Mapping the classical cross-bridge theory and backward steps in a three bead laser trap setup

According to the cross-bridge theory (Huxley, 1957) [1], the interaction between myosin and actin is governed by a deterministic process where the myosin molecule pulls the actin filament in one specific direction only. However, studies on single myosin-actin interactions produced displacements of actin not only in the preferred but also in the opposite direction. This phenomenon is typically referred to as backward steps by the myosin head. Molloy et al. (1995) [2] speculated that these backward steps are not caused by the molecular interactions of actin with myosin but are an artifact of the Brownian motion associated with these molecular level experiments. The aim of this study was to investigate, whether a theoretical model can support Molloy’s speculation. We therefore developed a theoretical model of actin-myosin based muscle contraction that was strictly based on Huxley’s assumption of one stepping direction only, but incorporated Brownian motion, as observed in single cross-bridge-actin interactions. The mathematical model is based on Langevin equations describing the classical three-bead laser trap setup and uses a novel semi-analytical approach to study the percentage of backward steps. We analyzed the effects of different initial actin attachment site distribution and laser trap stiffness on the ratio of forward to backward steps. Our results demonstrate that backward steps and the classical cross-bridge theory are perfectly compatible in a three-bead laser trap setup.     

An unexpectedly large working stroke from chymotryptic fragments of myosin II

Recent structural evidence indicates that the light chain domain of the myosin head (LCD) bends on the motor domain (MD) to move actin. Structural models usually assume that the actin-MD interface remains static and the possibility that part of the myosin working stroke might be produced by rotation about the acto-myosin interface has been neglected. We have used an optical trap to measure the movement produced by proteolytically shortened single rabbit skeletal muscle myosin heads (S-1(A1) and S-1(A2)). The working stroke produced by these shortened heads was more than that which the MD-LCD bend mechanism predicts from the full-length (papain) S-1’s working stroke obtained under similar conditions. This result indicates that part of the working stroke may be caused by motor action at the actin-MD interface.     

An integrative approach for the optical sequencing of single DNA molecules

A new approach for optically sequencing ensembles of single DNA molecules using DNA polymerase to mediate the consecutive incorporation of fluorochrome-labeled nucleotides into an array of large single DNA molecules is presented. The approach utilizes cycles of labeled fluorochrome addition, detection to count incorporations, and bleaching to reset the counter. These additions are imaged and analyzed to estimate the number of labeled additions and to correlate them on a per-locus basis along DNA backbones. Initial studies used precisely labeled polymerase chain reaction products to aid the development and validation of simple models of fluorochrome point spread functions within the imaging system. In complementary studies, nucleotides labeled with the fluorochrome R110 were incorporated into surface-elongated lambda DNA, and fluorescent signals corresponding to the addition of R110-dUTP were counted and assigned precise loci along DNA backbones. The labeled DNAs were then subjected to photobleaching and to a second cycle of addition of R110-labeled nucleotides-a second round of additions was correlated with the first to establish strings of addition histories among the ensemble of largely double-stranded templates. These results confirm the basic operational validity of this approach and point the way to the development of a practical system for optical sequencing.     

Single molecule techniques in DNA repair: A primer

A powerful new approach has become much more widespread and offers insights into aspects of DNA repair unattainable with billions of molecules. Single molecule techniques can be used to image, manipulate or characterize the action of a single repair protein on a single strand of DNA. This allows search mechanisms to be probed, and the effects of force to be understood. These physical aspects can dominate a biochemical reaction, where at the ensemble level their nuances are obscured. In this paper we discuss some of the many technical advances that permit study at the single molecule level. We focus on DNA repair to which these techniques are actively being applied. DNA repair is also a process that encompasses so much of what single molecule studies benefit – searching for targets, complex formation, sequential biochemical reactions and substrate hand-off to name just a few. We discuss how single molecule biophysics is poised to transform our understanding of biological systems, in particular DNA repair.     

