CNS Cell Distribution and Axon Orientation Determine Local Spinal Cord Mechanical Properties

Mechanical signaling plays an important role in cell physiology and pathology. Many cell types, including neurons and glial cells, respond to the mechanical properties of their environment. Yet, for spinal cord tissue, data on tissue stiffness are sparse. To investigate the regional and direction-dependent mechanical properties of spinal cord tissue at a spatial resolution relevant to individual cells, we conducted atomic force microscopy (AFM) indentation and tensile measurements on acutely isolated mouse spinal cord tissue sectioned along the three major anatomical planes, and correlated local mechanical properties with the underlying cellular structures. Stiffness maps revealed that gray matter is significantly stiffer than white matter irrespective of directionality (transverse, coronal, and sagittal planes) and force direction (compression or tension) (K_g= ∼130 Pa vs. K_w= ∼70 Pa); both matters stiffened with increasing strain. When all data were pooled for each plane, gray matter behaved like an isotropic material under compression; however, subregions of the gray matter were rather heterogeneous and anisotropic. For example, in sagittal sections the dorsal horn was significantly stiffer than the ventral horn. In contrast, white matter behaved transversely isotropic, with the elastic stiffness along the craniocaudal (i.e., longitudinal) axis being lower than perpendicular to it. The stiffness distributions we found under compression strongly correlated with the orientation of axons, the areas of cell nuclei, and cellular in plane proximity. Based on these morphological parameters, we developed a phenomenological model to estimate local mechanical properties of central nervous system (CNS) tissue. Our study may thus ultimately help predicting local tissue stiffness, and hence cell behavior in response to mechanical signaling under physiological and pathological conditions, purely based on histological data.     

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10^-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.     

Cirri of the stalked crinoid Metacrinus rotundus: neural elements and the effect of cholinergic agonists on mechanical properties

Sea lilies are enigmatic animals due to their scarcity and their biology is comparatively neglected. Cirri, arranged in whorls of five along the sea lily stalk, anchor and support the animal. They consist of ossicles interconnected by collagenous ligaments and by a central canal. Cirri have a well-developed nervous system but lack muscular cells. A light and electron microscopic study was performed to clarify the morphology of the nervous system of the cirri. Two cellular networks were found, one of neuron-like cells and one of cells filled with bullet-shaped organelles. Both networks ramify throughout the cirral ossicles up to the interossicle ligaments. Mechanical tests were performed to analyse the influence of cholinergic agonists on the mechanical properties of these ligaments. In the tests, the cirral ligaments softened after the application of acetylcholine, muscarinic agonists and nicotinic agonists. The reaction time to muscarinic agonists was much slower than to acetylcholine and nicotinic agonists. At low concentrations, muscarinic agonists caused active development of force. No reaction to stimuli was observed in anaesthetized cirri. The data clearly establish the existence of catch connective tissue which can change its mechanical properties under nervous control mediated via nerves with cholinergic receptors. The possible sources of the observed force production are discussed and it is concluded that active contraction of collagenous ligaments causes movement of cirri.     

Müller glia provide essential tensile strength to the developing retina

To investigate the cellular basis of tissue integrity in a vertebrate central nervous system (CNS) tissue, we eliminated Müller glial cells (MG) from the zebrafish retina. For well over a century, glial cells have been ascribed a mechanical role in the support of neural tissues, yet this idea has not been specifically tested in vivo. We report here that retinas devoid of MG rip apart, a defect known as retinoschisis. Using atomic force microscopy, we show that retinas without MG have decreased resistance to tensile stress and are softer than controls. Laser ablation of MG processes showed that these cells are under tension in the tissue. Thus, we propose that MG act like springs that hold the neural retina together, finally confirming an active mechanical role of glial cells in the CNS.     

