Dependence of the mechanical properties of actin/alpha-actinin gels on deformation rate

The cortical cytoplasm, including the cleavage furrow, is largely composed of a network of actin filaments that is rigid even as it is extensively deformed during cytokinesis. Here we address the question of how actin-filament networks such as those in the cortex can be simultaneously rigid (solid-like) and fluid-like. Conventional explanations are that actin filaments rearrange by some combination of depolymerization and repolymerization; fragmentation and annealing of filaments; and inactivation and reestablishment of crosslinks between filaments. We describe the mechanical properties of a model system consisting of actin filaments and Acanthamoeba alpha-actinin, one of several actin crosslinking proteins found in amoeba and other cells. The results suggest another molecular mechanism that may account for the paradoxical mechanical properties of the cortex. When deformed rapidly, these mixtures are 40 times more rigid than actin filaments without alpha-actinin, but when deformed slowly these mixtures were indistinguishable from filaments alone. These time-dependent mechanical properties can be explained by multiple, rapidly rearranging alpha-actinin crosslinks between the actin filaments, a mechanism proposed by Frey-Wyssling to account for the behaviour of cytoplasm long before the discovery of cytoplasmic actin or alpha-actinin. If other actin-filament crosslinking proteins behave like Acanthamoeba alpha-actinin, this mechanism may explain how the cortex recoils elastically from small rapid insults but deforms extensively when minute forces are applied over long periods of time.     

Homology of a yeast actin-binding protein to signal transduction proteins and myosin-I

In yeast, the cortical actin cytoskeleton seems to specify sites of growth of the cell surface. Because the actin-binding protein ABP1p is associated with the cortical cytoskeleton of Saccharomyces cerevisiae, it might be involved in the spatial organization of cell surface growth. ABP1p is localized to the cortical cytoskeleton and its overproduction causes assembly of the cortical actin cytoskeleton at inappropriate sites on the cell surface, resulting in delocalized surface growth. We have now cloned and sequenced the gene encoding ABP1p. ABP1p is a novel protein with a 50 amino-acid C-terminal domain that is very similar to the SH3 domain in the non-catalytic region of nonreceptor tyrosine kinases (including those encoded by the proto-oncogenes c-src and c-abl), in phospholipase C gamma and in alpha-spectrin. We also identified an SH3-related motif in the actin-binding tail domain of myosin-I. The identification of SH3 domains in a family of otherwise unrelated proteins that associate with the membrane cytoskeleton indicates that this domain might serve to bring together signal transduction proteins and their targets or regulators, or both, in the membrane cytoskeleton.     

Reversible stress softening of actin networks

The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes. Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension. Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks.     

Peptide mimetics of an actin-binding site on myosin span two functional domains on actin

The sites on the myosin heavy chain that interact with actin and are responsible for force generation are ill-defined: crosslinking and experiments with isolated domains of the myosin head implicate regions in both the 50K and 20K (molecular weights in thousands) domains of the myosin head (subfragment 1, S1) in this process. We have synthesized peptides from the sequence around the fast-reacting SH1 thiol residue in the 20K domain of S1 in order to delineate precisely an actin-binding site. We used a combination of 1H-NMR and enzyme inhibition assay and also assessed the effects of peptides on skinned rabbit psoas muscle fibres to show that the region of amino acids 690-725 contains an actin-binding site. Peptides from this region bind to actin, act as mixed inhibitors of the actin-stimulated S1 Mg2(+)-ATPase, and influence the contractile force developed in skinned fibres, whereas peptides flanking this sequence are without effect in our test systems. Remarkably, peptides from the N-terminal half of this segment 690-725 increase force development in skinned fibres at submaximal activating concentrations of Ca2+, that is, they behave as calcium-sensitizers; C-terminal peptides, however, inhibit force development without effecting sensitivity to calcium. These different responses indicate that this region is probably binding at two functionally distinct sites on actin.     

