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.     

李斯特菌诱导宿主细胞骨架重排与磷脂酶D

无论是免疫细胞对病原体的主动吞噬,还是病原体诱导非吞噬细胞的被动吞噬,均是不同细胞膜受体介导的细胞肌动蛋白骨架重排过程,受到单体G蛋白和肌动蛋白骨架相关蛋白的精密调控。细胞内重要信号蛋白,磷脂酰胆碱专一性磷脂酶D(PLD)的活性变化与细胞肌动蛋白骨架重排密切相关,其参与调节了由抗体受体(FcγR)及补体受体(CR3)介导的免疫细胞的主动吞噬,而细胞肌动蛋白骨架解聚蛋白cofilin被磷酸化后可与PLD结合并激活PLD,进而调节肌动蛋白骨架重排。另一方面,cofilin磷酸化状态严格调控李斯特菌感染细胞过程中的肌动蛋白骨架重排。因此,阐明PLD是否在李斯特菌感染细胞过程中被激活并参与调节肌动蛋白骨架重排,将有助于揭示PLD激活对感染发生的调控作用,对透彻理解细菌感染宿主细胞的分子机制具有重要意义。     

Toll样受体在烟曲霉侵染宿主细胞过程中作用的研究进展

烟曲霉侵染宿主细胞时伴有明显的细胞肌动蛋白骨架重排,而重要模式识别受体PRRs(pattern recognition receptors)之一,Toll样受体(Toll-like receptors,TLR)参与调节病原细菌诱导的宿主细胞肌动蛋白骨架重排,其中TLR2和TLR4两亚型可以识别烟曲霉的病原相关分子模式PAMP(pathogen-assosiated molecular patterns),并诱发炎症因子表达等一系列效应信号,在宿主细胞抗烟曲霉天然免疫中发挥重要作用,但在烟曲霉内化侵入过程中TLR能否特异性介导细胞肌动蛋白骨架重排尚不清楚。因此,研究揭示TLR激活在烟曲霉侵入宿主细胞的调控作用,对寻找可能的抗真菌药物作用靶点具有重要意义。     

力学刺激诱导细胞自噬的研究进展

自噬是细胞重要的自我保护机制,多种伤害性刺激激活的自噬具有维持细胞稳态和正常功能的作用.此外,自噬还参与调控恶性肿瘤、动脉粥样硬化等多种疾病的发生发展过程.体内细胞处于复杂的力学微环境中,力学刺激参与调控细胞自噬,如压力可诱导心肌细胞的自噬、牵张力调控运动系统多种细胞的自噬、流体剪切力可激活血管内皮细胞和肿瘤细胞的自噬.力学刺激诱导的细胞自噬依赖众多信号通路.细胞骨架作为重要的调节因子,不仅参与细胞力学信号转导,同时可参与调控细胞自噬.因此,细胞骨架与力学刺激诱导的细胞自噬密切相关.本文结合最新的研究成果,综述力学刺激对细胞自噬的影响及其分子机制,以期为研究力学刺激对细胞生物学行为的影响提供新的视角,进而为相关疾病的治疗提供新思路和分子靶点.     

机械力及力学信号转导影响干细胞命运的研究进展

干细胞作为一种未分化的祖细胞,目前已被广泛应用于开展组织损伤修复、再生以及干细胞特异谱系分化的研究.大量研究表明,干细胞所处的微环境对调控干细胞的生长和分化具有重要作用,多种溶液介质、细胞外基质和信号通路等参与了干细胞命运的调控.尽管已有大量研究证明,溶液介质(如激素和生长因子)在干细胞的生长和分化中发挥重要作用,但近年来越来越多的研究表明,机械力及力学信号转导同样在干细胞自我更新、分化、衰老和凋亡等细胞生理过程中起到重要的作用.本文将对机械应力响应的细胞基础、生物力学及力学信号调控干细胞自我更新和分化,以及生物力学调控干细胞命运可能的作用机制几个方面加以综述.     