神经元轴突生长的力生物学

神经系统作为一个复杂的体系,在其发育过程中轴突需要延伸较长的距离才能与下一级神经元或靶细胞形成突触。在这个复杂的移动过程中,神经元轴突在空间分布上形成了精确有序的结构。过去认为这种有序结构的形成主要由形态发生素的化学浓度梯度来指导,而最近的研究发现力学因素对调控轴突的延伸速度与方向发挥着重要的作用。因此,轴突的延伸本质上是一个力化学耦合过程。本文将结合自己过去的工作论述力学因素对轴突延伸的调控机制及相关的信号转导。这一领域的研究将为认识对神经系统疾病的发生以及神经再生提供重要的参考。     

The adhesion force of Notch with Delta and the rate of Notch signaling

Notch signaling is repeatedly used during animal development to specify cell fates. Using atomic force microscopy on live cells, chemical inhibitors, and conventional analyses, we show that the rate of Notch signaling is linked to the adhesion force between cells expressing Notch receptors and Delta ligand. Both the Notch extracellular and intracellular domains are required for the high adhesion force with Delta. This high adhesion force is lost within minutes, primarily due to the action of Presenilin on Notch. Reduced turnover or Delta pulling accelerate this loss. These data suggest that strong adhesion between Notch and Delta might serve as a booster for initiating Notch signaling at a high rate.     

Intermediate filaments in smooth muscle

The intermediate filament (IF) network is one of the three cytoskeletal systems in smooth muscle. The type III IF proteins vimentin and desmin are major constituents of the network in smooth muscle cells and tissues. Lack of vimentin or desmin impairs contractile ability of various smooth muscle preparations, implying their important role for smooth muscle force development. The IF framework has long been viewed as a fixed cytostructure that solely provides mechanical integrity for the cell. However, recent studies suggest that the IF cytoskeleton is dynamic in mammalian cells in response to various external stimulation. In this review, the structure and biological properties of IF proteins in smooth muscle are summarized. The role of IF proteins in the modulation of smooth muscle force development and redistribution/translocation of signaling partners (such as p130 Crk-associated substrate, CAS) is depicted. This review also summarizes our latest understanding on how the IF network may be regulated in smooth muscle. cytoskeleton; force development; vimentin; desmin     

Development of the enteric nervous system in chick embryonic duodenum in terms of the distribution of tubulin

An immunohistochemical method that uses anti-tubulin was utilized to observe the development of the enteric nervous system in chick embryonic duodenum. Neural crest cells, and enteric neuroblasts, or enteric ganglia, which derive from neural crest cells were clearly shown as sharp immunoreactive regions of tubulin. The distributions of enteric neuroblasts and enteric ganglia in chick duodena were in agreement with results of previous reports in which different techniques were used. The initial stage at which cells of neural crest origin were present in the duodenal walls (4-day-old embryos) was earlier than the initial stage (about 6-day-old embryos) reported earlier. This was verified by transmission electron microscopy. Also, the tubulin that is a component of the enteric nervous system was shown to be stable at a low temperature. This tubulin-immunostaining method provides a useful histochemical technique with which to study the development of the enteric ganglion and the function of tubulin as a component of the enteric nervous system.     

Simple display system of mechanical properties of cells and their dispersion

The mechanical properties of cells are unique indicators of their states and functions. Though, it is difficult to recognize the degrees of mechanical properties, due to small size of the cell and broad distribution of the mechanical properties. Here, we developed a simple virtual reality system for presenting the mechanical properties of cells and their dispersion using a haptic device and a PC. This system simulates atomic force microscopy (AFM) nanoindentation experiments for floating cells in virtual environments. An operator can virtually position the AFM spherical probe over a round cell with the haptic handle on the PC monitor and feel the force interaction. The Young's modulus of mesenchymal stem cells and HEK293 cells in the floating state was measured by AFM. The distribution of the Young's modulus of these cells was broad, and the distribution complied with a log-normal pattern. To represent the mechanical properties together with the cell variance, we used log-normal distribution-dependent random number determined by the mode and variance values of the Young's modulus of these cells. The represented Young's modulus was determined for each touching event of the probe surface and the cell object, and the haptic device-generating force was calculated using a Hertz model corresponding to the indentation depth and the fixed Young's modulus value. Using this system, we can feel the mechanical properties and their dispersion in each cell type in real time. This system will help us not only recognize the degrees of mechanical properties of diverse cells but also share them with others.     

