Force production by disassembling microtubules

Microtubules (MTs) are important components of the eukaryotic cytoskeleton: they contribute to cell shape and movement, as well as to the motions of organelles including mitotic chromosomes. MTs bind motor enzymes that drive many such movements, but MT dynamics can also contribute to organelle motility. Each MT polymer is a store of chemical energy that can be used to do mechanical work, but how this energy is converted to motility remains unknown. Here we show, by conjugating glass microbeads to tubulin polymers through strong inert linkages, such as biotin-avidin, that depolymerizing MTs exert a brief tug on the beads, as measured with laser tweezers. Analysis of these interactions with a molecular-mechanical model of MT structure and force production shows that a single depolymerizing MT can generate about ten times the force that is developed by a motor enzyme; thus, this mechanism might be the primary driving force for chromosome motion. Because even the simple coupler used here slows MT disassembly, physiological couplers may modulate MT dynamics in vivo.     

Bead movement by single kinesin molecules studied with optical tweezers

Kinesin, a mechanoenzyme that couples ATP hydrolysis to movement along microtubules, is thought to power vesicle transport and other forms of microtubule-based motility. Here, microscopic silica beads were precoated with carrier protein, exposed to low concentrations of kinesin, and individually manipulated with a single-beam gradient-force optical particle trap ('optical tweezers') directly onto microtubules. Optical tweezers greatly improved the efficiency of the bead assay, particularly at the lowest kinesin concentrations (corresponding to approximately 1 molecule per bead). Beads incubated with excess kinesin moved smoothly along a microtubule for many micrometres, but beads carrying from 0.17-3 kinesin molecules per bead, moved, on average, only about 1.4 microns and then spontaneously released from the microtubule. Application of the optical trap directly behind such moving beads often pulled them off the microtubule and back into the centre of the trap. This did not occur when a bead was bound by an AMP.PNP-induced rigor linkage, or when beads were propelled by several kinesin molecules. Our results are consistent with a model in which kinesin detaches briefly from the microtubule during a part of each mechanochemical cycle, rather than a model in which kinesin remains bound at all times.     

Steps and fluctuations of Listeria monocytogenes during actin-based motility

The actin-based motility of the bacterium, Listeria monocytogenes, is a model system for understanding motile cell functions involving actin polymerization. Although the biochemical and genetic aspects of Listeria motility have been intensely studied, biophysical data are sparse. Here we have used high-resolution laser tracking to follow the trailing ends of Listeria moving in the lamellae of COS7 cells. We found that pauses during motility occur frequently and that episodes of step-like motion often show pauses spaced at about 5.4 nm, which corresponds to the spatial periodicity of F-actin. We occasionally observed smaller steps (<3 nm), as well as periods of motion with no obvious pauses. Clearly, bacteria do not sense cytoplasmic viscoelasticity because they fluctuate 20 times less than adjacent lipid droplets. Instead, bacteria bind their own actin 'tails, and the anchoring proteins can 'step' along growing filaments within the actin tail. Because positional fluctuations are unusually small, the forces of association and propulsion must be very strong. Our data disprove the brownian ratchet models and limit alternative models, such as the 'elastic' brownian ratchet or the 'molecular' ratchet.     

单光刀与单光镊激光微束系统

提出了构建一种用于细胞非接触操作的激光微束系统。该系统由 Nd:YAG激光束经声压、热膨胀、汽化等综合效应实现的光刀和 He- Ne激光束经光学动力学效应实现的光镊组成。将两激光束耦合到显微镜中 ,实现了生物细胞的捕获、移动、翻转、打孔等一系列操作。在此基础上 ,分析了形成光镊所必需产生梯度力场的条件和形成光刀对能量的要求 ,进行了系统的总体设计、关键部件设计和选择 ,构建了一套激光微束操作实验系统 ,得到了预期的试验结果。在该系统上成功地实现了非接触细胞操作 ,并对染色体进行了切割。     

Simultaneous micromanipulation in multiple planes using a self-reconstructing light beam

