活细胞结构力学与荧光张力检测探针

微观力学效应决定着细胞形态结构变化,胞内动力蛋白、肌球蛋白和驱动蛋白等马达分子,构成了细胞微丝微管骨架结构组装的原始驱动力.而以细胞骨架结构为支架的力学感受器,也反馈性调节着细胞力学信号及其生物学效应,组成细胞结构调控必不可缺的力学基础,两者协同调控了机体生理和病理活动.本文从生物微观力学效应和信号入手,介绍了一种基于荧光共振能量转移(FRET)原理的活细胞结构力学检测新技术,将微观结构力学变化转换为光学信号,并采用克隆技术将其插入α辅肌动蛋白、β肌动蛋白及血影蛋白等骨架及相关蛋白质,观察活细胞、组织甚至动物整体水平结构力学变化,为癌细胞侵袭转移、分裂增殖、细胞极化、反分化以及太空失重等生物物理医学研究探寻新的途径.     

植物微丝骨架的研究进展

微丝骨架是细胞骨架的重要组成部分,在各种细胞活动中都发挥着重要作用。微丝骨架的主要组成部分是肌动蛋白和肌动蛋白结合蛋白,参与细胞形态建成、物质运输和信号转导等生命活动。通过鬼笔环肽标记或表达荧光融合蛋白等方法,国内外许多学者对植物微丝骨架的组成、功能等进行了大量的研究,并取得了一些成果。基于前人的研究,本研究从组成、功能及研究方法三个方面对植物微丝骨架的进行概述。     

模拟微重力诱导的细胞微丝变化影响COL1A1启动子活性

细胞骨架系统是细胞内的重力感受系统。已知微重力导致的细胞形态、功能、信号传导等多种变化均与细胞骨架系统变化有关,但微重力对相关基因调控的影响知之甚少。本研究以构建的基因工程细胞株（EGFP-ROS）为对象,以回转器模拟微重力效应,利用增强型绿色荧光蛋白（enhanced green fluorescence protein,EGFP）荧光半定量和细胞微丝荧光染色分析技术,探讨回转模拟微重力条件下,细胞微丝系统对Ⅰ型胶原α1链基因（collagen type Ialpha chain 1 gene,COL1A1）启动子活性的影响。空间飞行和回转模拟微重力后,细胞微丝解聚、张力纤维减少,表明微重力可降低细胞微丝结构的有序性,诱导细胞骨架重排。适合剂量的细胞松弛素B处理EGFP-ROS细胞诱导微丝骨架解聚,同时导致COL1A1启动子活性增加,细胞荧光强度增强,并呈现剂量依赖性。因此,一定程度的细胞微丝系统破坏将导致COL1A1启动子活性的增强,证明细胞微丝骨架系统参与了微重力对COL1A1启动子活性调节,且在微重力信号传导中起重要作用。     

细胞的荧光标记与融合检测细胞膜的流动性

细胞膜是细胞的物理屏障,也是在细胞生命活动中有复杂功能的重要结构.细胞膜把细胞包裹起来,使细胞能够保持相对的稳定性,维持正常的生命活动,保证必需养分的吸收和代谢产物的排出等.细胞膜结构具有共同特征:镶嵌性,蛋白质极性及流动性等.笔者使用同种细胞作为实验材料,用同种膜蛋白的抗体进行免疫,然后用带有不同荧光的二抗标记,最后对细胞进行融合,实验结果证明细胞膜是具有流动性的.     