The initiation of blood flow and flow induced events in early vascular development

Within a day of gastrulation, the embryonic heart begins to beat and creates blood flow in the developing cardiovascular system. The onset of blood flow completely changes the environment in which the cardiovascular system is forming. Flow provides physiological feedback such that the developing network adapts to cue provided by the flow. Targeted inactivation of genes that alter early blood fluid dynamics induce secondary defects in the heart and vasculature and therefore proper blood flow is known to be essential for vascular development. Though hemodynamics, or blood fluid dynamics, are known to activate signaling pathways in the mature cardiovascular system in pathologies ranging from artherosclerosis to angiogenesis, the role in development has not been as intensively studied. The question arises how blood vessels in the embryos, which initially lack cells types such as smooth muscle cells, differ in their response to mechanical signals from blood flow as compared to the more mature cardiovascular system. Many genes known to be regulated by hemodynamics in the adult are important for developmental angiogenesis. Therefore the onset of blood flow is of primary importance to vascular development. This review will focus on how blood flow initiates and the effects of the mechanical signals created by blood flow on cardiovascular development.     

Tracking mechanics and volume of globular cells with atomic force microscopy using a constant-height clamp

To understand the role of physical forces at a cellular level, it is necessary to track mechanical properties during cellular processes. Here we present a protocol that uses flat atomic force microscopy (AFM) cantilevers clamped at constant height, and light microscopy to measure the resistance force, mechanical stress and volume of globular animal cells under compression. We describe the AFM and cantilever setup, live cell culture in the AFM, how to ensure stability of AFM measurements during medium perfusion, integration of optical microscopy to measure parameters such as volume and track intracellular dynamics, and interpretation of the physical parameters measured. Although we use this protocol on trypsinized interphase and mitotic HeLa cells, it can also be applied to other cells with a relatively globular shape, especially animal cells in a low-adhesive environment. After a short setup phase, the protocol can be used to investigate approximately one cell per hour.     

erbb3 and erbb2 are essential for schwann cell migration and myelination in zebrafish

BACKGROUND: Myelin is critical for efficient axonal conduction in the vertebrate nervous system. Neuregulin (Nrg) ligands and their ErbB receptors are required for the development of Schwann cells, the glial cells that form myelin in the peripheral nervous system. Previous studies have not determined whether Nrg-ErbB signaling is essential in vivo for Schwann cell fate specification, proliferation, survival, migration, or the onset of myelination. RESULTS: In genetic screens for mutants with disruptions in myelinated nerves, we identified mutations in erbb3 and erbb2, which together encode a heteromeric tyrosine kinase receptor for Neuregulin ligands. Phenotypic analysis shows that both genes are essential for development of Schwann cells. BrdU-incorporation studies and time-lapse analysis reveal that Schwann cell proliferation and migration, but not survival, are disrupted in erbb3 mutants. We show that Schwann cells can migrate in the absence of DNA replication. This uncoupling of proliferation and migration indicates that erbb gene function is required independently for these two processes. Pharmacological inhibition of ErbB signaling at different stages reveals a continuing requirement for ErbB function during migration and also provides evidence that ErbB signaling is required after migration for proliferation and the terminal differentiation of myelinating Schwann cells. CONCLUSIONS: These results provide in vivo evidence that Neuregulin-ErbB signaling is essential for directed Schwann cell migration and demonstrate that this pathway is also required for the onset of myelination in postmigratory Schwann cells.     

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.     

