of thermocouples placed inside the computer. In addition, Pentium BGA packages were subjected to industry standard temperature cycles and also to in-situ measured temperature cycle profiles. Inelastic strain accumulation in each solder joint during thermal cycling was measured by high sensitivity Moire interferometry technique. Results indicate that fatigue life of the solder joint is not independent of the temperature cycle profile used. Industry standard temperature cycle profile leads to conservative fatigue life observations by underestimating the actual number of cycles to failure.