细胞的骨架系统

细胞骨架是一类复杂的蛋白质纤维结构,广泛地存在于动物细胞、植物细胞甚至一些原生动物与酵母中。细胞骨架按分布区域可分为胞质骨架和细胞核骨架,胞质骨架又具有三种类型:微管、微丝和中等纤维.胞质骨架和核骨架以及三种胞质骨架之间的结构、性质和功能上是有所区别的,但另一方面它们又协调地参予细胞的一系列生理活动,共同组成了细胞的骨架系统。六十年代初,波特(K·Porter)等第一次用电镜证明了细胞质中骨架结构的多样性,他们发现几乎每一个真核细胞的胞质中都存在三种类型的骨架结构,即微管、微丝和中等纤维。之后,对它们的结构、性质和功能进行了深入的研究。七十年代以来,在细胞核中又发现了一个形态类似于胞质骨架、蛋白质性质的网架结构——细胞核骨架(简称核骨架)对它可能的作用也有了初步的认识,这些发现丰富了骨架系统的内容。现在,已经证实胞质骨架和核骨架在结构与功能上是密切联系的,两者构成了统一的细胞骨架体系,对细胞生长、运动及细胞分化等过程起着重要的作用。     

绿色荧光蛋白基因结合鼠Talin基因蛋白标记转基因水稻活体生殖细胞及精细胞的微丝骨架

利用绿色荧光蛋白(GFP)基因结合鼠Talin基因表达技术及水稻(Oryza sativa L.)转基因技术,筛选出表达稳定和具等位基因型的第三代转基因水稻.在其活体花粉的4个发育阶段(Ⅰ.小孢子晚期;Ⅱ.二细胞早期;Ⅲ.二细胞晚期;Ⅳ.三细胞阶段),观察了细胞内微丝骨架的分布和结构形态的变化.发现在这4个花粉发育阶段,花粉内的营养核、生殖核、生殖细胞和精细胞都在不同的发育阶段出现位移.而这些位移与微丝骨架的结构变化和运动有密切关系.在胞质中央的微丝网络以及细胞周质的网络不断变化和互动,导致营养核、生殖核或生殖细胞和精细胞的定向位移.在活体生殖细胞和精细胞内,存有一股与细胞纵轴平行排列的微丝骨架.这些微丝骨架对生殖细胞及精细胞可以提供移动的动力,这对生殖细胞或精细胞在花管内以及胚囊内的运动(包括独自游动)提供了依据.     

结合微针及AFM的单细胞精准激励与力学特性同步测量

目的 细胞力学特性与细胞生理病理变化过程及机体健康状态密切相关,研究细胞力学特性对于揭示生命活动内在机制具有重要科学意义.原子力显微镜(AFM)的出现为单细胞研究提供了新的技术手段,它不仅可以在溶液环境下对单个活细胞的形貌结构进行高分辨率成像,还能够对细胞力学特性进行定量测量.基于AFM的单细胞力学特性研究在过去数十年...     

蛋白质亚细胞定位的生物信息学研究

细胞中蛋白质合成后被转运到特定的细胞器中,只有转运到正确的部位才能参与细胞的各种生命活动,如果定位发生偏差,将会对细胞功能甚至生命产生重大影响.蛋白质的亚细胞定位是蛋白质功能研究的重要方面,也是生物信息学中的热点问题,数据库的构建和亚细胞定位分析及预测加速了蛋白质结构和功能的研究.     

离子束注入对细胞有丝分裂微管骨架影响的研究

经能量为２０～３０Ｋｅｖ注入剂量为６×１０１５Ｎ＋／ｃｍ２和１０×１０１５Ｎ＋／ｃｍ２的Ｎ＋离子注入及免疫荧光抗体标记显微观察和统计,结果表明,在绿豆根尖细胞有丝分裂中,分裂细胞的形体、中后期细胞数目、细胞微管骨架以及纺缍体的排布方式均发生了异常变化。１２－１３％的分裂细胞个体增大,８－１０％有丝分裂中后期细胞数目高于对照组,间期细胞微管骨架荧光强度明显弱于对照组,且有２－４％的较强荧光小体和无荧光小体（微孔洞,ｍｉｃｒｏｏｐｅｎｉｎｇ）。３－１２％的中后期细胞中出现不对称和极向不同步移动排布的纺缍体。研究结果表明：２０～３０Ｋｅｖ的能量和剂量为６～１０×１０１５Ｎ＋／ｃｍ２的Ｎ＋离子束注入,对绿豆根尖细胞有丝分裂微管骨架可诱发产生异常变化,同时也可以初步认为这种变化结果与Ｎ＋离子束注入产生的染色体异常分配和运动功能活动有密切的相关性。     