Optical tweezers are commonly used for manipulating microscopic particles, with applications in cell manipulation, colloid research, manipulation of micromachines and studies of the properties of light beams. Such tweezers work by the transfer of momentum from a tightly focused laser to the particle, which refracts and scatters the light and distorts the profile of the beam. The forces produced by this process cause the particle to be trapped near the beam focus. Conventional tweezers use gaussian light beams, which cannot trap particles in multiple locations more than a few micrometres apart in the axial direction, because of beam distortion by the particle and subsequent strong divergence from the focal plane. Bessel beams, however, do not diverge and, furthermore, if part of the beam is obstructed or distorted the beam reconstructs itself after a characteristic propagation distance. Here we show how this reconstructive property may be utilized within optical tweezers to trap particles in multiple, spatially separated sample cells with a single beam. Owing to the diffractionless nature of the Bessel beam, secondary trapped particles can reside in a second sample cell far removed ( approximately 3 mm) from the first cell. Such tweezers could be used for the simultaneous study of identically prepared ensembles of colloids and biological matter, and potentially offer enhanced control of 'lab-on-a-chip' and optically driven microstructures.     

Massively parallel manipulation of single cells and microparticles using optical images

The ability to manipulate biological cells and micrometre-scale particles plays an important role in many biological and colloidal science applications. However, conventional manipulation techniques--including optical tweezers, electrokinetic forces (electrophoresis, dielectrophoresis, travelling-wave dielectrophoresis), magnetic tweezers, acoustic traps and hydrodynamic flows--cannot achieve high resolution and high throughput at the same time. Optical tweezers offer high resolution for trapping single particles, but have a limited manipulation area owing to tight focusing requirements; on the other hand, electrokinetic forces and other mechanisms provide high throughput, but lack the flexibility or the spatial resolution necessary for controlling individual cells. Here we present an optical image-driven dielectrophoresis technique that permits high-resolution patterning of electric fields on a photoconductive surface for manipulating single particles. It requires 100,000 times less optical intensity than optical tweezers. Using an incoherent light source (a light-emitting diode or a halogen lamp) and a digital micromirror spatial light modulator, we have demonstrated parallel manipulation of 15,000 particle traps on a 1.3 x 1.0 mm2 area. With direct optical imaging control, multiple manipulation functions are combined to achieve complex, multi-step manipulation protocols.     

Banach空间中时滞型多值积分微分包含解的拓扑性质

主要利用收缩核的相关理论,证明了时滞型积分微分包含的解集S（Φ,F）是Banach空间中一个凸子集的收缩.从而改进和推广了前人已有的结果。     

Actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling.

Studies of the actin-based motility of the intracellular pathogens Listeria monocytogenes and Shigella flexneri have provided important insight into the events occurring at the leading edges of motile cells. Like the bacteria Listeria and Shigella, vaccinia virus, a relative of the causative agent of smallpox, uses actin-based motility to spread between cells. In contrast to Listeria or Shigella, the actin-based motility of vaccinia is dependent on an unknown phosphotyrosine protein, but the underlying mechanism remains obscure. Here we show that phosphorylation of tyrosine 112 in the viral protein A36R by Src-family kinases is essential for the actin-based motility of vaccinia. Tyrosine phosphorylation of A36R results in a direct interaction with the adaptor protein Nck and the recruitment of the Ena/VASP family member N-WASP to the site of actin assembly. We also show that Nck and N-WASP are essential for the actin-based motility of vaccinia virus. We suggest that vaccinia virus spreads by mimicking the signalling pathways that are normally involved in actin polymerization at the plasma membrane.     

