Staurosporine and cytochalasin D induce chondrogenesis by regulation of actin dynamics in different way

Actin cytoskeleton has been known to control and/or be associated with chondrogenesis. Staurosporine and cytochalasin D modulate actin cytoskeleton and affect chondrogenesis. However, the underlying mechanisms for actin dynamics regulation by these agents are not known well. In the present study, we investigate the effect of staurosporine and cytochalasin D on the actin dynamics as well as possible regulatory mechanisms of actin cytoskeleton modulation. Staurosporine and cytochalasin D have different effects on actin stress fibers in that staurosporine dissolved actin stress fibers while cytochalasin D disrupted them in both stress forming cells and stress fiber-formed cells. Increase in the G-/F-actin ratio either by dissolution or disruption of actin stress fiber is critical for the chondrogenic differentiation. Cytochalasin D reduced the phosphorylation of cofilin, whereas staurosporine showed little effect on cofilin phosphorylation. Either staurosporine or cytochalasin D had little effect on the phosphorylation of myosin light chain. These results suggest that staurosporine and cytochalasin D employ different mechanisms for the regulation of actin dynamics and provide evidence that removal of actin stress fibers is crucial for the chondrogenic differentiation.     

Influence of simulated microgravity on the activation of the small GTPase Rho involved in cytoskeletal formation – molecular cloning and sequencing of bovine leukemia-associated guanine nucleotide exchange factor

Background  

The irregular formation of cytoskeletal fibers in spaceflown experimental cells has been observed, but the disorganization process of fibers is still poorly understood. It is well known that the activation of the small GTPase Rho leads to actin stress fibers assembly. This study was performed to evaluate the effect of simulated microgravity on the activation of Rho that is involved in actin fiber remodeling in cells.     

Chemically encapsulated structural elements for probing the mechanical responses of biologically inspired systems

A living cell has a crowded environment with a dense distribution of molecules that requires structured organization for its efficient functioning. One component of this structure, the actin cytoskeleton, is essential for providing mechanical support and facilitating many response activities, including the contraction of muscle cells and chemotaxis. Whereas many investigations have provided insight into the mechanical response from either an in vivo or in vitro perspective, a significant gap exists in determining how the living cell response and the polymer physics response are bridged. The understanding of these systems involves studying their components, including the individual cytoskeletal elements versus the higher-order organism organization in a living cell. Here, we leverage this organization in nature by using a chemistry-based approach to mimic the cytoskeleton in an artificial environment composed of spherically distributed lipid bilayers. This construct bears similarities to the cell membrane. To create a structurally regulated environment, we encapsulate G-actin into giant unilamellar vesicles and then polymerize actin filaments within individual liposomes. We visualize these vesicles with epifluorescence microscopy and confocal microscopy. Atomic force microscopy is then used to probe the mechanical properties of these artificial cells. This polymer cytoskeletal network appears to connect with the lipid bilayer and span the internal space within the liposomes in a manner similar to what is observed in living cells. This work will have implications in a variety of fields, including chemistry, polymer physics, structural biology, and engineering mechanics.     

热场效应下聚酰亚胺纤维力学性质有限元分析

利用COMSOL Multiphysics 5.3软件构建了聚酰亚胺纤维三维有限元模型。 该模型实现了固体传热和表面对表面辐射传热产生的温度场中聚酰亚胺纤维固体力学的计算,重点分析了孔洞的大小、位置和热膨胀系数的差异对聚酰亚胺纤维力学性能的影响。 结果表明,聚酰亚胺(PI)纤维在两端固定约束的条件下,在固体传热和表面对表面辐射传热产生的温度场中呈现相似的应力变化趋势,即聚酰亚胺纤维出现孔洞,使纤维的力学性能降低,孔洞越大,应力分布越不均衡,越不利于纤维性质的稳定;温度越高,应力越大;但随着负轴向热膨胀系数的增加,应力逐渐减小。     

A multichannel dampened flow system for studies on shear stress-mediated mechanotransduction

Shear stresses are powerful regulators of cellular function and potent mediators of the development of vascular disease. We have designed and optimized a system allowing the application of flow to cultured cells in a multichannel format. By using a multichannel peristaltic pump, flow can be driven continuously in the system for long-term studies in multiple isolated flow loops. A key component of the system is a dual-chamber pulse dampener that removes the pulsatility of the flow without the need for having an open system or elevated reservoir. We optimized the design parameters of the pulse dampening chambers for the maximum reduction in flow pulsation while minimizing the fluid needed for each isolated flow channel. Human umbilical vein endothelial cells (HUVECs) were exposed to steady and pulsatile shear stress using the system. We found that cells under steady flow had a marked increased production of eNOS and formation of actin stress fibers in comparison to those under pulsatile flow conditions. Overall, the results confirm the utility of the device as a practical means to apply shear stress to cultured cells in the multichannel format and provide steady, long term flow to microfluidic devices.     