可变剪接与细胞凋亡调控

细胞凋亡(apoptosis)是多细胞生物的一种基本生命活动,在机体的生长发育、免疫调节及维持内环境稳定等各方面扮演着重要的角色.遗传和生化研究表明,细胞凋亡受到复杂而精细的调控.转录水平、翻译后水平等各种层次的调控,构成了一个复杂的凋亡调控网络.     

Single-Molecule Force Spectroscopy of Membrane Protein Folding

Single-molecule force spectroscopy is a unique method that can probe the structural changes of single proteins at a high spatiotemporal resolution while mechanically manipulating them over a wide force range. Here, we review the current understanding of membrane protein folding learned by using the force spectroscopy approach. Membrane protein folding in lipid bilayers is one of the most complex biological processes in which diverse lipid molecules and chaperone proteins are intricately involved. The approach of single protein forced unfolding in lipid bilayers has produced important findings and insights into membrane protein folding. This review provides an overview of the forced unfolding approach, including recent achievements and technical advances. Progress in the methods can reveal more interesting cases of membrane protein folding and clarify general mechanisms and principles.     

Simple models for extracting mechanical work from the ATP hydrolysis cycle

According to the binding-zipper model, the RecA class of ATPase motors converts chemical energy into mechanical force by the progressive annealing of hydrogen bonds between the nucleotide and the catalytic pocket. The role of hydrolysis is to weaken the binding of products, allowing them to be released so that the cycle can repeat. Molecular dynamics can be used to study the unbinding process, but the binding process is more complex, so that inferences about it are made indirectly from structural, mutation, and biochemical studies. Here we present a series of models of varying complexity that illustrate the basic processes involved in force production during ATP binding. These models reveal the role of solvent and geometry in determining the amount of mechanical work that can be extracted from the binding process.     

Integration of protein motions with molecular networks reveals different mechanisms for permanent and transient interactions

The integration of molecular networks with other types of data, such as changing levels of gene expression or protein-structural features, can provide richer information about interactions than the simple node-and-edge representations commonly used in the network community. For example, the mapping of 3D-structural data onto networks enables classification of proteins into singlish- or multi-interface hubs (depending on whether they have >2 interfaces). Similarly, interactions can be classified as permanent or transient, depending on whether their interface is used by only one or by multiple partners. Here, we incorporate an additional dimension into molecular networks: dynamic conformational changes. We parse the entire PDB structural databank for alternate conformations of proteins and map these onto the protein interaction network, to compile a first version of the Dynamic Structural Interaction Network (DynaSIN). We make this network available as a readily downloadable resource file, and we then use it to address a variety of downstream questions. In particular, we show that multi-interface hubs display a greater degree of conformational change than do singlish-interface ones; thus, they show more plasticity which perhaps enables them to utilize more interfaces for interactions. We also find that transient associations involve smaller conformational changes than permanent ones. Although this may appear counterintuitive, it is understandable in the following framework: as proteins involved in transient interactions shuttle between interchangeable associations, they interact with domains that are similar to each other and so do not require drastic structural changes for their activity. We provide evidence for this hypothesis through showing that interfaces involved in transient interactions bind fewer classes of domains than those in a control set.     

