An all-optical approach for probing microscopic flows in living embryos

Living systems rely on fluid dynamics from embryonic development to adulthood. To visualize biological fluid flow, devising the proper labeling method compatible with both normal biology and in vivo imaging remains a major experimental challenge. Here, we describe a simple strategy for probing microscopic fluid flows in vivo that meets this challenge. An all-optical procedure combining femtosecond laser ablation, fast confocal microscopy and 3D-particle tracking was devised to label, image and quantify the flow. This approach is illustrated by studying the flow generated within a micrometer scale ciliated vesicle located deep inside the zebrafish embryo and involved in breaking left-right embryonic symmetry. By mapping the velocity field within the vesicle and surrounding a single beating cilium, we show this method can address the dynamics of cilia-driven flows at multiple length scales, and can validate the flow features as predicted from previous simulations. This approach provides new experimental access to questions of microscopic fluid dynamics in vivo.     

Fluid shear stress and the vascular endothelium: for better and for worse

As blood flows, the vascular wall is constantly subjected to physical forces, which regulate important physiological blood vessel responses, as well as being implicated in the development of arterial wall pathologies. Changes in blood flow, thus generating altered hemodynamic forces are responsible for acute vessel tone regulation, the development of blood vessel structure during embryogenesis and early growth, as well as chronic remodeling and generation of adult blood vessels. The complex interaction of biomechanical forces, and more specifically shear stress, derived by the flow of blood and the vascular endothelium raise many yet to be answered questions:How are mechanical forces transduced by endothelial cells into a biological response, and is there a "shear stress receptor"?Are "mechanical receptors" and the final signaling pathways they evoke similar to other stimulus-response transduction systems?How do vascular endothelial cells differ in their response to physiological or pathological shear stresses?Can shear stress receptors or shear stress responsive genes serve as novel targets for the design of diagnostic and therapeutic modalities for cardiovascular pathologies?The current review attempts to bring together recent findings on the in vivo and in vitro responses of the vascular endothelium to shear stress and to address some of the questions raised above.     

干细胞发育的生物力学调控

生物力学是采用力学方法对生物系统的结构和功能进行的研究,与生物化学信号一起是调节胚胎发育、干细胞发育分化和组织器官形成的重要因素。近年来,随着学科交叉的深入,生物力学因素越来越受到研究者的重视。目前的研究表明：在心血管和造血系统,血流产生的流体剪切力对于血管内皮和造血细胞的发育分化至关重要;此外,对于广泛研究的间充质干细胞,由细胞外基质物理特性诱导的细胞张力对于干细胞功能及其向不同子代细胞的分化也扮演了重要的角色;而在肝脏等上皮组织来源的器官,也有研究提示生物力学因素,如基质弹性等在疾病的发生发展过程中起到了不可忽视的作用。总之,在干细胞发育分化过程中,生物力学调控与生物化学信号通路怎样协同发挥作用将成为今后研究的重点。     

MAP kinase and calcium signaling mediate fluid flow-induced human mesenchymal stem cell proliferation

Mechanical signals are important regulators of skeletal homeostasis, and strain-induced oscillatory fluid flow is a potent mechanical stimulus. Although the mechanisms by which osteoblasts and osteocytes respond to fluid flow are being elucidated, little is known about the mechanisms by which bone marrow-derived mesenchymal stem cells respond to such stimuli. Here we show that the intracellular signaling cascades activated in human mesenchymal stem cells by fluid flow are similar to those activated in osteoblastic cells. Oscillatory fluid flow inducing shear stresses of 5, 10, and 20 dyn/cm² triggered rapid, flow rate-dependent increases in intracellular calcium that pharmacological studies suggest are inositol trisphosphate mediated. The application of fluid flow also induced the phosphorylation of extracellular signal-regulated kinase-1 and -2 as well as the activation of the calcium-sensitive protein phosphatase calcineurin in mesenchymal stem cells. Activation of these signaling pathways combined to induce a robust increase in cellular proliferation. These data suggest that mechanically induced fluid flow regulates not only osteoblastic behavior but also that of mesenchymal precursors, implying that the observed osteogenic response to mechanical loading may be mediated by alterations in the cellular behavior of multiple members of the osteoblast lineage, perhaps by a common signaling pathway. mechanotransduction; bone; marrow     

Integration of Biochemical and Biomechanical Signals Regulating Endothelial Barrier Function

