Optical trapping reveals propulsion forces, power generation and motility efficiency of the unicellular parasites Trypanosoma brucei brucei

NASA Astrophysics Data System (ADS)

Stellamanns, Eric; Uppaluri, Sravanti; Hochstetter, Axel; Heddergott, Niko; Engstler, Markus; Pfohl, Thomas

2014-10-01

Unicellular parasites have developed sophisticated swimming mechanisms to survive in a wide range of environments. Cell motility of African trypanosomes, parasites responsible for fatal illness in humans and animals, is crucial both in the insect vector and the mammalian host. Using millisecond-scale imaging in a microfluidics platform along with a custom made optical trap, we are able to confine single cells to study trypanosome motility. From the trapping characteristics of the cells, we determine the propulsion force generated by cells with a single flagellum as well as of dividing trypanosomes with two fully developed flagella. Estimates of the dissipative energy and the power generation of single cells obtained from the motility patterns of the trypanosomes within the optical trap indicate that specific motility characteristics, in addition to locomotion, may be required for antibody clearance. Introducing a steerable second optical trap we could further measure the force, which is generated at the flagellar tip. Differences in the cellular structure of the trypanosomes are correlated with the trapping and motility characteristics and in consequence with their propulsion force, dissipative energy and power generation.

Optical trapping reveals propulsion forces, power generation and motility efficiency of the unicellular parasites Trypanosoma brucei brucei.

PubMed

Stellamanns, Eric; Uppaluri, Sravanti; Hochstetter, Axel; Heddergott, Niko; Engstler, Markus; Pfohl, Thomas

2014-10-01

Unicellular parasites have developed sophisticated swimming mechanisms to survive in a wide range of environments. Cell motility of African trypanosomes, parasites responsible for fatal illness in humans and animals, is crucial both in the insect vector and the mammalian host. Using millisecond-scale imaging in a microfluidics platform along with a custom made optical trap, we are able to confine single cells to study trypanosome motility. From the trapping characteristics of the cells, we determine the propulsion force generated by cells with a single flagellum as well as of dividing trypanosomes with two fully developed flagella. Estimates of the dissipative energy and the power generation of single cells obtained from the motility patterns of the trypanosomes within the optical trap indicate that specific motility characteristics, in addition to locomotion, may be required for antibody clearance. Introducing a steerable second optical trap we could further measure the force, which is generated at the flagellar tip. Differences in the cellular structure of the trypanosomes are correlated with the trapping and motility characteristics and in consequence with their propulsion force, dissipative energy and power generation.

Active mechanics in living oocytes reveal molecular-scale force kinetics

NASA Astrophysics Data System (ADS)

Ahmed, Wylie; Fodor, Etienne; Almonacid, Maria; Bussonnier, Matthias; Verlhac, Marie-Helene; Gov, Nir; Visco, Paolo; van Wijland, Frederic; Betz, Timo

Unlike traditional materials, living cells actively generate forces at the molecular scale that change their structure and mechanical properties. This nonequilibrium activity is essential for cellular function, and drives processes such as cell division. Single molecule studies have uncovered the detailed force kinetics of isolated motor proteins in-vitro, however their behavior in-vivo has been elusive due to the complex environment inside the cell. Here, we quantify active forces and intracellular mechanics in living oocytes using in-vivo optical trapping and laser interferometry of endogenous vesicles. We integrate an experimental and theoretical framework to connect mesoscopic measurements of nonequilibrium properties to the underlying molecular- scale force kinetics. Our results show that force generation by myosin-V drives the cytoplasmic-skeleton out-of-equilibrium (at frequencies below 300 Hz) and actively softens the environment. In vivo myosin-V activity generates a force of F ~ 0 . 4 pN, with a power-stroke of length Δx ~ 20 nm and duration τ ~ 300 μs, that drives vesicle motion at vv ~ 320 nm/s. This framework is widely applicable to characterize living cells and other soft active materials.

Tissue architecture, cell traction, deformable scaffolds, and the forces that shape the embryo during morphogenesis.

NASA Astrophysics Data System (ADS)

Davidson, Lance

2005-03-01

Morphogenesis is the process of constucting form and shape. Morphogenesis during early development of the embryo involves orchestrated movements of cells and tissues. These morphogenetic movements establish the body plan and organs of the early embryo. The rates and trajectories of these movements depend on three physical features of the early embryo: 1) the forces generated by cells, 2) the mechanical properties of the tissues, and 3) the architecture of the tissues. These three mechanical features of the embryo are some of the earliest phenotypic features generated by the genome. We are taking an interdisciplinary approach combining biophysical, cell biological, and classical embryological techniques to understand the mechanics of morphogenesis. Using nanoNewton-sensitive force transducers we can apply forces and measure time dependent elastic modulii of tissue fragments 100 micrometers across. Using traction-force microscopy we can measure forces generated by cells on their environment. We use drugs and chimeric proteins to investigate the localization and function of molecular complexes responsible for force generation and the modulus. We use microsurgery to take-apart and construct novel tissues to investigate the role of geometry and architecture in the mechanics of morphogenesis. Together with simulation techniques these quantitative approaches will provide us with a practical nuts-and-bolts understanding of how the genome encodes the shapes and forms of life.