机械力对间充质干细胞向成骨细胞分化的力学响应机制研究

间充质干细胞的分化受多种因素的影响,其中力学是重要因素之一.为了探讨力学信号在间充质干细胞分化中的传导机制,利用力学加载装置在成骨细胞诱导体系条件下,对小鼠骨髓间充质干细胞系(D1细胞)加载不同拉伸应变,运用RT-PCR方法、Flou-3-AMCa2 染色技术、激光共聚焦显微镜技术及5种信号阻断剂,SB203580(p38MAPK特异抑制剂)、PD98059(MEK-1/2MAPK特异抑制剂)、LY294002(PI3Ks特异抑制剂)、细胞松弛素B(微丝结构阻断剂)、EGTA(Ca2 螯合剂),探讨力学信号的基本传导途径.结果显示:3%的拉伸应变能明显提高细胞内Ca2 水平;细胞微丝结构破坏后,延迟了3%拉伸应变对细胞内Ca2 水平增加的影响;细胞外Ca2 螯合后,拉伸应变不能促进细胞内Ca2 水平的升高,该结果提示,拉伸应变对细胞内Ca2 水平的影响主要通过细胞外Ca2 的内流实现.5种信号阻断剂能完全阻断干细胞向成骨细胞分化过程中关键基因骨钙蛋白(osteocalcin,OCN)和OSX mRNA的表达.p38MAPK途径、MEK-1/2MAPK途径被阻断后,拉伸应变激活了OCN和Osterix(OSX,与成骨细胞分化相关的关键基因)mRNA的表达;PI3Ks途径阻断后,拉伸应变部分激活了OCN和OSX mRNA的表达;细胞微丝破坏及胞外Ca2 螯合后,拉伸应变不能促进OCN和OSXmRNA的表达.上述结果表明:力学信号通过Ca2 信号、细胞微丝结构以及PI3Ks信号途径引起细胞的应答反应和生物学效应.     

仿生黏弹性高分子水凝胶及其生物医学应用

天然细胞外基质和生物体软组织固有的黏弹性是调控细胞行为和组织修复与再生过程的关键因素.基于动态建构化学反应交联得到的动态高分子水凝胶材料可有效模拟在体细胞或组织的黏弹性力学微环境,为体外调控细胞命运、揭示其力学生物学响应机制提供了重要工具,也为组织修复与再生提供了仿生支架材料.本综述在介绍天然细胞外基质及生物体软组织黏弹性的基础上,重点对仿生黏弹性水凝胶材料的设计思路、性能表征及影响因素等进行了概括和总结,并揭示了黏弹性水凝胶调控细胞、组织行为的规律及机制,最后,分析了目前该领域研究中所存在的问题并对未来发展方向进行了展望.本综述将有助于启发高分子水凝胶的仿生功能化设计思路及材料生物学效应研究,进一步拓展高分子水凝胶材料的生物医学应用.     

微图案技术研究MSCs几何形状对YAP定位的影响

已有研究证明,一些物理因素,如基底的硬度、几何约束等对干细胞的凋亡、增殖及分化有影响.转录因子YAP已被证实在胞外力学刺激通过细胞骨架向细胞核内传递过程中起着非常重要的作用.本研究通过微图案技术控制骨髓间充质干细胞(MSCs)的几何形状,探讨了相同面积下,7种几何形状的MSC单细胞中YAP定位情况.结果显示,当微图案限制细胞的生长时,这种几何约束促进YAP出核.进一步观察发现,在细胞骨架肌动蛋白纤维束较明显、收缩性较强的细胞内,YAP核定位仍较明显;反之,丝状肌动蛋白(F-Actin)排列松散的几何结构细胞中,YAP出核,主要定位在细胞质.通过用不同几何图案限制细胞铺展,进一步证实了细胞的几何约束可以通过肌动蛋白收缩性对YAP活性进行调控,肌动蛋白骨架对YAP的胞质、胞核转移有分子开关的作用.     

模拟微重力效应通过微丝骨架调控成骨细胞BMP2-Smad信号

细胞微丝骨架在力信号传导和基因表达调控中起重要作用。为了研究微丝骨架在模拟微重力效应调控成骨细胞BMP2-Smad信号中的作用,作者通过构建反映Smad活性的报告基因载体转染MC3T3-E1细胞,并通过报告基因活性分析、Western blot等方法检测了微丝骨架解聚剂和回转模拟微重力效应对BMP2诱导Samd磷酸化、核质分布和转录活性的作用。结果显示,构建的报告基因载体在成骨细胞中正确表达并响应BMP2;破坏微丝骨架会抑制BMP2诱导的Smad1/5/8蛋白磷酸化、入核及转录活性;回转抑制Smad1/5/8磷酸化、入核及其转录活性,而微丝骨架稳定剂可对抗回转的抑制作用。因此,认为回转模拟微重力效应可通过解聚微丝骨架抑制BMP2-Smad信号传导。     

宿主细胞丝切蛋白(cofilin)与病原微生物感染

摘要:病原微生物侵入宿主细胞是其引发有效感染的必要环节,该过程依赖于宿主细胞内肌动蛋白骨架的重排。丝切蛋白(cofilin)是细胞内一种重要的肌动蛋白解聚因子,参与多种病毒、细菌及真菌的感染过程。病原微生物感染可诱导宿主细胞肌动蛋白发生两相变化,同时伴随cofilin的磷酸化水平改变。通过突变、抑制或过表达改变cofilin 的活性均能有效的抑制病原微生物的感染。本文将对宿主细胞cofilin在病原微生物感染过程中的具体变化及可能的调控机制进行综述。     