Endothelial barrier function is critical for tissue homeostasis throughout the body. Disruption of the endothelial monolayer leads to edema, vascular diseases and even cancer metastasis among other pathological conditions. Breakdown of the endothelial barrier integrity triggered by cytokines (e.g.IL-8,IL-1β) and growth factors (e.g.VEGF) is well documented. However, endothelial cells are subject to major biomechanical forces that affect their behavior. Due to their unique location at the interface between circulating blood and surrounding tissues, endothelial cells experience shear stress, strain and contraction forces. More than three decades ago, it was already appreciated that shear flow caused endothelial cells alignment in the direction of the flow. After that observation, it took around 20 years to begin to uncover some of the mechanisms used by the cells for mechanotransduction. In this review, we describe mechanosensors on the endothelium identified to date and the associated signaling pathways that integrate biochemical and biomechanical inputs into biological responses and how they modulate the integrity of the endothelial barrier.     

Dual echo steady state (dess) pulse sequence in the evaluation of brain cerebrospinal fluid flow disturbances

The authors present their experience with the Dual Echo Steady State pulse sequence used to evaluate brain cerebrospinal fluid (CSF) flow by low-field magnetic resonance imaging. Normal and pathologic CSF flow patterns are discussed. The capacities of the technique as an auxiliary tool to have additional diagnostic information via visualization of flows in the brain CSF pathways are shown.     

Chondrocyte calcium signaling in response to fluid flow is regulated by matrix adhesion in 3-D alginate scaffolds

The interaction between chondrocytes and their surrounding extracellular matrix plays an important role in regulating cartilage metabolism in response to environmental cues. This study characterized the role of cell adhesion on the calcium signaling response of chondrocytes to fluid flow. Bovine chondrocytes were suspended in alginate hydrogels functionalized with RGD at concentrations of 0–400 μM. The hydrogels were perfused and the calcium signaling response of the cells was measured over a range of fluid velocities from 0 to 68 μm/s. Attachment to RGD-alginate doubled the sensitivity of chondrocytes to flows in the range of 8–13 μm/s, but at higher fluid velocities, the contribution of cell adhesion to the observed calcium signaling response was no longer apparent. The enhanced sensitivity to flow was dependent on the density of RGD-ligand present in the scaffolds. The RGD-enhanced sensitivity to flow was completely inhibited by the addition of soluble RGD which acted as a competitive inhibitor. The results of this study indicate a role for matrix adhesion in regulating chondrocyte response to fluid flow through a calcium dependent mechanism.     

Groundwater and pore water inputs to the coastal zone

Both terrestrial and marine forces drive underground fluid flows in the coastal zone. Hydraulic gradients on land result in groundwater seepage near shore and may contribute to flows further out on the shelf from confined aquifers. Marine processes such as tidal pumping and current-induced pressure gradients may induce interfacial fluid flow anywhere on the shelf where permeable sediments are present. The terrestrial and oceanic forces overlap spatially so measured fluid advection through coastal sediments may be a result of composite forcing. We thus define “submarine groundwater discharge” (SGD) as any and all flow of water on continental margins from the seabed to the coastal ocean, regardless of fluid composition or driving force. SGD is typically characterized by low specific flow rates that make detection and quantification difficult. However, because such flows occur over very large areas, the total flux is significant. Discharging fluids, whether derived from land or composed of re-circulated seawater, will react with sediment components. These reactions may increase substantially the concentrations of nutrients, carbon, and metals in the fluids. These fluids are thus a source of biogeochemically important constituents to the coastal ocean. Terrestrially-derived fluids represent a pathway for new material fluxes to the coastal zone. This may result in diffuse pollution in areas where contaminated groundwaters occur. This paper presents an historical context of SGD studies, defines the process in a form that is consistent with our current understanding of the driving forces as well as our assessment techniques, and reviews the estimated global fluxes and biogeochemical implications. We conclude that to fully characterize marine geochemical budgets, one must give due consideration to SGD. New methodologies, technologies, and modeling approaches are required to discriminate among the various forces that drive SGD and to evaluate these fluxes more precisely.     