丙烯酸十六酯与丙烯酸羟乙酯共聚物凝胶的合成及其性能

合成了丙烯酸十六酯(HDA)与丙烯酸羟乙酯(HEA)的共聚物凝胶,研究了共聚组成对共聚物凝胶性能的影响.结果表明:经二甲亚砜(DMSO)平衡溶胀后的凝胶,在HDA∶HEA(摩尔比)为1∶1时,具有较高的规整性和较高的溶胀倍率.溶剂的极性对聚合物凝胶的规整性有较大的影响.DSC及X射线衍射表明共聚物凝胶具有液晶性能.     

Mechanical strain in actin networks regulates FilGAP and integrin binding to filamin A

Mechanical stresses elicit cellular reactions mediated by chemical signals. Defective responses to forces underlie human medical disorders such as cardiac failure and pulmonary injury. The actin cytoskeleton's connectivity enables it to transmit forces rapidly over large distances, implicating it in these physiological and pathological responses. Despite detailed knowledge of the cytoskeletal structure, the specific molecular switches that convert mechanical stimuli into chemical signals have remained elusive. Here we identify the actin-binding protein filamin A (FLNA) as a central mechanotransduction element of the cytoskeleton. We reconstituted a minimal system consisting of actin filaments, FLNA and two FLNA-binding partners: the cytoplasmic tail of β-integrin, and FilGAP. Integrins form an essential mechanical linkage between extracellular and intracellular environments, with β-integrin tails connecting to the actin cytoskeleton by binding directly to filamin. FilGAP is an FLNA-binding GTPase-activating protein specific for RAC, which in vivo regulates cell spreading and bleb formation. Using fluorescence loss after photoconversion, a novel, high-speed alternative to fluorescence recovery after photobleaching, we demonstrate that both externally imposed bulk shear and myosin-II-driven forces differentially regulate the binding of these partners to FLNA. Consistent with structural predictions, strain increases β-integrin binding to FLNA, whereas it causes FilGAP to dissociate from FLNA, providing a direct and specific molecular basis for cellular mechanotransduction. These results identify a molecular mechanotransduction element within the actin cytoskeleton, revealing that mechanical strain of key proteins regulates the binding of signalling molecules.     

Identification of an actin-binding protein from Dictyostelium as elongation factor 1a

Indirect evidence has implicated an interaction between the cytoskeleton and the protein synthetic machinery. Two recent reports have linked the elongation factor 1a (EF-1a) which is involved in protein synthesis, with the microtubular cytoskeleton. In situ hybridization has, however, revealed that the messages for certain cytoskeletal proteins are preferentially associated with actin filaments. ABP-50 is an abundant actin filament bundling protein of native relative molecular mass 50,000 (50K) isolated from Dictyostelium discoideum. Immunofluorescence studies show that ABP-50 is present in filopodia and other cortical regions that contain actin filament bundles. In addition, ABP-50 binds to monomeric actin in the cytosol of unstimulated cells and the association of ABP-50 with the actin cytoskeleton is regulated during chemotaxis. Through complementary DNA sequencing and subsequent functional analysis, we have identified ABP-50 as D. discoideum EF-1a. The ability of EF-1a to bind reversibly to the actin cytoskeleton upon stimulation could provide a mechanism for spatially and temporally regulated protein synthesis in eukaryotic cells.     

Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins

Most nucleated cells crawl about by extending a pseudopod that is driven by the polymerization of actin filaments in the cytoplasm behind the leading edge of the plasma membrane. These actin filaments are linked into a network by Y-branches, with the pointed end of each filament attached to the side of another filament and the rapidly growing barbed end facing forward. Because Arp2/3 complex nucleates actin polymerization and links the pointed end to the side of another filament in vitro, a dendritic nucleation model has been proposed in which Arp2/3 complex initiates filaments from the sides of older filaments. Here we report, by using a light microscopy assay, many new features of the mechanism. Branching occurs during, rather than after, nucleation by Arp2/3 complex activated by the Wiskott-Aldrich syndrome protein (WASP) or Scar protein; capping protein and profilin act synergistically with Arp2/3 complex to favour branched nucleation; phosphate release from aged actin filaments favours dissociation of Arp2/3 complex from the pointed ends of filaments; and branches created by Arp2/3 complex are relatively rigid. These properties result in the automatic assembly of the branched actin network after activation by proteins of the WASP/Scar family and favour the selective disassembly of proximal regions of the network.     