Probing Cell Structure Responses Through a Shear and Stretching Mechanical Stimulation Technique

Cells are complex, dynamic systems that respond to various in vivo stimuli including chemical, mechanical, and scaffolding alterations. The influence of mechanics on cells is especially important in physiological areas that dictate what modes of mechanics exist. Complex, multivariate physiological responses can result from multi-factorial, multi-mode mechanics, including tension, compression, or shear stresses. In this study, we present a novel device based on elastomeric materials that allowed us to stimulate NIH 3T3 fibroblasts through uniaxial strip stretching or shear fluid flow. Cell shape and structural response was observed using conventional approaches such as fluorescent microscopy. Cell orientation and actin cytoskeleton alignment along the direction of applied force were observed to occur after an initial 3 h time period for shear fluid flow and static uniaxial strip stretching experiments although these two directions of alignment were oriented orthogonal relative to each other. This response was then followed by an increasingly pronounced cell and actin cytoskeleton alignment parallel to the direction of force after 6, 12, and 24 h, with 85% of the cells aligned along the direction of force after 24 h. These results indicate that our novel device could be implemented to study the effects of multiple modes of mechanical stimulation on living cells while probing their structural response especially with respect to competing directions of alignment and orientation under these different modes of mechanical stimulation. We believe that this will be important in a diversity of fields including cell mechanotransduction, cell–material interactions, biophysics, and tissue engineering.     

Trans-scale Granular Modelling of Cytoskeleton: a Mini-Review

Living cells are the functional unit of organs that controls reactions to their exterior. However, the mechanics of living cells can be difficult to characterize due to the crypticity of their microscale structures and associated dynamic cellular processes. Fortunately, multiscale modelling provides a powerful simulation tool that can be used to study the mechanical properties of these soft hierarchical, biological systems. This paper reviews recent developments in hierarchical multiscale modeling technique that aimed at understanding cytoskeleton mechanics. Discussions are expanded with respects to cytoskeletal components including: intermediate filaments, microtubules and microfilament networks. The mechanical performance of difference cytoskeleton components are discussed with respect to their structural and material properties. Explicit granular simulation methods are adopted with different coarse-grained strategies for these cytoskeleton components and the simulation details are introduced in this review.     

Mechanical aspects of cell shape regulation and signaling

Physical forces play a critical role in cell integrity and development, but little is known how cells convert mechanical signals into biochemical responses. This mini-review examines potential molecular mediators like integrins, focal adhesion proteins, and the cytoskeleton in the context of a complex cell structure. These molecules-when activated by cell binding to the extracellular matrix-associate with the skeletal scaffold via the focal adhesion complex. Vinculin is presented as a mechanical coupling protein that contributes to the integrity of the cytoskeleton and cell shape control, and examples are given of how mechanical signals converge into biochemical responses through force-dependent changes in cell geometry and molecular mechanics.     

Microtubules can bear enhanced compressive loads in living cells because of lateral reinforcement

Cytoskeletal microtubules have been proposed to influence cell shape and mechanics based on their ability to resist large-scale compressive forces exerted by the surrounding contractile cytoskeleton. Consistent with this, cytoplasmic microtubules are often highly curved and appear buckled because of compressive loads. However, the results of in vitro studies suggest that microtubules should buckle at much larger length scales, withstanding only exceedingly small compressive forces. This discrepancy calls into question the structural role of microtubules, and highlights our lack of quantitative knowledge of the magnitude of the forces they experience and can withstand in living cells. We show that intracellular microtubules do bear large-scale compressive loads from a variety of physiological forces, but their buckling wavelength is reduced significantly because of mechanical coupling to the surrounding elastic cytoskeleton. We quantitatively explain this behavior, and show that this coupling dramatically increases the compressive forces that microtubules can sustain, suggesting they can make a more significant structural contribution to the mechanical behavior of the cell than previously thought possible.     

Actin fusion proteins alter the dynamics of mechanically induced cytoskeleton rearrangement

Mechanical forces can regulate various functions in living cells. The cytoskeleton is a crucial element for the transduction of forces in cell-internal signals and subsequent biological responses. Accordingly, many studies in cellular biomechanics have been focused on the role of the contractile acto-myosin system in such processes. A widely used method to observe the dynamic actin network in living cells is the transgenic expression of fluorescent proteins fused to actin. However, adverse effects of GFP-actin fusion proteins on cell spreading, migration and cell adhesion strength have been reported. These shortcomings were shown to be partly overcome by fusions of actin binding peptides to fluorescent proteins. Nevertheless, it is not understood whether direct labeling by actin fusion proteins or indirect labeling via these chimaeras alters biomechanical responses of cells and the cytoskeleton to forces. We investigated the dynamic reorganization of actin stress fibers in cells under cyclic mechanical loading by transiently expressing either egfp-Lifeact or eyfp-actin in rat embryonic fibroblasts and observing them by means of live cell microscopy. Our results demonstrate that mechanically-induced actin stress fiber reorganization exhibits very different kinetics in EYFP-actin cells and EGFP-Lifeact cells, the latter showing a remarkable agreement with the reorganization kinetics of non-transfected cells under the same experimental conditions.     