Optical trapping reveals propulsion forces, power generation and motility efficiency of the unicellular parasites Trypanosoma brucei brucei

PubMed Central

Stellamanns, Eric; Uppaluri, Sravanti; Hochstetter, Axel; Heddergott, Niko; Engstler, Markus; Pfohl, Thomas

2014-01-01

Unicellular parasites have developed sophisticated swimming mechanisms to survive in a wide range of environments. Cell motility of African trypanosomes, parasites responsible for fatal illness in humans and animals, is crucial both in the insect vector and the mammalian host. Using millisecond-scale imaging in a microfluidics platform along with a custom made optical trap, we are able to confine single cells to study trypanosome motility. From the trapping characteristics of the cells, we determine the propulsion force generated by cells with a single flagellum as well as of dividing trypanosomes with two fully developed flagella. Estimates of the dissipative energy and the power generation of single cells obtained from the motility patterns of the trypanosomes within the optical trap indicate that specific motility characteristics, in addition to locomotion, may be required for antibody clearance. Introducing a steerable second optical trap we could further measure the force, which is generated at the flagellar tip. Differences in the cellular structure of the trypanosomes are correlated with the trapping and motility characteristics and in consequence with their propulsion force, dissipative energy and power generation. PMID:25269514

Spatial distribution of filament elasticity determines the migratory behaviors of a cell

PubMed Central

Harn, Hans I-Chen; Hsu, Chao-Kai; Wang, Yang-Kao; Huang, Yi-Wei; Chiu, Wen-Tai; Lin, Hsi-Hui; Cheng, Chao-Min; Tang, Ming-Jer

2016-01-01

ABSTRACT Any cellular response leading to morphological changes is highly tuned to balance the force generated from structural reorganization, provided by actin cytoskeleton. Actin filaments serve as the backbone of intracellular force, and transduce external mechanical signal via focal adhesion complex into the cell. During migration, cells not only undergo molecular changes but also rapid mechanical modulation. Here we focus on determining, the role of spatial distribution of mechanical changes of actin filaments in epithelial, mesenchymal, fibrotic and cancer cells with non-migration, directional migration, and non-directional migration behaviors using the atomic force microscopy. We found 1) non-migratory cells only generated one type of filament elasticity, 2) cells generating spatially distributed two types of filament elasticity showed directional migration, and 3) pathologic cells that autonomously generated two types of filament elasticity without spatial distribution were actively migrating non-directionally. The demonstration of spatial regulation of filament elasticity of different cell types at the nano-scale highlights the coupling of cytoskeletal function with physical characters at the sub-cellular level, and provides new research directions for migration related disease. PMID:26919488

From static to animated: Measuring mechanical forces in tissues

PubMed Central

2017-01-01

Cells are physical objects that exert mechanical forces on their surroundings as they migrate and take their places within tissues. New techniques are now poised to enable the measurement of cell-generated mechanical forces in intact tissues in vivo, which will illuminate the secret dynamic lives of cells and change our current perception of cell biology. PMID:28003332

Active cell mechanics: Measurement and theory.

PubMed

Ahmed, Wylie W; Fodor, Étienne; Betz, Timo

2015-11-01

Living cells are active mechanical systems that are able to generate forces. Their structure and shape are primarily determined by biopolymer filaments and molecular motors that form the cytoskeleton. Active force generation requires constant consumption of energy to maintain the nonequilibrium activity to drive organization and transport processes necessary for their function. To understand this activity it is necessary to develop new approaches to probe the underlying physical processes. Active cell mechanics incorporates active molecular-scale force generation into the traditional framework of mechanics of materials. This review highlights recent experimental and theoretical developments towards understanding active cell mechanics. We focus primarily on intracellular mechanical measurements and theoretical advances utilizing the Langevin framework. These developing approaches allow a quantitative understanding of nonequilibrium mechanical activity in living cells. This article is part of a Special Issue entitled: Mechanobiology. Copyright © 2015. Published by Elsevier B.V.

Quantification of cellular penetrative forces using lab-on-a-chip technology and finite element modeling

PubMed Central

Sanati Nezhad, Amir; Naghavi, Mahsa; Packirisamy, Muthukumaran; Bhat, Rama; Geitmann, Anja

2013-01-01

Tip-growing cells have the unique property of invading living tissues and abiotic growth matrices. To do so, they exert significant penetrative forces. In plant and fungal cells, these forces are generated by the hydrostatic turgor pressure. Using the TipChip, a microfluidic lab-on-a-chip device developed for tip-growing cells, we tested the ability to exert penetrative forces generated in pollen tubes, the fastest-growing plant cells. The tubes were guided to grow through microscopic gaps made of elastic polydimethylsiloxane material. Based on the deformation of the gaps, the force exerted by the elongating tubes to permit passage was determined using finite element methods. The data revealed that increasing mechanical impedance was met by the pollen tubes through modulation of the cell wall compliance and, thus, a change in the force acting on the obstacle. Tubes that successfully passed a narrow gap frequently burst, raising questions about the sperm discharge mechanism in the flowering plants. PMID:23630253

Endogenous Sheet-Averaged Tension Within a Large Epithelial Cell Colony.