交联聚合物中蛋白质含量的测定

蛋白质含量的测定是蛋白质研究中必不可少的手段.一般可利用蛋白质的物理性质,如折射率、比重、紫外吸收等进行初步的测定.进而采用化学方法进行严格的定量分析.以往的化学分析方法,如定氮法、双缩脲法[1]、Folin-酚试剂法[2~3]、考马斯亮蓝法[4]等,均是对蛋白质的溶液进行测量     

羧基聚苯乙烯微球共价连接蛋白的定量分析

通过微球表面的羧基在有催化剂的条件下与蛋白质氨基共价连接,并且对微球上包被的蛋白质含量进行测定,探讨了初始加入蛋白量和混匀反应时间对微球蛋白包被率的影响。结果表明羧基微球包被上的蛋白可以精确定量;在蛋白初始加入量为400~1200μg范围内,初始加入蛋白量越多则微球包被蛋白量会增加;确定了混匀反应时间为1h是微球包被蛋白实验的最佳选择。     

ICPA水凝胶的电致溶胀/溶蚀行为研究

以2-丙烯酰胺-2-甲基丙磺酸(AMPS)和甲基丙烯酸二乙胺基乙酯(DEAEM)形成的离子复合物,通过自由基共聚反应制备了新型离子交联聚两性电解质(ICPA),并获得ICPA水凝胶.ICPA水凝胶在电场作用下具有溶胀/溶蚀的双重响应行为,该行为受电压、电解质种类和浓度等多种因素影响.扫描电镜表征在溶胀阶段,凝胶网络保持完整,不发生溶蚀;而在溶蚀阶段,凝胶的网络结构解体.实验结果表明:凝胶的溶蚀行为与溶胀行为响应程度呈同一变化趋势,凝胶网络的紧密程度(即交联的密度和强度)是其溶胀/溶蚀行为的主要决定因素.     

Disruption of the actin cytoskeleton in yeast capping protein mutants

Capping protein controls the addition of actin subunits to the barbed end of actin filaments and nucleates actin polymerization in vitro. Capping protein has been identified in all eukaryotic cells examined so far; it is a heterodimer with subunits of relative molecular masses 32,000-36,000 (alpha-subunit) and 28,000-32,000 (beta-subunit). In skeletal muscle, capping protein (CapZ) probably binds the barbed ends of actin filaments at the Z line. The in vivo role of this protein in non-muscle cells is not known. We report here the characterization of CAP2, the single gene encoding the beta-subunit of capping protein in Saccharomyces cerevisiae. Yeast cells in which the CAP2 gene was disrupted by an insertion or a deletion had an abnormal actin distribution, including the loss of actin cables. The mutant cells were round and large, with a heterogeneous size distribution, and, although viable, grew more slowly than congenic wild-type cells. Chitin, a cell wall component restricted to the mother-bud junction in wild-type budding yeast, was found on the entire mother cell surface in the mutants. The phenotype of CAP2 disruption resembled that of temperature-sensitive mutations in the yeast actin gene ACT1, indicating that capping protein regulates actin-filament distribution in vivo.     

盐诱导乳清蛋白冷凝胶对双歧杆菌的保护作用

探讨盐诱导乳清分离蛋白（whey protein isolates, WPI）形成的包含双歧杆菌的乳清蛋白冷凝胶（cold-set gels of whey protein containing bifidobacteria,CGWPCB）对双歧杆菌的保护作用。借助扫描电子显微镜和物性仪观察凝胶结构,评价以氯化钙为凝胶剂,形成的CGWPCB对双歧杆菌的保护作用。结果表明,7.5mmol/L CaCl2诱导8% WPI形成的网络状冷凝在pH 1.5的酸环境下对双歧杆菌的保护性最好,作用120min后菌落数下降3个数量级,菌体存活率达到0.42%。说明制备的乳清蛋白冷凝胶对双歧杆菌有良好的保护性,为益生菌产品的开发和应用提供了参考。     