Nanotopographic control of cytoskeletal organization

Growth of 3T3-L1 preadipocytes on a nanoscalar poly(ethylene terephthalate) (PET) surface produced an absence of the intracellular stress fibers characteristic of cell growth on "normal" planar surfaces. This phenomenon was consistently observed from time zero throughout 3 days of culture and was accompanied by changes in paxillin expression along with an approximately 50% decrease in the number of adherent cells in response to 500 dynes/cm(2) of shear stress. This suggests that the cytoskeleton in cells adherent to nanofibrillar surfaces does indeed form, but at a smaller, more difficult to observe scale. We propose a novel mechanism by which the growth and clustering of integrin-associated focal adhesions on surface nanofibrils regulates cytoskeletal development. The width of the extracellular matrix contacts is constrained by the width of the nanofibrils and the absence of any surface between them. The limited dimensions of these point contacts then constrain receptor polymerization and the associated aggregation of actin filaments. The existence of a topographic mechanism leading to growth-limited integrin clustering is hypothesized.     

Using a TEMPO-based fluorescent probe for monitoring oxidative stress in living cells

Generation of too many reactive oxygen species (ROS) in relation to available antioxidants in living cells can cause oxidative stress, which is involved in the development and progression of several serious diseases. 2',7'-Dichlorodihydrofluorescein (DCFH) and its diacetate form, DCFH-DA, are widely used probes for monitoring general oxidative stress in cells, but DCFH oxidation is not always related to ROS. We report here a new method for quantifying cellular oxidative stress using a 2,2,6,6-tetramethyl- piperidine-1-oxyl (TEMPO)-based probe. We tested and verified the probe both in cell-free solutions and in living cells under conditions of increased or reduced oxidative stress. The probe revealed the oxidative stress status in living cells and may be a useful complement to DCFH fluorescent probes.     

Micro-well arrays for 3D shape control and high resolution analysis of single cells

In addition to rigidity, matrix composition, and cell shape, dimensionality is now considered an important property of the cell microenvironment which directs cell behavior. However, available tools for cell culture in two-dimensional (2D) versus three-dimensional (3D) environments are difficult to compare, and no tools exist which provide 3D shape control of single cells. We developed polydimethylsiloxane (PDMS) substrates for the culture of single cells in 3D arrays which are compatible with high-resolution microscopy. Cell adhesion was limited to within microwells by passivation of the flat upper surface through 'wet-printing' of a non-fouling polymer and backfilling of the wells with specific adhesive proteins or lipid bilayers. Endothelial cells constrained within microwells were viable, and intracellular features could be imaged with high resolution objectives. Finally, phalloidin staining of actin stress fibers showed that the cytoskeleton of cells in microwells was 3D and not limited to the cell-substrate interface. Thus, microwells can be used to produce microenvironments for large numbers of single cells with 3D shape control and can be added to a repertoire of tools which are ever more sought after for both fundamental biological studies as well as high throughput cell screening assays.     

Imaging of vascular smooth muscle cells with soft X-ray spectromicroscopy

Using X-ray microscopy and spectromicroscopy, vascular smooth muscle cells (VSMCs) were imaged, prepared without using additional embedding material or staining, but by applying simple, noncryo fixation techniques. The cells were imaged with a compact source transmission X-ray microscope and a scanning transmission X-ray microscope (STXM). With the STXM, spectromicroscopy was performed at the C K-edge and the Ca L(III,II)-edges. VSMCs were chosen because of their high amount of actin stress fibers, so that the actin cytoskeleton should be visible. Other parts of the cell, such as the nucleus and organelles, were also identified from the micrographs. Both in the spectra and the images, the effects of the different preparation procedures were observable. Furthermore, Ca hotspots were detected and their density is determined.     

