Computational models for mechanics of morphogenesis

In the developing embryo, tissues differentiate, deform, and move in an orchestrated manner to generate various biological shapes driven by the complex interplay between genetic, epigenetic, and environmental factors. Mechanics plays a key role in regulating and controlling morphogenesis, and quantitative models help us understand how various mechanical forces combine to shape the embryo. Models allow for the quantitative, unbiased testing of physical mechanisms, and when used appropriately, can motivate new experimentaldirections. This knowledge benefits biomedical researchers who aim to prevent and treat congenital malformations, as well as engineers working to create replacement tissues in the laboratory. In this review, we first give an overview of fundamental mechanical theories for morphogenesis, and then focus on models for specific processes, including pattern formation, gastrulation, neurulation, organogenesis, and wound healing. The role of mechanical feedback in development is also discussed. Finally, some perspectives aregiven on the emerging challenges in morphomechanics and mechanobiology. Birth Defects Research (Part C) 96:132–152, 2012. © 2012 Wiley Periodicals, Inc.     

Coupling traction force patterns and actomyosin wave dynamics reveals mechanics of cell motion

Motile cells can use and switch between different modes of migration. Here, we use traction force microscopy and fluorescent labeling of actin and myosin to quantify and correlate traction force patterns and cytoskeletal distributions in Dictyostelium discoideum cells that move and switch between keratocyte‐like fan‐shaped, oscillatory, and amoeboid modes. We find that the wave dynamics of the cytoskeletal components critically determine the traction force pattern, cell morphology, and migration mode. Furthermore, we find that fan‐shaped cells can exhibit two different propulsion mechanisms, each with a distinct traction force pattern. Finally, the traction force patterns can be recapitulated using a computational model, which uses the experimentally determined spatiotemporal distributions of actin and myosin forces and a viscous cytoskeletal network. Our results suggest that cell motion can be generated by friction between the flow of this network and the substrate.     

An affine micro-sphere-based constitutive model,accounting for junctional sliding,can capture F-actin network mechanics

Actin filaments are a major component of the cytoskeleton and play a crucial role in cell mechanotransduction. F-actin networks can be reconstituted in vitro and their mechanical behaviour has been studied experimentally. Constitutive models that assume an idealised network structure, in combination with a non-affine network deformation, have been successful in capturing the elastic response of the network. In this study, an affine network deformation is assumed, in which we propose an alternative 3D finite strain constitutive model. The model makes use of a micro-sphere to calculate the strain energy density of the network, which is represented as a continuous distribution of filament orientations in space. By incorporating a simplified sliding mechanism at the filament-to-filament junctions, premature filament locking, inherent to affine network deformation, could be avoided. The model could successfully fit experimental shear data for a specific cross-linked F-actin network, demonstrating the potential of the novel model.     

Models of morphogenesis: the mechanisms and mechanics of cell rearrangement

The directional rearrangement of cells is a key mechanism for reshaping embryos. Despite substantial recent progress in understanding the basic signal transduction pathways that allow cells to orient themselves in space, the extrinsic cues that activate these pathways are just beginning to be understood. Even less-well understood are the physical mechanisms cells use to change position, especially when those cells are epithelial, and how mechanical forces within the embryo affect those movements. Recent studies are providing clues regarding how this fundamental process occurs with such remarkable reliability.     

Cell traction force and measurement methods

Cell traction forces (CTFs) are crucial to many biological processes such as inflammation, wound healing, angiogenesis, and metastasis. CTFs are generated by actomyosin interactions and actin polymerization and regulated by intracellular proteins such as alpha-smooth muscle actin (α-SMA) and soluble factors such as transforming growth factor-β (TGF-β). Once transmitted to the extracellular matrix (ECM) through stress fibers via focal adhesions, which are assemblies of ECM proteins, transmembrane receptors, and cytoplasmic structural and signaling proteins (e.g., integrins), CTFs direct many cellular functions, including cell migration, ECM organization, and mechanical signal generation. Various methods have been developed over the years to measure CTFs of both populations of cells and of single cells. At present, cell traction force microscopy (CTFM) is among the most efficient and reliable method for determining CTF field of an entire cell spreading on a two-dimensional (2D) substrate surface. There are currently three CTFM methods, each of which is unique in both how displacement field is extracted from images and how CTFs are subsequently estimated. A detailed review and comparison of these methods are presented. Future research should improve CTFM methods such that they can automatically track dynamic CTFs, thereby providing new insights into cell motility in response to altered biological conditions. In addition, research effort should be devoted to developing novel experimental and theoretical methods for determining CTFs in three-dimensional (3D) matrix, which better reflects physiological conditions than 2D substrate used in current CTFM methods.     

