细胞结构动力学——实时调控细胞力学的因素分析

机械力普遍存在于活细胞的生命活动中,而细胞内力学活动必须依赖骨架结构传递,这种独特的力学形式被称为细胞结构力学.单位时间内细胞结构力学变化受多因素调控,如外力、渗透压、动力分子、张力敏感性离子通道、胞内力学感受器及骨架组装等,构成了细胞结构动力学研究的重要内容.基于荧光共振能量转移(FRET)原理开发的荧光张力探针能整合到细胞骨架内,将细胞结构力学变化转化为光学信号,可能带来细胞力学研究的革命.随着细胞结构动力学研究内容的不断深入,特别是太空时代细胞力学稳态的打破,细胞结构动力学将在生命及医学研究领域显露出越来越重要的地位.     

Modulation of coupling in the Escherichia coli ATP synthase by ADP and Pi: Role of the ε subunit C-terminal domain

The ε-subunit of ATP-synthase is an endogenous inhibitor of the hydrolysis activity of the complex and its α-helical C-terminal domain (εCTD) undergoes drastic changes among at least two different conformations. Even though this domain is not essential for ATP synthesis activity, there is evidence for its involvement in the coupling mechanism of the pump. Recently, it was proposed that coupling of the ATP synthase can vary as a function of ADP and P_i concentration. In the present work, we have explored the possible role of the εCTD in this ADP- and P_i-dependent coupling, by examining an εCTD-lacking mutant of Escherichia coli. We show that the loss of P_i-dependent coupling can be observed also in the εCTD-less mutant, but the effects of P_i on both proton pumping and ATP hydrolysis were much weaker in the mutant than in the wild-type. We also show that the εCTD strongly influences the binding of ADP to a very tight binding site (half-maximal effect ≈ 1 nM); binding at this site induces higher coupling in EF_OF₁ and increases responses to P_i. It is proposed that one physiological role of the εCTD is to regulate the kinetics and affinity of ADP/P_i binding, promoting ADP/P_i-dependent coupling.     

Ocellatin‐PT antimicrobial peptides: High‐resolution microscopy studies in antileishmania models and interactions with mimetic membrane systems

Although the mechanism of action of antimicrobial peptides (AMPs) is not clear, they can interact electrostatically with the cell membranes of microorganisms. New ocellatin‐PT peptides were recently isolated from the skin secretion of Leptodactylus pustulatus. The secondary structure of these AMPs and their effect on Leishmania infantum cells, and on different lipid surface models was characterized in this work. The results showed that all ocellatin‐PT peptides have an α‐helix structure and five of them (PT3, PT4, PT6 to PT8) have leishmanicidal activity; PT1 and PT2 affected the cellular morphology of the parasites and showed greater affinity for leishmania and bacteria‐mimicking lipid membranes than for those of mammals. The results show selectivity of ocellatin‐PTs to the membranes of microorganisms and the applicability of biophysical methods to clarify the interaction of AMPs with cell membranes.     

Probing the effect of glycosaminoglycan depletion on integrin interactions with collagen I fibrils in the native extracellular matrix environment

Fibrillar collagen–integrin interactions in the extracellular matrix (ECM) regulate a multitude of cellular processes and cell signalling. Collagen I fibrils serve as the molecular scaffolding for connective tissues throughout the human body and are the most abundant protein building blocks in the ECM. The ECM environment is diverse, made up of several ECM proteins, enzymes, and proteoglycans. In particular, glycosaminoglycans (GAGs), anionic polysaccharides that decorate proteoglycans, become depleted in the ECM with natural aging and their mis-regulation has been linked to cancers and other diseases. The impact of GAG depletion in the ECM environment on collagen I protein interactions and on mechanical properties is not well understood. Here, we integrate ELISA protein binding assays with liquid high-resolution atomic force microscopy (AFM) to assess the effects of GAG depletion on the interaction of collagen I fibrils with the integrin α2I domain using separate rat tails. ELISA binding assays demonstrate that α2I preferentially binds to GAG-depleted collagen I fibrils in comparison to native fibrils. By amplitude modulated AFM in air and in solution, we find that GAG-depleted collagen I fibrils retain structural features of the native fibrils, including their characteristic D-banding pattern, a key structural motif. AFM fast force mapping in solution shows that GAG depletion reduces the stiffness of individual fibrils, lowering the indentation modulus by half compared to native fibrils. Together these results shed new light on how GAGs influence collagen I fibril–integrin interactions and may aid in strategies to treat diseases that result from GAG mis-regulation.     

Cargo properties play a critical role in myosin Va-driven cargo transport along actin filaments

High-resolution experiments revealed that a single myosin-Va motor can transport micron-sized cargo on actin filaments in a stepwise manner. However, intracellular cargo transport is mediated through the dense actin meshwork by a team of myosin Va motors. The mechanism of how motors interact mechanically to bring about efficient cargo transport is still poorly understood. This study describes a stochastic model where a quantitative understanding of the collective behaviors of myosin Va motors is developed based on cargo stiffness. To understand how cargo properties affect the overall cargo transport, we have designed a model in which two myosin Va motors were coupled by wormlike chain tethers with persistence length ranging from 10 to 80 nm and contour length from 100 to 200 nm, and predicted distributions of velocity, run length, and tether force. Our analysis showed that these parameters are sensitive to both the contour and persistence length of cargo. While the velocity of two couple motors is decreased compared to a single motor (from 531 ± 251 nm/s to as low as 318 ± 287 nm/s), the run length (716 ± 563 nm for a single motor) decreased for short, rigid tethers (to as low as 377 ± 187 μm) and increased for long, flexible tethers (to as high as 1.74 ± 1.50 μm). The sensitivity of processive properties to tether rigidity (persistence length) was greatest for short tethers, which caused the motors to exhibit close, yet anti-cooperative coordination. Motors coupled by longer tethers stepped more independently regardless of tether rigidity. Therefore, the properties of the cargo or linkage must play an essential role in motor-motor communication and cargo transport.     