TD1修饰绿色荧光蛋白透皮功能的研究

TD1作为一种含有11个氨基酸的短肽,具有良好的促进蛋白类大分子透皮的功能.过去的研究显示TD1可以有效协助胰岛素通过皮肤进入循环并最终降低血糖.在本研究中我们构建了一种TD1 N端修饰的GFP融合蛋白(TGFP). 我们的实验表明, 与TD1与GFP蛋白的混合物相比, TGFP具有更加良好的透皮功能.这一发现为透皮给药研究提供了一条新的途径,并对解释TD1透皮功能具有指导意义.     

低Ca/Si比的C-S-H凝胶产物在抑制AAR中的作用

碱是混凝土发生碱-集料(AAR)反应的重要因素之一.许多资料报道低Ca/Si比的C-S-H凝胶对碱有强烈的吸附作用.从C-S-H凝胶的组成出发,指出低Ca/Si比的C-S-H凝胶在抑制AAR中的作用,解释了低Ca/Si的C-S-H凝胶对碱的吸附能力强的原因及其影响因素.为在实际工程应用中掺加混合材来抑制AAR反应,提高混凝土耐久性有重大指导意义.     

Spatial control of actin organization at adherens junctions by a synaptotagmin-like protein Btsz

Epithelial tissues maintain a robust architecture during development. This fundamental property relies on intercellular adhesion through the formation of adherens junctions containing E-cadherin molecules. Localization of E-cadherin is stabilized through a pathway involving the recruitment of actin filaments by E-cadherin. Here we identify an additional pathway that organizes actin filaments in the apical junctional region (AJR) where adherens junctions form in embryonic epithelia. This pathway is controlled by Bitesize (Btsz), a synaptotagmin-like protein that is recruited in the AJR independently of E-cadherin and is required for epithelial stability in Drosophila embryos. On loss of btsz, E-cadherin is recruited normally to the AJR, but is not stabilized properly and actin filaments fail to form a stable continuous network. In the absence of E-cadherin, actin filaments are stable for a longer time than they are in btsz mutants. We identify two polarized cues that localize Btsz: phosphatidylinositol (4,5)-bisphosphate, to which Btsz binds; and Par-3. We show that Btsz binds to the Ezrin-Radixin-Moesin protein Moesin, an F-actin-binding protein that is localized apically and is recruited in the AJR in a btsz-dependent manner. Expression of a dominant-negative form of Ezrin that does not bind F-actin phenocopies the loss of btsz. Thus, our data indicate that, through their interaction, Btsz and Moesin may mediate the proper organization of actin in a local domain, which in turn stabilizes E-cadherin. These results provide a mechanism for the spatial order of actin organization underlying junction stabilization in primary embryonic epithelia.     

Ca2+-sensitive gelation of actin filaments by a new protein factor

Two protein factors which bind to, and induce gelation of, actin filaments were purified from Ehrlich tumour cells. Filamin induced Ca2+-insensitive gelation, whereas a new protein factor ('actinogelin') was found to induce Ca2+-sensitive gelation.     

交联羧甲基罗望子胶对Cd2+的吸附研究

以罗望子胶原粉(TKP)为基料,氯乙酸钠(SMCA)为羧甲基醚化剂,环氧氯丙烷(ECH)为交联剂制备了取代度(DS)为0.42,0.64和0.88的3种交联羧甲基罗望子胶(CCMTKP),探究其对水溶液中Cd2+的吸附行为。结果表明:适宜吸附的pH值范围为2~6;吸附剂较佳用量为0.5%(质量分数);3种CCMTKP对Cd2+的吸附在15min内达到平衡,遵从二级动力学方程;吸附符合Langmuir等温吸附,CCMTKP对Cd2+的最大吸附量为64.10mg/g;再生后的CCMTKP吸附性能良好,脱吸附率高,有望作为Cd2+的吸附剂使用。