Single molecule measurements and biological motors

Recent technological advances in lasers and optical detectors have enabled a variety of new, single molecule technologies to be developed. Using intense and highly collimated laser light sources in addition to super-sensitive cameras, the fluorescence of single fluorophores can now be imaged in aqueous solution. Also, laser optical tweezers have enabled the piconewton forces produced by pair of interacting biomolecules to be measured directly. However, for a researcher new to the field to begin to use such techniques in their own research might seem a daunting prospect. Most of the equipment that is in use is custom-built. However, most of the equipment is essence fairly simple and the aim of this article is to provide an entry point to the field for a newcomer. It focuses mainly on those practical aspects which are not particularly well covered in the literature, and aims to provide an overview of the field as a whole with references and web links to more detailed sources elsewhere. Indeed, the opportunity to publish an article such as this on the Internet affords many new opportunities (and more space!) for presenting scientific ideas and information. For example, we have illustrated the nature of optical trap data with an interactive Java simulation; provided links to relevant web sites and technical documents, and included a large number of colour figures and plots. Our group’s research focuses on molecular motors, and the bias of this article reflects this. It turns out that molecular motors have been a paradigm (or prototype) for single molecule research and the field has seen a rapid development in the techniques. It is hoped that the methods described here will be broadly applicable to other biological systems.This is an interactive contribution, which can be accessed at:     

光镊和拉曼光镊在生物医学研究中的应用

光镊是由美国科学家Arthur Ashkin于1986年发明的,是一种利用高度汇聚的激光束产生的三维梯度势阱来俘获、操纵微小粒子的技术。因其可俘获、操纵单个细胞,并在细胞和亚细胞层次上为生物医学研究提供方便,近年来,已越来越多地被应用于生物医学研究中。本文在介绍光镊的原理和特点的基础上,阐述了光镊(尤其是拉曼光镊)技术在生物医学领域中的研究进展、现状和展望。     

Thermal noise limitations on micromechanical experiments

Thermal motions of microscopic probes limit the possibilities of experiments that are designed to resolve single-macromolecule dynamics in aqueous conditions. We investigate theoretical strategies for maximizing signal-to-noise ratios or resolution in typical situations, illustratin+g our discussion with examples from optical tweezers and atomic force microscopy experiments. A central result is that the viscous drag on a micromechanical probe is more important than the compliance of the probe. Within limits, increased stiffness of an AFM cantilever or of an optical trap does not increase resolution, and decreased stiffness does not provide the possibility of less invasive measurements. Received: 15 August 1997 / Accepted: 11 September 1997     

On-chip single-cell microcultivation assay for monitoring environmental effects on isolated cells

We have developed a on-chip single-cell microcultivation assay as a means of observing the adaptation process of single bacterial cells during nutrient concentration changes. This assay enables the direct observation of single cells captured in microchambers made on thin glass slides and having semipermeable membrane lids, in which cells were kept isolated with optical tweezers. After changing a medium of 0.2% (w/v) glucose concentration to make it nutrient-free 0.9% NaCl medium, the growth of all cells inserted into the medium stopped within 20 min, irrespective of their cell cycles. When a nutrient-rich medium was restored, the cells started to grow again, even after the medium had remained nutrient-free for 42 h. The results indicate that the cell's growth and division are directly related to their nutrient condition. The growth curve also indicates that the cells keep their memory of what their growth and division had been before they stopped growing.     

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

Here we report on a method that extends the study of the mechanical behavior of single proteins to the low force regime of optical tweezers. This experimental approach relies on the use of DNA handles to specifically attach the protein to polystyrene beads and minimize the non-specific interactions between the tethering surfaces. The handles can be attached to any exposed pair of cysteine residues. Handles of different lengths were employed to mechanically manipulate both monomeric and polymeric proteins. The low spring constant of the optical tweezers enabled us to monitor directly refolding events and fluctuations between different molecular structures in quasi-equilibrium conditions. This approach, which has already yielded important results on the refolding process of the protein RNase H (Cecconi et al. in Science 309: 2057-2060, 2005), appears robust and widely applicable to any protein engineered to contain a pair of reactive cysteine residues. It represents a new strategy to study protein folding at the single molecule level, and should be applicable to a range of problems requiring tethering of protein molecules.     