Mechanically unfolding the small, topologically simple protein L

beta-sheet proteins are generally more able to resist mechanical deformation than alpha-helical proteins. Experiments measuring the mechanical resistance of beta-sheet proteins extended by their termini led to the hypothesis that parallel, directly hydrogen-bonded terminal beta-strands provide the greatest mechanical strength. Here we test this hypothesis by measuring the mechanical properties of protein L, a domain with a topology predicted to be mechanically strong, but with no known mechanical function. A pentamer of this small, topologically simple protein is resistant to mechanical deformation over a wide range of extension rates. Molecular dynamics simulations show the energy landscape for protein L is highly restricted for mechanical unfolding and that this protein unfolds by the shearing apart of two structural units in a mechanism similar to that proposed for ubiquitin, which belongs to the same structural class as protein L, but unfolds at a significantly higher force. These data suggest that the mechanism of mechanical unfolding is conserved in proteins within the same fold family and demonstrate that although the topology and presence of a hydrogen-bonded clamp are of central importance in determining mechanical strength, hydrophobic interactions also play an important role in modulating the mechanical resistance of these similar proteins.     

Probing protein mechanics: residue-level properties and their use in defining domains

It is becoming clear that, in addition to structural properties, the mechanical properties of proteins can play an important role in their biological activity. It nevertheless remains difficult to probe these properties experimentally. Whereas single-molecule experiments give access to overall mechanical behavior, notably the impact of end-to-end stretching, it is currently impossible to directly obtain data on more local properties. We propose a theoretical method for probing the mechanical properties of protein structures at the single-amino acid level. This approach can be applied to both all-atom and simplified protein representations. The probing leads to force constants for local deformations and to deformation vectors indicating the paths of least mechanical resistance. It also reveals the mechanical coupling that exists between residues. Results obtained for a variety of proteins show that the calculated force constants vary over a wide range. An analysis of the induced deformations provides information that is distinct from that obtained with measures of atomic fluctuations and is more easily linked to residue-level properties than normal mode analyses or dynamic trajectories. It is also shown that the mechanical information obtained by residue-level probing opens a new route for defining so-called dynamical domains within protein structures.     

Mechanical unfolding pathway and origin of mechanical stability of proteins of ubiquitin family: An investigation by steered molecular dynamics simulation

Like the muscle protein Titin, proteins of the ubiquitin family exhibit a parallel strand arrangement, but otherwise having a distinctly different fold and not involved in an obvious load‐bearing function, exhibit high resistance to mechanical unfolding. We have applied all‐atom molecular dynamics simulation technique in implicit solvent to present a deep insight into the force‐induced unfolding pathway of three proteins—ubiquitin, NEDD8, and SUMO‐2—all having almost similar structural features. Two intermediates evolve in the unfolding pathway of each of the three proteins. The first intermediate, which has already been identified in case of ubiquitin by earlier simulation results, is similar for ubiquitin and NEDD8, but different in SUMO‐2. We have found a new intermediate with β3–β4 hairpin and some residual α‐helical character; and this intermediate is common for all the three proteins. Thus, proteins of the ubiquitin family pass through a well‐defined conformation in their force‐induced unfolding pathway. Reason behind the higher mechanical stability of the proteins with parallel strand structures like Titin has also been identified. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