Emergent morphogenesis: Elastic mechanics of a self-deforming tissue

Multicellular organisms are generated by coordinated cell movements during morphogenesis. Convergent extension is a key tissue movement that organizes mesoderm, ectoderm, and endoderm in vertebrate embryos. The goals of researchers studying convergent extension, and morphogenesis in general, include understanding the molecular pathways that control cell identity, establish fields of cell types, and regulate cell behaviors. Cell identity, the size and boundaries of tissues, and the behaviors exhibited by those cells shape the developing embryo; however, there is a fundamental gap between understanding the molecular pathways that control processes within single cells and understanding how cells work together to assemble multicellular structures. Theoretical and experimental biomechanics of embryonic tissues are increasingly being used to bridge that gap. The efforts to map molecular pathways and the mechanical processes underlying morphogenesis are crucial to understanding: (1) the source of birth defects, (2) the formation of tumors and progression of cancer, and (3) basic principles of tissue engineering. In this paper, we first review the process of tissue convergent extension of the vertebrate axis and then review models used to study the self-organizing movements from a mechanical perspective. We conclude by presenting a relatively simple “wedge-model” that exhibits key emergent properties of convergent extension such as the coupling between tissue stiffness, cell intercalation forces, and tissue elongation forces.     

Identifying constitutive and context-specific molecular-tension-sensitive protein recruitment within focal adhesions

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A primer to traction force microscopy

Traction force microscopy (TFM) has emerged as a versatile technique for the measurement of single-cell-generated forces. TFM has gained wide use among mechanobiology laboratories, and several variants of the original methodology have been proposed. However, issues related to the experimental setup and, most importantly, data analysis of cell traction datasets may restrain the adoption of TFM by a wider community. In this review, we summarize the state of the art in TFM-related research, with a focus on the analytical methods underlying data analysis. We aim to provide the reader with a friendly compendium underlying the potential of TFM and emphasizing the methodological framework required for a thorough understanding of experimental data. We also compile a list of data analytics tools freely available to the scientific community for the furtherance of knowledge on this powerful technique.     

Single cell rigidity sensing: A complex relationship between focal adhesion dynamics and large-scale actin cytoskeleton remodeling

ABSTRACT

Many physiological and pathological processes involve tissue cells sensing the rigidity of their environment. In general, tissue cells have been shown to react to the stiffness of their environment by regulating their level of contractility, and in turn applying traction forces on their environment to probe it. This mechanosensitive process can direct early cell adhesion, cell migration and even cell differentiation. These processes require the integration of signals over time and multiple length scales. Multiple strategies have been developed to understand force- and rigidity-sensing mechanisms and much effort has been concentrated on the study of cell adhesion complexes, such as focal adhesions, and cell cytoskeletons. Here, we review the major biophysical methods used for measuring cell-traction forces as well as the mechanosensitive processes that drive cellular responses to matrix rigidity on 2-dimensional substrates.     

斑马鱼管腔器官空腔形态发生及分子机制

后生动物复杂的体内结构和器官结构多以网络状的管道系统出现。中空的管腔作为这个系统的重要结构单元承担了运输物质、区分器官不同部位功能、分隔机体和外环境等诸多重要的生理功能。管腔的发育障碍将致使相关器官形态发生畸形、功能紊乱。管腔型器官形态发生易被直接观察以及各种相关突变鱼和荧光转基因鱼的出现, 使得斑马鱼(Danio rerio)成为管道器官研究的优秀模式动物。斑马鱼血管、神经管、小肠、胰腺外分泌腺、前肾管等几种重要的器官的形态发生都伴随着典型的腔道发育过程, 是研究管腔形成的重要器官模型。管腔形成由胞外信号诱导、细胞极性化、胞内物质定向运输、腔内液体形成和胞内细胞骨架重构等相关管腔细胞内外发生的结构功能变化过程所构成, 而这些结构与功能的变化过程是通过精确而复杂的分子调控网络来实现, 最终形成管道器官。文章对斑马鱼4种典型管腔型器官的空腔形态发生过程进行了综述, 并总结了此过程中的分子机制, 为今后的相关研究提供了参考。     