Combining mechanical and optical approaches to dissect cellular mechanobiology

Mechanical force modulates a wide array of cell physiological processes. Cells sense and respond to mechanical stimuli using a hierarchy of structural complexes spanning multiple length scales, including force-sensitive molecules and cytoskeletal networks. Understanding mechanotransduction, i.e., the process by which cells convert mechanical inputs into biochemical signals, has required the development of novel biophysical tools that allow for probing of cellular and subcellular components at requisite time, length, and force scales and technologies that track the spatio-temporal dynamics of relevant biomolecules. In this review, we begin by discussing the underlying principles and recent applications of atomic force microscopy, magnetic twisting cytometry, and traction force microscopy, three tools that have been widely used for measuring the mechanical properties of cells and for probing the molecular basis of cellular mechanotransduction. We then discuss how such tools can be combined with advanced fluorescence methods for imaging biochemical processes in living cells in the context of three specific problem spaces. We first focus on fluorescence resonance energy transfer, which has enabled imaging of intra- and inter-molecular interactions and enzymatic activity in real time based on conformational changes in sensor molecules. Next, we examine the use of fluorescence methods to probe force-dependent dynamics of focal adhesion proteins. Finally, we discuss the use of calcium ratiometric signaling to track fast mechanotransductive signaling dynamics. Together, these studies demonstrate how single-cell biomechanical tools can be effectively combined with molecular imaging technologies for elucidating mechanotransduction processes and identifying mechanosensitive proteins.     

Substrate viscosity impairs temozolomide-mediated inhibition of glioblastoma cells' growth

BackgroundThe mechanical state of the extracellular environment of the brain cells considerably affects their phenotype during the development of central nervous system (CNS) pathologies, and when the cells respond to drugs. The reports on the evaluation of the viscoelastic properties of different brain tumors have shown that both tissue stiffness and viscosity can be altered during cancer development. Although a compelling number of reports established the role of substrate stiffness on the proliferation, motility, and drug sensitivity of brain cancer cells, there is a lack of parallel data in terms of alterations in substrate viscosity.MethodsBased on viscoelasticity measurements of rat brain samples using strain rheometry, polyacrylamide (PAA) hydrogels mimicking elastic and viscous parameters of the tissues were prepared. Optical microscopy and flow cytometry were employed to assess the differences in glioblastoma cells morphology, proliferation, and cytotoxicity of anticancer drug temozolomide (TMZ) due to increased substrate viscosity.ResultsOur results indicate that changes in substrate viscosity affect the proliferation of untreated glioma cells to a lesser extent, but have a significant impact on the apoptosis-associated depolarization of mitochondria and level of DNA fragmentation. This suggests that viscosity sensing and stiffness sensing machinery can activate different signaling pathways in glioma cells.ConclusionCollected data indicate that viscosity should be considered an important parameter in in vitro polymer-based cell culture systems used for drug screening.     

On the Molecular Basis for Mechanotransduction

Much is currently known about the signaling pathways that are excited when cells are subjected to a mechanical stimulus, yet we understand little of the process by which the mechanical perturbation is transformed into a biochemical signal. Numerous theories have been proposed, and each has merit. While cells may possess many different ways of responding to stress, the existence of a single unifying principle has much appeal. Here we propose the hypothesis that cells sense mechanical force through changes in protein conformation, leading to altered binding affinities of proteins, ultimately initiating an intracellular signaling cascade or producing changes in the proteins localized to regions of high stress. More generally, this represents an alternative to transmembrane signaling through receptor-ligand interactions providing the cell with a means of reacting to changes in its mechanical, as opposed to biochemical, environment. One example is presented showing how the binding affinity between the focal adhesion targeting domain of focal adhesion kinase and the LD motif of paxillin is influenced by externally applied force.     