Asymmetric elastic properties of Dictyostelium discoideum in relation to chemotaxis

In this study we used an AFM to investigate the cytoskeletal properties of live Dictyostelium discoideum cells by measuring the local stiffness across individual living cells. We have examined differences in elastic properties of polarized and unpolarized AX3 wild type and the mutant DAip1- cells, as well as the differences in the front and rear of the cells in relation to organization of the actin cytoskeleton. We found that the average Young's modulus increases upon polarization for the thin regions of the cell and that in polarized cells, the cell front was stiffer than the cell back. We also found that AX3 cells were stiffer than DAip1- cells. This finding suggests that actin polymerization is one of the major determinants of cell motility in Dictyostelium. In addition, a thin agarose film was studied as a model system to examine the influence of the substrate of thin materials probed with the AFM.     

Amphidinolide h, a potent cytotoxic macrolide, covalently binds on actin subdomain 4 and stabilizes actin filament

The actin-targeting toxins have not only proven to be invaluable tools in studies of actin cytoskeleton structure and function but they also served as a foundation for a new class of anticancer drugs. Here, we describe that amphidinolide H (AmpH) targets actin cytoskeleton. AmpH induced multinucleated cells by disrupting actin organization in the cells, and the hyperpolymerization of purified actin into filaments of apparently normal morphology in vitro. AmpH covalently binds on actin, and the AmpH binding site is determined as Tyr200 of actin subdomain 4 by mass spectrometry and halo assay using the yeast harboring site-directed mutagenized actins. Time-lapse analyses showed that AmpH stimulated the formation of small actin-patches, followed by F-actin rearrangement into aggregates via the retraction of actin fibers. These results indicate that AmpH is a novel actin inhibitor that covalently binds on actin.     

分子量分布对等规聚丙烯卷绕丝织构形成的影响

本文研究了分子量分布对聚丙烯卷绕丝结构和性能的影响。结果表明,聚丙烯树脂中的高分子量尾端对卷绕丝的结构有明显影响。在通常的纺丝条件下,用控制降解的聚丙烯树脂纺得的卷绕丝具有低取向的次晶结构;而在相同的粘均分子量和纺丝条件下,高分子量尾端的存在使聚丙烯易于在纺丝线上生成结晶性较高的α-晶型,卷绕丝的取向也随高分子量尾端而显著增大。高分子量尾端对卷绕丝结构的影响,导致卷绕丝牵伸性能和成品纤维力学性能变差。     

碳纳米管改性聚苯硫醚熔纺纤维的结构与性能研究

将多壁碳纳米管(MWCNTs)和聚苯硫醚(PPS)经过熔融挤出后制备成复合材料切片,并采用熔融纺丝法制得碳纳米管改性聚苯硫醚复合纤维.采用扫描电镜(SEM)、拉曼光谱、示差扫描量热分析(DSC)、动态机械分析(DMA)以及力学性能测试等表征手段研究了复合纤维中碳管的分散状态,与基体的界面作用,复合纤维的结晶性能以及力学性能,从而探讨了聚苯硫醚/碳纳米管复合纤维体系的微观结构与宏观性能之间的关系.研究表明,聚苯硫醚分子结构与碳纳米管之间具有的π-π共轭作用使碳管较为均匀的分散在基体中,界面结合较为紧密.同时熔融纺丝过程中的拉伸作用使碳管进一步解缠并使碳管沿纤维拉伸方向取向.另一方面,拉曼光谱显示拉伸作用有效地增强了界面作用,有利于外界应力的传递.碳管的良好分散以及强的界面作用使复合纤维力学性能得到大幅度的提高,当碳管含量达到5 wt%时,复合纤维的模量有了明显的提高,拉伸强度较纯PPS纤维提高了近220%.     

Effects of fiber orientation on the stress distribution in model composites

A Raman-mechanical technique was used to study the relationship between stress distribution and fiber orientation in model composites. Our experimental data generally were consistent with most simplistic mechanical models. A more complete analysis, using the Eshelby equivalent inclusion method, fitted our experimental data exceptionally well. For large applied strains, compressive failure occurred for fibers which were oriented at high angles relative to the draw direction. This occurred because of the lateral shrinkage associated with the matrix when the sample was stretched. The effect of fiber end geometry on the stress distribution for these misaligned fibers was the same as observed earlier. Tapered-end fibers generally carried loads more efficiently in composites than blunt-end fibers.     