Optical trapping and manipulation of single cells using infrared laser beams

Use of optical traps for the manipulation of biological particles was recently proposed, and initial observations of laser trapping of bacteria and viruses with visible argon-laser light were reported. We report here the use of infrared (IR) light to make much improved laser traps with significantly less optical damage to a variety of living cells. Using IR light we have observed the reproduction of Escherichia coli within optical traps at power levels sufficient to give manipulation at velocities up to approximately 500 micron s-1. Reproduction of yeast cells by budding was also achieved in IR traps capable of manipulating individual cells and clumps of cells at velocities of approximately micron s-1. Damage-free trapping and manipulation of suspensions of red blood cells of humans and of organelles located within individual living cells of spirogyra was also achieved, largely as a result of the reduced absorption of haemoglobin and chlorophyll in the IR. Trapping of many types of small protozoa and manipulation of organelles within protozoa is also possible. The manipulative capabilities of optical techniques were exploited in experiments showing separation of individual bacteria from one sample and their introduction into another sample. Optical orientation of individual bacterial cells in space was also achieved using a pair of laser-beam traps. These new manipulative techniques using IR light are capable of producing large forces under damage-free conditions and improve the prospects for wider use of optical manipulation techniques in microbiology.     

微粒在飞秒激光光场中的受力特性分析

在分析微粒处于连续高斯光束光场和飞秒脉冲激光光场中轴向光阱力特性的基础上,用数值方法计算了两种情况下微粒所受的轴向力,比较了微粒在两种光场中所受轴向力和热效应的异同,导出了飞秒激光光镊实现对微粒捕获的条件．     

橙色粘球菌四型菌毛膜复合物的发现

细菌四型菌毛(T4P)不仅在生物膜形成、DNA吸收和侵染致病等生物过程中发挥着重要的作用,同样也能够介导固体界面的滑动运动。尽管T4P具有非常重要的作用,但橙色粘球菌T4P系统中蛋白间组装机制的研究仍处于初级阶段。T4P系统pil N/O/P/Q单基因的敲除与回补实验显示敲除菌株导致橙色粘球菌群体运动完全丧失,而pil N/O/P/Q相应基因回补则恢复突变株群体运动。Pil N/O/P/Q蛋白通过酵母双杂交(Y2H)检测蛋白间的相互作用发现,定位于周质空间的Pil P不仅与外膜蛋白Pil Q的C末端相互作用,也与内膜蛋白Pil O相互作用。遗传学与酵母双杂实验结果显示着Pil P蛋白是联通内外膜的桥梁,暗示着Pil N/O/P/Q蛋白形成跨越内膜、细胞周质和外膜的整合复合物结构,并在菌毛延伸和收缩过程中发挥着重要的作用,为阐明橙色粘球菌菌毛的延伸与收缩的分子机制奠定坚实基础。     

海藻酸钠包埋脱氮菌株工艺研究

选用海藻酸钠作为包埋剂,4%CaC l2作为交联剂.通过正交试验和单因素实验,研究了包菌量,海藻酸钠(SA)质量分数,交联时间和小球直径4个因素对海藻酸钠包埋菌株的脱氮性能的影响,以氨氮去除率和总氮去除率为指标,优选包埋条件.在实验范围内的最佳包埋条件下,包埋的脱氮菌株的脱氮性能与其游离状态下的脱氮性能相当.     

海藻酸钠作为固定化细胞包埋剂的研究

通过正交实验和单因素实验,研究了包菌量,海藻酸钠(SA)浓度,交联时间和小球直径四个因素对海藻酸钠包埋菌株的脱氮性能的影响,并优选出最佳包埋条件.在最佳包埋条件下包埋的脱氮菌株的脱氮性能优于其游离状态下的脱氮性能.     