RNAi在甲壳动物中的研究进展

RNA干扰（RNA interference,RNAi）是一类在真核生物中广泛存在的,由双链RNA介导的转录后基因沉默机制。作为一项研究基因功能的有力工具,RNAi技术已经被广泛应用在线虫、果蝇、斑马鱼和小鼠等生物的基因组学研究中。近来在甲壳动物中,通过RNAi技术取得了众多的科研成果。文章从免疫、生长发育、蜕皮、生殖、性别调控、渗透压调节和代谢等几个方面进行了综述。进而对RNAi技术在甲壳动物中的研究前景进行了展望,旨在为以后更好地研究甲壳动物的基因功能和调控网络提供参考。     

干旱胁迫诱导下植物基因的表达与调控

干旱胁迫能够诱导植物表达大量的基因 ,研究这些基因的表达与调控 ,为植物抗旱的定向育种创造条件。本文系统介绍了在干旱胁迫条件下 ,植物体内渗透调节物质和可溶性糖合成有关的基因、离子和水分通道及Lea蛋白基因的表达 ,以及与这些基因表达相关的调控元件和因子 ,干旱胁迫信号转导等方面的最新研究进展。     

应用光学微操作技术分选单条水稻染色体

报道了一种基于光镊技术的实用的单条染色体分选技术。具体介绍了用光镊与光刀结合、并辅以微吸管分选水稻单条染色体的过程。通过该方法得到的水稻单条染色体样品经过分子克隆,制备出了染色体特异的DNA片段并用于水稻基因组测序工作。还将光学微操作技术与现有的几种分选单条染色体的方法(如玻璃微针挑取、激光弹射以及流式细胞仪等)进行了比较。与这些方法相比,光学微操作方法具有液相环境中分离、操作简易、对染色体损伤小、选择性高、无污染等优点。     

Observing structure,function and assembly of single proteins by AFM

Single molecule experiments provide insight into the individuality of biological macromolecules, their unique function, reaction pathways, trajectories and molecular interactions. The exceptional signal-to-noise ratio of the atomic force microscope allows individual proteins to be imaged under physiologically relevant conditions at a lateral resolution of 0.5–1 nm and a vertical resolution of 0.1–0.2 nm. Recently, it has become possible to observe single molecule events using this technique. This capability is reviewed on various water-soluble and membrane proteins. Examples of the observation of function, variability, and assembly of single proteins are discussed. Statistical analysis is important to extend conclusions derived from single molecule experiments to protein species. Such approaches allow the classification of protein conformations and movements. Recent developments of probe microscopy techniques allow simultaneous measurement of multiple signals on individual macromolecules, and greatly extend the range of experiments possible for probing biological systems at the molecular level. Biologists exploring molecular mechanisms will benefit from a burgeoning of scanning probe microscopes and of their future combination with molecular biological experiments.     

活细胞染色体切割（光刀）和光捕捉（光钳）的研究

本文报道了光捕捉活细胞染色体的最新实验结果。对ＰＴＫ＿２有丝分裂细胞的染色体先围激光刀切割,再用光钳捕捉使该切割的染色体片断的行为发生改变。光捕捉中期切割的染色体片断有可能使它们整合到同一个子细胞中或丢失在分裂沟中。光捕捉后期切割的染色体可使该切割片断或掺入相反的细胞中或丢失在分裂沟中或回到原有的相应子细胞中。光捕捉操纵染色体去水螈肺上支子细胞中不仅同样有效,还可以在纺缍体的边缘,即纺缍体和间丝笼之间的细胞质清澈区域内用光钳操纵染色体片断移动,旋转。根据细胞和染色体形态和行为,对７００－８４０ｎｍ波长范围内的各种波长的光捕捉进行了比较,结果表明,７００ｎｍ或８００－８２０ｎｍ波长操纵的细胞,出现最少的异常细胞百分率,７６０ｎｍ则诱发百分之百的异常细胞率。根据各方面的综合比较,７００ｎｍ为最佳波长,共次为１０６０和８００ｎｍ。７６０ｎｍ损伤细胞最严重,应避免使用。文中并讨论了光捕捉染色体的应用前景。     

光钳捕获,操纵,分离和模拟提取酵母细胞

运用光陷阱技术进行对酵母细胞的捕获、操纵、分离和模拟提取。