Biomechanical force in blood development: Extrinsic physical cues drive pro-hematopoietic signaling

The hematopoietic system is dynamic during development and in adulthood, undergoing countless spatial and temporal transitions during the course of one's life. Microenvironmental cues in the many unique hematopoietic niches differ, characterized by distinct soluble molecules, membrane-bound factors, and biophysical features that meet the changing needs of the blood system. Research from the last decade has revealed the importance of substrate elasticity and biomechanical force in determination of stem cell fate. Our understanding of the role of these factors in hematopoiesis is still relatively poor; however, the developmental origin of blood cells from the endothelium provides a model for comparison. Many endothelial mechanical sensors and second messenger systems may also determine hematopoietic stem cell fate, self renewal, and homing behaviors. Further, the intimate contact of hematopoietic cells with mechanosensitive cell types, including osteoblasts, endothelial cells, mesenchymal stem cells, and pericytes, places them in close proximity to paracrine signaling downstream of mechanical signals. The objective of this review is to present an overview of the sensors and intracellular signaling pathways activated by mechanical cues and highlight the role of mechanotransductive pathways in hematopoiesis.     

Application of atomic force microscopy in cancer research

Atomic force microscopy (AFM) allows for nanometer-scale investigation of cells and molecules. Recent advances have enabled its application in cancer research and diagnosis. The physicochemical properties of live cells undergo changes when their physiological conditions are altered. These physicochemical properties can therefore reflect complex physiological processes occurring in cells. When cells are in the process of carcinogenesis and stimulated by external stimuli, their morphology, elasticity, and adhesion properties may change. AFM can perform surface imaging and ultrastructural observation of live cells with atomic resolution under near-physiological conditions, collecting force spectroscopy information which allows for the study of the mechanical properties of cells. For this reason, AFM has potential to be used as a tool for high resolution research into the ultrastructure and mechanical properties of tumor cells. This review describes the working principle, working mode, and technical points of atomic force microscopy, and reviews the applications and prospects of atomic force microscopy in cancer research.     

Synaptic signaling by lipids in the life and death of neurons

Synaptic activity promotes the regulated formation of lipid messengers through phospholipase-mediated cleavage of specific phospholipid reservoirs from membranes. Multiple effectors trigger the formation of lipid messengers, including neurotransmitters, membrane depolarization, ion channels, cytokines, and neurotrophic factors. Lipid messengers in turn modulate and interact with other signaling cascades, contributing to the development, differentiation, function (e.g., long-term potentiation [LTP] and memory), protection, and repair of cells in the nervous system. These relationships with other signaling cascades remain largely to be investigated. Oxidative stress disrupts lipid signaling, enhances lipid peroxidation, and initiates and propagates neurodegeneration. There is growing evidence that lipid messengers participate in the extensive interactions among neurons, astrocytes, oligodendrocytes, microglia, cells of the microvasculature, and other cells. This article provides an example of how signaling by lipids regulates critical events essential for neuronal survival and reviews the recent identification of a novel endogenous neuroprotective signaling pathway involving a docosahexaneoic acid-derived mediator.     

Components of the Corticotropin-Releasing-Factor (CRF) Signaling System in Snail Atria

The vertebrate CRF signaling system consists of corticotropin-releasing factor (CRF), two types of receptors to CRF (CRF-R1 and CRF-R2), and CRF-binding protein (CRF-BP). The aim of this study was to investigate the presence and localization of CRF, CRF-R2, and CRF-BP in the snail atrial neuroendocrine complexes, which include granular cells (GCs) and tightly connected nerve fibers and gliointerstitial cells. Immunofluorescence assay and immunogold electron microscopy using polyclonal antibodies against these proteins revealed immunoreactivity in the granules of all these cells. Western-blot analysis of the snailatria lysate using rabbit anti-CRF-R2 polyclonal antibodies revealed a specific band with a weight of 56 kids. These are the first data on the molecular weight of this receptor in mollusks. Furthermore, to clarify the possible functions of CRF in the neuroendocrine complexes, the hormone was added to GCs isolated from the snail atrium. The proportion of degranulated GCs after CRF addition almost doubled (45.5 vs. 24.5% in control, p < 0.05). The results indicate the presence of all three components of the CRF signaling system in the neuroendocrine complexes of snail atrium. In addition, their presence in both secretory and nervous components of the complexes suggests that the CRF signaling system participates in the nervous regulation of secretory activity of GCs and transfer of information from GCs to the CNS.