Swimming performance is reduced by reflective markers intended for the analysis of swimming kinematics

The present study aimed to clarify whether swimming performance is affected by reflective markers being attached to the swimmer’s body, as is required for a kinematic analysis of swimming. Fourteen well-trained male swimmers (21.1 ± 1.7 yrs) performed maximal 50 m front crawl swimming with (W) and without (WO) 25 reflective markers attached to their skin and swimwear. This number represents the minimum required to estimate the body’s center of mass. Fifty meter swimming time, mid-pool swimming velocity, stroke rate, and stroke length were determined using video analysis. We found swimming time to be 3.9 ± 1.6% longer for W condition. Swimming velocity (3.3 ± 1.8%), stroke rate (1.2 ± 2.0%), and stroke length (2.1 ± 2.7%) were also significantly lower for W condition. To elucidate whether the observed reduction in performance was potentially owing to an additional drag force induced by the reflective markers, measured swimming velocity under W condition was compared to a predicted velocity that was calculated based on swimming velocity obtained under WO condition and an estimate of the additional drag force induced by the reflective markers. The mean prediction error and ICC (2,1) for this analysis of measured and predicted velocities was 0.014 m s⁻¹ and 0.894, respectively. Reducing the drag force term led to a decrease in the degree of agreement between the velocities. Together, these results suggest that the reduction in swimming performance resulted, at least in part, from an additional drag force produced by the reflective markers.     

Pushing, pulling and trapping--modes of motor protein supported protein translocation

Protein translocation across the cellular membranes is an ubiquitous and crucial activity of cells. This process is mediated by translocases that consist of a protein conducting channel and an associated motor protein. Motor proteins interact with protein substrates and utilize the free energy of ATP binding and hydrolysis for protein unfolding, translocation and unbinding. Since motor proteins are found either at the cis- or trans-side of the membrane, different mechanisms for translocation have been proposed. In the Power stroke model, cis-acting motors are thought to push, while trans-motors pull on the substrate protein during translocation. In the Brownian ratchet model, translocation occurs by diffusion of the unfolded polypeptide through the translocation pore while directionality is achieved by trapping and refolding. Recent insights in the structure and function of the molecular motors suggest that different mechanisms can be employed simultaneously.     

Poly(dG)-poly(dC) DNA appears shorter than poly(dA)-poly(dT) and possibly adopts an A-related conformation on a mica surface under ambient conditions

Three types of DNA: approximately 2700 bp polydeoxyguanylic olydeoxycytidylic acid [poly(dG)-poly(dC)], approximately 2700 bp polydeoxyadenylic polydeoxythymidylic acid [poly(dA)-poly(dT)] and 2686 bp linear plasmid pUC19 were deposited on a mica surface and imaged by atomic force microscopy. Contour length measurements show that the average length of poly(dG)-poly(dC) is approximately 30% shorter than that of poly(dA)-poly(dT) and the plasmid. This led us to suggest that individual poly(dG)-poly(dC) molecules are immobilized on mica under ambient conditions in a form which is likely related to the A-form of DNA in contrast to poly(dA)-poly(dT) and random sequence DNA which are immobilized in a form that is related to the DNA B-form.     

Sexual dimorphism in rat left atrial function and response to adrenergic stimulation

A number of investigations in humans and animals suggest that there may be intrinsic sex-associated differences in cardiac function. Using left atrial preparations from male and female rat hearts, we examined differences in myocardial function and response to adrenergic agonists. Contractile parameters were measured in isolated atria by conventional isometric methods in the absence or presence of isoproterenol or phenylephrine. Responsiveness to Ca²⁺ was measured in detergent-skinned atrial fibers and actomyosin ATPase activity was measured in isolated myofibrils. Tetanic contractions were generated by treating the atrium with ryanodine followed by high frequency stimulation. Developed force was greater and maximal rates of contraction and relaxation were more rapid in the female atrium. The relationship between Ca²⁺ concentration and force in both intact atria and detergent-skinned atrial fibers in females fell to the left of that for males. At low Ca²⁺ concentrations, skinned fibers from female atria generated more force and myofibrils from female atria had higher myosin ATPase activity than males. Tetanic contraction in the presence of high extracellular Ca²⁺ was greater in female atria. Male atrium had larger inotropic responses to isoproterenol and to phenylephrine, but drug-elicited cAMP and inositol phosphate production did not differ between sexes. The results demonstrate sex-related differences in atrial function that can be partially explained by greater myofibrillar Ca²⁺-sensitivity in females. A potential contribution of sarcolemmal Ca2+ influx is suggested by greater tetanic contraction in ryanodine-treated female atrium. The larger response of males to adrenergic stimulation does not appear to be explained by higher production of relevant second messengers. Future studies will investigate the role of sex hormones in these sexually dimorphic responses and may indicate a need for gender-specific therapeutic interventions for myocardial dysfunction.     

一种基于单分子纳米操纵的有序化测序策略

尽管包括人类在内的许多生物物种的基因组测序工作已经完成,但由于现有测序技术的限制,大部分复杂基因组还存在很多大大小小的缺口.缺口的填补以及对其他重复序列区域的测序迫切需要全新的思路和技术.基于在DNA单分子定位切割和拾取方面的实验进展,提出了一种基于原子力显微镜纳米操纵技术的单分子有序化测序策略.计算机模拟的结果表明,这一方法和策略是可行的,有助于解决目前测序工作中所遇到的一些棘手问题.