Cytoskeletal architecture and mechanical behavior of living cells

Conventional continuum mechanics models considering living cells as viscous fluid balloons are unable to explain some recent experimental observations. In contrast, new microstructural models provide the desirable explanations. These models emphasize the role of the cell cytoskeleton built of struts-microtubules and cables-microfilaments. A specific architectural model of the cytoskeletal framework called "tensegrity" deserved wide attention recently. Tensegrity models particularly account for the phenomenon of linear stiffening of living cells. These models are discussed from the structural mechanics perspective. Classification of structural assemblies is given and the meaning of "tensegrity" is pinpointed. Possible sources of non-linearity leading to cell stiffening are emphasized. The role of local buckling of microtubules and overall stability of the cytoskeleton is stressed. Computational studies play a central role in the development of the microstructural theoretical framework allowing for the prediction of the cell behavior from "first principles". Algorithms of computer analysis of the cytoskeleton that consider unilateral response of microfilaments and deep postbuckling of microtubules are addressed.     

Mechanical models for living cells--a review

As physical entities, living cells possess structural and physical properties that enable them to withstand the physiological environment as well as mechanical stimuli occurring within and outside the body. Any deviation from these properties will not only undermine the physical integrity of the cells, but also their biological functions. As such, a quantitative study in single cell mechanics needs to be conducted. In this review, we will examine some mechanical models that have been developed to characterize mechanical responses of living cells when subjected to both transient and dynamic loads. The mechanical models include the cortical shell-liquid core (or liquid drop) models which are widely applied to suspended cells; the solid model which is generally used for adherent cells; the power-law structural damping model which is more suited for studying the dynamic behavior of adherent cells; and finally, the biphasic model which has been widely used to study musculoskeletal cell mechanics. Based upon these models, future attempts can be made to develop even more detailed and accurate mechanical models of living cells once these three factors are adequately addressed: structural heterogeneity, appropriate constitutive relations for each of the distinct subcellular regions and components, and active forces acting within the cell. More realistic mechanical models of living cells can further contribute towards the study of mechanotransduction in cells.     

The Mechanochemical Basis of Cell and Tissue Regulation

This article is a summary of a lecture presented at a symposium on "Mechanics and Chemistry of Biosystems' in honor of Professor Y.C. Fung that convened at the University of California, Irvine in February 2004. The article reviews work from our laboratory that focuses on the mechanism by which mechanical and chemical signals interplay to control how individual cells decide whether to grow, differentiate, move, or die, and thereby promote pattern formation during tissue morphogenesis. Pursuit of this challenge has required development and application of new microtechnologies, theoretical formulations, computational models and bioinformatics tools. These approaches have been used to apply controlled mechanical stresses to specific cell surface molecules and to measure mechanical and biochemical responses; to control cell shape independently of chemical factors; and to handle the structural, hierarchical and informational complexity of living cells. Results of these studies have changed our view of how cells and tissues control their shape and mechanical properties, and have led to the discovery that integrins and the cytoskeleton play a central role in cellular mechanotransduction. Recognition of these critical links between mechanics and cellular biochemistry should lead to novel strategies for the development of new drugs and engineered tissues, as well as biomimetic microdevices and nanotechnologies that more effectively function within the context of living tissues.     

Intracellular Mechanics and Activity of Breast Cancer Cells Correlate with Metastatic Potential

Mechanics of cancer cells are directly linked to their metastatic potential, or ability to produce a secondary tumor at a distant site. Metastatic cells survive in the circulatory system in a non-adherent state, and can squeeze through barriers in the body. Such considerable structural changes in cells rely on rapid remodeling of internal structure and mechanics. While external mechanical measurements have demonstrated enhanced pliability of cancer cells with increased metastatic potential, little is known about dynamics of their interior and we expect that to change significantly in metastatic cells. We perform a comparative study, using particle-tracking to evaluate the intracellular mechanics of living epithelial breast cells with varying invasiveness. Particles in all examined cell lines exhibit super-diffusion with a scaling exponent of 1.4 at short lag times, likely related to active transport by fluctuating microtubules and their associated molecular motors. Specifics of probe-particle transport differ between the cell types, depending on the cytoskeleton network-structure and interactions with it. Our study shows that the internal microenvironment of the highly metastatic cells evaluated here is more pliable and their cytoskeleton is less dense than the poorly metastatic and benign cells. We thus reveal intracellular structure and mechanics that can support the unique function and invasive capabilities of highly metastatic cells.