单个红细胞的拉曼光谱研究

研究了不同物种的血红细胞的拉曼光谱,采用一个波长为785nm的半导体激光束来囚禁血红细胞和激发血红细胞的拉曼光谱。结果显示不同物种血红细胞的拉曼光谱在1603cm^-1苯丙氨酸和色氨酸的C=C平行振动模及1616cm^-1酪氨酸和色氨酸的C=C拉伸模有明显的差异,这可以用来作为不同种血红细胞鉴定的特征标记。表明激光镊子拉曼光谱技术可以成为血红细胞分析的有效手段。     

Compliance of bacterial flagella measured with optical tweezers

The development of the gradient force optical particle trap ('optical tweezers') has made it possible to manipulate biological materials using a single beam of laser light. Optical traps can produce forces in the microdyne range on intact cells without causing overt damage: such forces are sufficient to arrest actively swimming bacteria and can overcome torque generated by the flagellar motor of a bacterium tethered to a glass surface by a flagellar filament. By calibrating the trapping force against Stokes' drag and measuring the twist that is sustained by this force, we determined the torsional compliance of flagella in tethered Escherichia coli and a motile Streptococcus. Flagella behaved as linear torsion springs for roughly half a revolution, but became much more rigid when turned beyond this point in either direction.     

Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein

The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport and membrane fusion. The AAA+ ATPase motor cytoplasmic dynein regulates ciliary trafficking, mitotic spindle formation and organelle transport, and dissecting its precise functions has been challenging because of its rapid timescale of action and the lack of cell-permeable, chemical modulators. Here we describe the discovery of ciliobrevins, the first specific small-molecule antagonists of cytoplasmic dynein. Ciliobrevins perturb protein trafficking within the primary cilium, leading to their malformation and Hedgehog signalling blockade. Ciliobrevins also prevent spindle pole focusing, kinetochore-microtubule attachment, melanosome aggregation and peroxisome motility in cultured cells. We further demonstrate the ability of ciliobrevins to block dynein-dependent microtubule gliding and ATPase activity in vitro. Ciliobrevins therefore will be useful reagents for studying cellular processes that require this microtubule motor and may guide the development of additional AAA+ ATPase superfamily inhibitors.     

Banach空间中的逼近问题

设E为实Banach空间,C为E上的非空闭凸子集且为E上的收缩核,P:E→C的保核收缩映象,文章在文献[2]的基础上,对带误差的迭代序列进行了修改,并证明了序列{xn}收敛于T1,T2,…,TN的公共不动点的充分必要条件为:limn→∞inf d(xn,F)=0,最后给出了在此基础上的两个推论.     

三元多项式环中超越次数为2的收缩

设R为k［x,y,z］的收缩且其对应收缩同态为φ. 证明了如果R的超越次数为2, 且满足下列条件之一 , 则存在p,q∈R, 使得R=k［p,q］:   1) R为inert子代数, 不含坐标, 并且φ为某多项式的梯度; 2) R为2 赋值代数.     

基于电动现象的细胞光镊阱力测量

提出了一种新的细胞光阱力标定的实验方法，利用细胞电泳原理，设计并研制了一套符合用光镊测力的电动样品池系统，用它替代单光镊操作系统中的普通样品池和高精密压电位移驱动平台，可以用电压调控细胞运动速度，系统相对简单、经济．实验测量结果为：在2～18V／cm电压梯度范围内，酵母细胞的运动速度与电压梯度成正线性关系，即在皮牛量级以下，最大光阱力与光镊光源的功率成线性增加关系．测量结果表明，该方法可实现对细胞光阱力的测量，还可用来测量细胞表面电量．     

Fibronectin binding mediated by a novel class of surface organelles on Escherichia coli

Gram-negative bacteria are known to produce two types of surface organelles: flagella, which are required for motility and chemotaxis, and pili (fimbriae), which play a part in the interaction of bacteria with other bacteria and with eukaryotic host cells. Here we report a third class of E. coli surface organelles for which we propose the name curli. Curli are coiled surface structures composed of a single type of subunit, the curlin, which differs from all known pilin proteins and is synthesized in the absence of a cleavable signal peptide. Although the gene encoding this structural subunit, crl, is present and transcribed in most natural isolates of E. coli, only certain strains are able to assemble the subunit protein into curli. This assembly process occurs preferentially at growth temperatures below 37 degrees C. The ability of curli to mediate binding to fibronectin may be a virulence-associated property for wound colonization and for the colonization of fibronectin-coated surfaces.