Mechanics of cell spreading within 3D-micropatterned environments

Most tissue cells evolve in vivo in a three-dimensional (3D) microenvironment including complex topographical patterns. Cells exert contractile forces to adhere and migrate through the extracellular matrix (ECM). Although cell mechanics has been extensively studied on 2D surfaces, there are too few approaches that give access to the traction forces that are exerted in 3D environments. Here, we describe an approach to measure dynamically the contractile forces exerted by fibroblasts while they spread within arrays of large flexible micropillars coated with ECM proteins. Contrary to very dense arrays of microposts, the density of the micropillars has been chosen to promote cell adhesion in between the pillars. Cells progressively impale onto the micropatterned substrate. They first adhere on the top of the pillars without applying any detectable forces. Then, they spread along the pillar sides, spanning between the elastic micropillars and applying large forces on the substrate. Interestingly, the architecture of the actin cytoskeleton and the adhesion complexes vary over time as cells pull on the pillars. In particular, we observed less stress fibers than for cells spread on flat surfaces. However, prominent actin stress fibers are observed at cell edges surrounding the micropillars. They generate increasing contractile forces during cell spreading. Cells treated with blebbistatin, a myosin II inhibitor, relax their internal tension, as observed by the release of pillar deformations. Moreover, cell spreading on pillars coated with ECM proteins only on their tops are not able to generate significant traction forces. Taken together, these findings highlight the dynamic relationship between cellular forces and acto-myosin contractility in 3D environments, the influence of cytoskeletal network mechanics on cell shape, as well as the importance of cell-ECM contact area in the generation of traction forces.     

Kinetic Insights into the Elongation Reaction of Actin Filaments as a Function of Temperature,Pressure, and Macromolecular Crowding

Actin polymerization is an essential process in eukaryotic cells that provides a driving force for motility and mechanical resistance for cell shape. By using preformed gelsolin–actin nuclei and applying stopped‐flow methodology, we quantitatively studied the elongation kinetics of actin filaments as a function of temperature and pressure in the presence of synthetic and protein crowding agents. We show that the association of actin monomers to the pointed end of double‐stranded helical actin filaments (F‐actin) proceeds via a transition state that requires an activation energy of 56 kJ mol^?1 for conformational and hydration rearrangements, but exhibits a negligible activation volume, pointing to a compact transition state that is devoid of packing defects. Macromolecular crowding causes acceleration of the F‐actin elongation rate and counteracts the deteriorating effect of pressure. The results shed new light on the combined effect of these parameters on the polymerization process of actin, and help us understand the temperature and pressure sensitivity of actin polymerization under extreme conditions.     

Fiber spinning from the nematic melt. V. Flow instabilities in the 75:25 copolyester of p-hydroxybenzoic acid and 2-hydroxy-6-naphthoic acid

We have investigated the rheological properties of the Celanese copolyester with the composition 75 mol% p-hydroxybenzoic acid and 25 mol% 2-hydroxy-6-naphthoic acid (designated as 75HBA/25HNA). Three different samples having inherent viscosities 3.0, 6.0, and 9.2 dL/g were studied. A flow instability is observed at low shear stress which produces an irregularity in the fiber diameter. The surface irregularity becomes less pronounced above a minimum shear stress, indicating that the flow instability originates in the capillary. For these nematic melts, the minimum shear stress marking the onset of more regular flow is found to decrease with increasing temperature and with decreasing inherent viscosity of the copolyester. The die swell ratio of extrudates decreases with increasing shear stress. Fibers were spun from the samples having η_inh = 9.2 and 3.0 dL/g. The initial modulus and tenacity to break for 75HBA/25HNA fibers spun at sufficiently high shear stress to produce smooth filaments are significantly lower than the values we previously reported for fibers of the 58HBA/42HNA copolyester. Moreover, the optimum properties are obtained at relatively low spin-draw ratios. The 75HBA/25HNA polyester also exhibits a yield stress which decreases with increasing temperature. This observation indicates the presence of crystallities at the test temperatures. We believe that the higher content of HBA in the present copolymer gives rise to crystallization of HBA blocks in the thread line and that defects are introduced at higher spin-draw ratios which cause the mechanical properties to become worse.     

有机蒙脱土改性聚氨酯纺丝研究

无机纳米颗粒如二氧化硅、二氧化钛和有机改性层状硅酸盐添加到聚合物如塑料和橡胶基质中,可以提高复合材料的某些力学性能、使用性能和热学性能,如强度、模量、热变形温度、阻隔性能和阻燃性能等等.近年来,将无机纳米颗粒加入到纤维基质中,以期获得纳米复合纤维也成为国内关注