钙调蛋白结合蛋白及其磷酸化对癌细胞迁移能力的影响研究

轻链钙调蛋白结合蛋白(light-chain caldesmon,l-CaD)是一种肌动蛋白结合蛋白,它通过与肌动蛋白结合而稳定胞内微丝结构,在磷酸化作用下则能从微丝上脱离.在众多非转移性癌细胞以及永生化的正常细胞系中,l-CaD的表达量很低甚至没有,但在高迁移活性的转移性癌细胞中,l-CaD表达量显著上升,因此l-CaD可能是维持转移性癌细胞高迁移能力的重要因素.为了探索l-CaD如何调节转移性癌细胞迁移活性及其所处地位,以人源转移性乳腺癌细胞MDAMB-231作为载体,一方面,在胞内高表达外源野生型l-CaD及其磷酸化突变株,干扰胞内l-CaD的磷酸化进程,从而考察l-CaD磷酸化对细胞迁移的调节,另一方面,利用siRNA技术,抑制l-CaD在MDAMB-231细胞内的表达量,检测l-CaD对转移性癌细胞迁移活性的总体影响.通过细胞骨架荧光染色、细胞迁移小室、单细胞层次上的牵张力测定以及细胞基底脱黏附能力测定,结果显示:a.阻断MDAMB-231胞内l-CaD的磷酸化进程将显著抑制细胞的迁移能力,细胞骨架调整受阻,基底牵张力增加,细胞基底脱附能力下降;b.l-CaD表达抑制的MDAMB-231细胞失去了完...     

Functionalization of liposomes: microscopical methods for preformulative screening

Abstract

The development of smart delivery systems able to deliver and target a drug to the site of action is one of the major challenges in the field of pharmaceutical technology. The surface modification of nanocarriers, such as liposomes, is widely investigated either for increasing the blood circulation time (by pegylation) or for interacting with specific tissues or cells (by conjugation of a selective ligand as a monoclonal antibody, mAb). Microscopical analysis thereby is a useful approach to evaluate the morphology and the size owing to resolution and versatility in defining either surface modification or the architecture and the internal structure of liposomes. This contribution aims to connect the outputs obtained by transmission electron (TEM) and atomic force (AFM) microscopical techniques for identifying the modifications on the liposomal surface. To reach this objective, we prepared liposomes applying two different pegylation technologies and further modifying the surface by mAb conjugation. This work demonstrates the feasibility to apply the combined approach (TEM and AFM analysis) in the evaluation of the efficacy of a surface engineering process.     

Fatigue loading of tendon results in collagen kinking and denaturation but does not change local tissue mechanics

Fatigue loading is a primary cause of tendon degeneration, which is characterized by the disruption of collagen fibers and the appearance of abnormal (e.g., cartilaginous, fatty, calcified) tissue deposits. The formation of such abnormal deposits, which further weakens the tissue, suggests that resident tendon cells acquire an aberrant phenotype in response to fatigue damage and the resulting altered mechanical microenvironment. While fatigue loading produces clear changes in collagen organization and molecular denaturation, no data exist regarding the effect of fatigue on the local tissue mechanical properties. Therefore, the objective of this study was to identify changes in the local tissue stiffness of tendons after fatigue loading. We hypothesized that fatigue damage would reduce local tissue stiffness, particularly in areas with significant structural damage (e.g., collagen denaturation). We tested this hypothesis by identifying regions of local fatigue damage (i.e., collagen fiber kinking and molecular denaturation) via histologic imaging and by measuring the local tissue modulus within these regions via atomic force microscopy (AFM). Counter to our initial hypothesis, we found no change in the local tissue modulus as a consequence of fatigue loading, despite widespread fiber kinking and collagen denaturation. These data suggest that immediate changes in topography and tissue structure – but not local tissue mechanics – initiate the early changes in tendon cell phenotype as a consequence of fatigue loading that ultimately culminate in tendon degeneration.     