Interstitial Fluid Flow Increases Hepatocellular Carcinoma Cell Invasion through CXCR4/CXCL12 and MEK/ERK Signaling

Hepatocellular carcinoma (HCC) is the most common form of liver cancer (~80%), and it is one of the few cancer types with rising incidence in the United States. This highly invasive cancer is very difficult to detect until its later stages, resulting in limited treatment options and low survival rates. There is a dearth of knowledge regarding the mechanisms associated with the effects of biomechanical forces such as interstitial fluid flow (IFF) on hepatocellular carcinoma invasion. We hypothesized that interstitial fluid flow enhanced hepatocellular carcinoma cell invasion through chemokine-mediated autologous chemotaxis. Utilizing a 3D in vitro invasion assay, we demonstrated that interstitial fluid flow promoted invasion of hepatocellular carcinoma derived cell lines. Furthermore, we showed that autologous chemotaxis influences this interstitial fluid flow-induced invasion of hepatocellular carcinoma derived cell lines via the C-X-C chemokine receptor type 4 (CXCR4)/C-X-C motif chemokine 12 (CXCL12) signaling axis. We also demonstrated that mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling affects interstitial fluid flow-induced invasion; however, this pathway was separate from CXCR4/CXCL12 signaling. This study demonstrates, for the first time, the potential role of interstitial fluid flow in hepatocellular carcinoma invasion. Uncovering the mechanisms that control hepatocellular carcinoma invasion will aid in enhancing current liver cancer therapies and provide better treatment options for patients.     

Biological impact of mechanical stimuli on tumor metastasis

Increasing evidence suggests tumor cell exposure to mechanical stimuli during the perioperative period as well as throughout the normal disease process may have a discernable impact on tumor metastasis and patient outcome. In vitro studies have demonstrated that transient exposure to increased extracellular pressure and shear forces modulates integrin binding affinity and stimulates cancer cell adhesion through a cytoskeleton- and focal adhesion complex-dependent signaling mechanism. More prolonged exposure to elevated pressures stimulates tumor cell proliferation by a distinct signaling pathway. Whether pressure effects on cell adhesion and proliferation pose biological ramifications in vivo remained unknown. We recently reported that pressure activation of malignant cells does indeed have a biological impact on surgical wound implantation, tumor development, and tumor-free survival in a murine colon tumor model. Moreover, this effect can be disrupted by preoperative administration of colchicine. Taken together with previous work from our laboratory and others, these findings suggest that further

elucidation of the mechanical signaling pathways governing pressure-stimulated tumor cell adhesion and proliferation may identify novel therapeutic targets for the treatment and prevention of tumor metastasis.     

Effects of membrane cholesterol depletion and GPI-anchored protein reduction on osteoblastic mechanotransduction

We previously demonstrated that oscillatory fluid flow activates MC3T3-E1 osteoblastic cell calcium signaling pathways via a mechanism involving ATP releases and P2Y(2) puringeric receptors. However, the molecular mechanisms by which fluid flow initiates cellular responses are still unclear. Accumulating evidence suggests that lipid rafts, one of the important membrane structural components, may play an important role in transducing extracellular fluid shear stress to intracellular responses. Due to the limitations of current techniques, there is no direct approach to study the role of lipid rafts in transmitting fluid shear stress. In this study, we targeted two important membrane components associated with lipid rafts, cholesterol, and glycosylphosphatidylinositol-anchored proteins (GPI-anchored proteins), to disrupt the integrity of cell membrane structures. We first demonstrated that membrane cholesterol depletion with the treatment of methyl-β-cyclodextrin inhibits oscillatory fluid flow induced intracellular calcium mobilization and ERK1/2 phosphorylation in MC3T3-E1 osteoblastic cells. Secondly, we used a novel approach to decrease the levels of GPI-anchored proteins on cell membranes by overexpressing glycosylphosphatidylinositol-specific phospholipase D in MC3T3-E1 osteoblastic cells. This resulted in significant inhibition of intracellular calcium mobilization and ERK1/2 phosphorylation in response to oscillatory fluid flow. Finally, we demonstrated that cholesterol depletion inhibited oscillatory fluid flow induced ATP releases, which were responsible for the activation of calcium signaling pathways in MC3T3-E1 osteoblastic cells. Our findings suggest that cholesterol and GPI-anchored proteins, two membrane structural components related to lipid rafts, may play an important role in osteoblastic cell mechanotransduction.     

A tridomain model for potassium clearance in optic nerve of Necturus

Complex fluids flow in complex ways in complex structures. Transport of water and various organic and inorganic molecules in the central nervous system are important in a wide range of biological and medical processes. However, the exact driving mechanisms are often not known. In this work, we investigate flows induced by action potentials in an optic nerve as a prototype of the central nervous system. Different from traditional fluid dynamics problems, flows in biological tissues such as the central nervous system are coupled with ion transport. They are driven by osmosis created by concentration gradient of ionic solutions, which in turn influence the transport of ions. Our mathematical model is based on the known structural and biophysical properties of the experimental system used by the Harvard group Orkand et al. Asymptotic analysis and numerical computation show the significant role of water in convective ion transport. The full model (including water) and the electrodiffusion model (excluding water) are compared in detail to reveal an interesting interplay between water and ion transport. In the full model, convection due to water flow dominates inside the glial domain. This water flow in the glia contributes significantly to the spatial buffering of potassium in the extracellular space. Convection in the extracellular domain does not contribute significantly to spatial buffering. Electrodiffusion is the dominant mechanism for flows confined to the extracellular domain.     