The allosteric role of the Ca2+ switch in adhesion and elasticity of C-cadherin

Modular proteins such as titin, fibronectin, and cadherin are ubiquitous components of living cells. Often involved in signaling and mechanical processes, their architecture is characterized by domains containing a variable number of heterogeneous “repeats” arranged in series, with either flexible or rigid linker regions that determine their elasticity. Cadherin repeats arranged in series are unique in that linker regions also feature calcium-binding motifs. While it is well known that the extracellular repeats of cadherin proteins mediate cell-cell adhesion in a calcium-dependent manner, the molecular mechanisms behind the influence of calcium in adhesion dynamics and cadherin's mechanical response are not well understood. Here we show, using molecular dynamics simulations, how calcium ions control the structural integrity of cadherin's linker regions, thereby affecting cadherin's equilibrium dynamics, the availability of key residues involved in cell-cell adhesion, and cadherin's mechanical response. The all-atom, multi-nanosecond molecular dynamics simulations involved the entire C-cadherin extracellular domain solvated in water (a 345,000 atom system). Equilibrium simulations show that the extracellular domain maintains its crystal conformation (elongated and slightly curved) when calcium ions are present. In the absence of calcium ions, however, it assumes a disordered conformation. The conserved residue Trp², which is thought to insert itself into a hydrophobic pocket of another cadherin molecule (thereby providing the basis for cell-cell adhesion), switches conformation from exposed to intermittently buried upon removal of calcium ions. Furthermore, the overall mechanical response of C-cadherin's extracellular domain is characterized at low force by changes in shape (tertiary structure elasticity), and at high force by unraveling of secondary structure elements (secondary structure elasticity). This mechanical response is modulated by calcium ions at both low and high force, switching from a stiff, rod-like to a soft, spring-like behavior upon removal of ions. The simulations provide an unprecedented molecular view of calcium-mediated allostery in cadherins, also illustrating the general principles of linker-mediated elasticity of modular proteins relevant not only for cell-cell adhesion and sound transduction, but also muscle elasticity.     

Recombination of protein fragments: a promising approach toward engineering proteins with novel nanomechanical properties

Combining single molecule atomic force microscopy (AFM) and protein engineering techniques, here we demonstrate that we can use recombination-based techniques to engineer novel elastomeric proteins by recombining protein fragments from structurally homologous parent proteins. Using I27 and I32 domains from the muscle protein titin as parent template proteins, we systematically shuffled the secondary structural elements of the two parent proteins and engineered 13 hybrid daughter proteins. Although I27 and I32 are highly homologous, and homology modeling predicted that the hybrid daughter proteins fold into structures that are similar to that of parent protein, we found that only eight of the 13 daughter proteins showed beta-sheet dominated structures that are similar to parent proteins, and the other five recombined proteins showed signatures of the formation of significant alpha-helical or random coil-like structure. Single molecule AFM revealed that six recombined daughter proteins are mechanically stable and exhibit mechanical properties that are different from the parent proteins. In contrast, another four of the hybrid proteins were found to be mechanically labile and unfold at forces that are lower than the approximately 20 pN, as we could not detect any unfolding force peaks. The last three hybrid proteins showed interesting duality in their mechanical unfolding behaviors. These results demonstrate the great potential of using recombination-based approaches to engineer novel elastomeric protein domains of diverse mechanical properties. Moreover, our results also revealed the challenges and complexity of developing a recombination-based approach into a laboratory-based directed evolution approach to engineer novel elastomeric proteins.     

Structural comparison and classification of alpha‐helical transmembrane domains based on helix interaction patterns

Structural classification of membrane proteins is still in its infancy due to the relative paucity of available three‐dimensional structures compared with soluble proteins. However, recent technological advances in protein structure determination have led to a significant increase in experimentally known membrane protein folds, warranting exploration of the structural universe of membrane proteins. Here, a new and completely membrane protein specific structural classification system is introduced that classifies α‐helical membrane proteins according to common helix architectures. Each membrane protein is represented by a helix interaction graph depicting transmembrane helices with their pairwise interactions resulting from individual residue contacts. Subsequently, proteins are clustered according to similarities among these helix interaction graphs using a newly developed structural similarity score called HISS. As HISS scores explicitly disregard structural properties of loop regions, they are more suitable to capture conserved transmembrane helix bundle architectures than other structural similarity scores. Importantly, we are able to show that a classification approach based on helix interaction similarity closely resembles conventional structural classification databases such as SCOP and CATH implying that helix interactions are one of the major determinants of α‐helical membrane protein folds. Furthermore, the classification of all currently available membrane protein structures into 20 recurrent helix architectures and 15 singleton proteins demonstrates not only an impressive variability of membrane helix bundles but also the conservation of common helix interaction patterns among proteins with distinctly different sequences. Proteins 2010. © 2010 Wiley‐Liss, Inc.