Nanoscale structures and mechanics of barnacle cement

Polymerized barnacle glue was studied by atomic force microscopy (AFM), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and chemical staining. Nanoscale structures exhibiting rod-shaped, globular and irregularly-shaped morphologies were observed in the bulk cement of the barnacle Amphibalanus amphitrite (=Balanus amphitrite) by AFM. SEM coupled with energy dispersive X-ray (EDX) provided chemical composition information, making evident the organic nature of the rod-shaped nanoscale structures. FTIR spectroscopy gave signatures of β-sheet and random coil conformations. The mechanical properties of these nanoscale structures were also probed using force spectroscopy and indentation with AFM. Indentation data yielded higher elastic moduli for the rod-shaped structures when compared with the other structures in the bulk cement. Single molecule AFM force-extension curves on the matrix of the bulk cement often exhibited a periodic sawtooth-like profile, observed in both the extend and retract portions of the force curve. Rod-shaped structures stained with amyloid protein-selective dyes (Congo red and thioflavin-T) revealed that about 5% of the bulk cement were amyloids. A dominant 100 kDa cement protein was found to be mechanically agile, using repeating hydrophobic structures that apparently associate within the same protein or with neighbors, creating toughness on the 1–100 nm length scale.     

Generic physical mechanisms of tissue morphogenesis: A common basis for development and evolution

Morphological evolution is usually considered to occur by the selection of small heritable variations in the expression of anatomical traits, on the basis of improved adaptation to new environmental conditions. An alternative mode of morphological evolution is proposed here: the production of a spectrum of forms by the action of intrinsic physical properties of cell aggregates, followed by intense selection for biochemical mechanisms that make the generation of a subset of viable morphologies, and pathways of transition between morphologies, more reliable. This view provides an account of the origins of important features of metazoan body plans and organ forms, including gastrulation and other types of tissue multilayering, lumen formation, and segmentation. It also implies that most major morphological innovations would occur early in phylogeny, often more than once, with much subsequent genetic selection being of a stabilizing or canalizing variety. Consistent with recent findings, this view predicts that functional redundancy among developmentally important genes and genetic circuits should be prevalent.     

Toward correlating structure and mechanics of platelets

ABSTRACT

The primary physiological function of blood platelets is to seal vascular lesions after injury and form hemostatic thrombi in order to prevent blood loss. This task relies on the formation of strong cellular-extracellular matrix interactions in the subendothelial lesions. The cytoskeleton of a platelet is key to all of its functions: its ability to spread, adhere and contract. Despite the medical significance of platelets, there is still no high-resolution structural information of their cytoskeleton. Here, we discuss and present 3-dimensional (3D) structural analysis of intact platelets by using cryo-electron tomography (cryo-ET) and atomic force microscopy (AFM). Cryo-ET provides in situ structural analysis and AFM gives stiffness maps of the platelets. In the future, combining high-resolution structural and mechanical techniques will bring new understanding of how structural changes modulate platelet stiffness during activation and adhesion.     

Spatial distribution of the transverse stiffness of relaxed and activated rat soleus muscle fibers

The transverse stiffness of glycerinated and demembranated fibers from the soleus muscle of Wistar rats in different functional states was measured by atomic force microscopy. It was demonstrated that the transverse stiffness of relaxed fibers near the Z disk is approximately twofold higher as compared with the M-line region. However, the stiffness of glycerinated fibers in the Z-disk and M-line regions is considerably lower than that of demembranated fibers. The values of mechanical parameters of activated fibers are significantly higher as compared with the relaxed fibers. However, the stiffness of activated glycerinated fibers near the Z disk approximately doubled as compared with the relaxed state, whereas the stiffness of the Z-disk region in demembranated fibers increased more than fourfold. The stiffness of both glycerinated and demembranized fibers near the M-line increased approximately threefold.     

SnoN in regulation of embryonic development and tissue morphogenesis

SnoN (Ski-novel protein) plays an important role in embryonic development, tumorigenesis and aging. Past studies largely focused on its roles in tumorigenesis. Recent studies of its expression patterns and functions in mouse models and mammalian cells have revealed that SnoN interacts with multiple signaling molecules at different cellular levels to modulate the activities of several signaling pathways in a tissue context and developmental stage dependent manner. These studies suggest that SnoN may have broad functions in the embryonic development and tissue morphogenesis.     

Cell surface mechanics and the control of cell shape, tissue patterns and morphogenesis

Embryonic morphogenesis requires the execution of complex mechanisms that regulate the local behaviour of groups of cells. The orchestration of such mechanisms has been mainly deciphered through the identification of conserved families of signalling pathways that spatially and temporally control cell behaviour. However, how this information is processed to control cell shape and cell dynamics is an open area of investigation. The framework that emerges from diverse disciplines such as cell biology, physics and developmental biology points to adhesion and cortical actin networks as regulators of cell surface mechanics. In this context, a range of developmental phenomena can be explained by the regulation of cell surface tension.