Assembly and mechanosensory function of focal contacts.

Focal contacts, focal complexes and related extracellular matrix adhesions are used by cells to explore their environment. These sites act as mechanosensory 'devices', where internal contractile forces or externally applied force can regulate the assembly of the adhesion site and trigger adhesion-dependent signaling involving Rho-family small G-proteins and other signaling pathways. The molecular mechanisms underlying these processes are discussed.     

Fluid‐driven interfacial instabilities and turbulence in bacterial biofilms

Biofilms are thin layers of bacteria embedded within a slime matrix that live on surfaces. They are ubiquitous in nature and responsible for many medical and dental infections, industrial fouling and are also evident in ancient fossils. A biofilm structure is shaped by growth, detachment and response to mechanical forces acting on them. The main contribution to biofilm versatility in response to physical forces is the matrix that provides a platform for the bacteria to grow. The interaction between biofilm structure and hydrodynamics remains a fundamental question concerning biofilm dynamics. Here, we document the appearance of ripples and wrinkles in biofilms grown from three species of bacteria when subjected to high‐velocity fluid flows. Linear stability analysis suggested that the ripples were Kelvin–Helmholtz Instabilities. The analysis also predicted a strong dependence of the instability formation on biofilm viscosity explaining the different surface corrugations observed. Turbulence through Kelvin–Helmholtz instabilities occurring at the interface demonstrated that the biofilm flows like a viscous liquid under high flow velocities applied within milliseconds. Biofilm fluid‐like behavior may have important implications for our understanding of how fluid flow influences biofilm biology since turbulence will likely disrupt metabolite and signal gradients as well as community stratification.     

Flow mechanotransduction regulates traction forces, intercellular forces, and adherens junctions

Endothelial cells respond to fluid shear stress through mechanotransduction responses that affect their cytoskeleton and cell-cell contacts. Here, endothelial cells were grown as monolayers on arrays of microposts and exposed to laminar or disturbed flow to examine the relationship among traction forces, intercellular forces, and cell-cell junctions. Cells under laminar flow had traction forces that were higher than those under static conditions, whereas cells under disturbed flow had lower traction forces. The response in adhesion junction assembly matched closely with changes in traction forces since adherens junctions were larger in size for laminar flow and smaller for disturbed flow. Treating the cells with calyculin-A to increase myosin phosphorylation and traction forces caused an increase in adherens junction size, whereas Y-27362 cause a decrease in their size. Since tugging forces across cell-cell junctions can promote junctional assembly, we developed a novel approach to measure intercellular forces and found that these forces were higher for laminar flow than for static or disturbed flow. The size of adherens junctions and tight junctions matched closely with intercellular forces for these flow conditions. These results indicate that laminar flow can increase cytoskeletal tension while disturbed flow decreases cytoskeletal tension. Consequently, we found that changes in cytoskeletal tension in response to shear flow conditions can affect intercellular tension, which in turn regulates the assembly of cell-cell junctions.     

Force-induced Unfolding of the Focal Adhesion Targeting Domain and the Influence of Paxillin Binding

Membrane-bound integrin receptors are linked to intracellular signaling pathways through focal adhesion kinase (FAK). FAK tends to colocalize with integrin receptors at focal adhesions through its C-terminal focal adhesion targeting (FAT) domain. Through recruitment and binding of intracellular proteins, FAs transduce signals between the intracellular and extracellular regions that regulate a variety of cellular processes including cell migration, proliferation, apoptosis and detachment from the ECM. The mechanism of signaling through the cell is of interest, especially the transmission of mechanical forces and subsequent transduction into biological signals. One hypothesis relates mechanotransduction to conformational changes in intracellular proteins in the force transmission pathway, connecting the extracellular matrix with the cytoskeleton through FAs. To assess this hypothesis, we performed steered molecular dynamics simulations to mechanically unfold FAT and monitor how force-induced changes in the molecular conformation of FAT affect its binding to paxillin.