PubMed

Dumbali, Sandeep P; Mei, Lanju; Qian, Shizhi; Maruthamuthu, Venkat

2017-10-01

Epithelial cells form quasi-two-dimensional sheets that function as contractile media to effect tissue shape changes during development and homeostasis. Endogenously generated intrasheet tension is a driver of such changes, but has predominantly been measured in the presence of directional migration. The nature of epithelial cell-generated forces transmitted over supracellular distances, in the absence of directional migration, is thus largely unclear. In this report, we consider large epithelial cell colonies which are archetypical multicell collectives with extensive cell-cell contacts but with a symmetric (circular) boundary. Using the traction force imbalance method (TFIM) (traction force microscopy combined with physical force balance), we first show that one can determine the colony-level endogenous sheet forces exerted at the midline by one half of the colony on the other half with no prior assumptions on the uniformity of the mechanical properties of the cell sheet. Importantly, we find that this colony-level sheet force exhibits large variations with orientation-the difference between the maximum and minimum sheet force is comparable to the average sheet force itself. Furthermore, the sheet force at the colony midline is largely tensile but the shear component exhibits significantly more variation with orientation. We thus show that even an unperturbed epithelial colony with a symmetric boundary shows significant directional variation in the endogenous sheet tension and shear forces that subsist at the colony level.

Grip and slip of L1-CAM on adhesive substrates direct growth cone haptotaxis

PubMed Central

Abe, Kouki; Katsuno, Hiroko; Toriyama, Michinori; Baba, Kentarou; Mori, Tomoyuki; Hakoshima, Toshio; Kanemura, Yonehiro; Watanabe, Rikiya; Inagaki, Naoyuki

2018-01-01

Chemical cues presented on the adhesive substrate direct cell migration, a process termed haptotaxis. To migrate, cells must generate traction forces upon the substrate. However, how cells probe substrate-bound cues and generate directional forces for migration remains unclear. Here, we show that the cell adhesion molecule (CAM) L1-CAM is involved in laminin-induced haptotaxis of axonal growth cones. L1-CAM underwent grip and slip on the substrate. The ratio of the grip state was higher on laminin than on the control substrate polylysine; this was accompanied by an increase in the traction force upon laminin. Our data suggest that the directional force for laminin-induced growth cone haptotaxis is generated by the grip and slip of L1-CAM on the substrates, which occur asymmetrically under the growth cone. This mechanism is distinct from the conventional cell signaling models for directional cell migration. We further show that this mechanism is disrupted in a human patient with L1-CAM syndrome, suffering corpus callosum agenesis and corticospinal tract hypoplasia. PMID:29483251

Cell force mapping using a double-sided micropillar array based on the moiré fringe method

NASA Astrophysics Data System (ADS)

Zhang, F.; Anderson, S.; Zheng, X.; Roberts, E.; Qiu, Y.; Liao, R.; Zhang, X.

2014-07-01

The mapping of traction forces is crucial to understanding the means by which cells regulate their behavior and physiological function to adapt to and communicate with their local microenvironment. To this end, polymeric micropillar arrays have been used for measuring cell traction force. However, the small scale of the micropillar deflections induced by cell traction forces results in highly inefficient force analyses using conventional optical approaches; in many cases, cell forces may be below the limits of detection achieved using conventional microscopy. To address these limitations, the moiré phenomenon has been leveraged as a visualization tool for cell force mapping due to its inherent magnification effect and capacity for whole-field force measurements. This Letter reports an optomechanical cell force sensor, namely, a double-sided micropillar array (DMPA) made of poly(dimethylsiloxane), on which one side is employed to support cultured living cells while the opposing side serves as a reference pattern for generating moiré patterns. The distance between the two sides, which is a crucial parameter influencing moiré pattern contrast, is predetermined during fabrication using theoretical calculations based on the Talbot effect that aim to optimize contrast. Herein, double-sided micropillar arrays were validated by mapping mouse embryo fibroblast contraction forces and the resulting force maps compared to conventional microscopy image analyses as the reference standard. The DMPA-based approach precludes the requirement for aligning two independent periodic substrates, improves moiré contrast, and enables efficient moiré pattern generation. Furthermore, the double-sided structure readily allows for the integration of moiré-based cell force mapping into microfabricated cell culture environments or lab-on-a-chip devices.

Time-Dependent Measure of a Nano-Scale Force-Pulse Driven by the Axonemal Dynein Motors in Individual Live Sperm Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allen, M J; Rudd, R E; McElfresh, M W

Nano-scale mechanical forces generated by motor proteins are crucial to normal cellular and organismal functioning. The ability to measure and exploit such forces would be important to developing motile biomimetic nanodevices powered by biological motors for Nanomedicine. Axonemal dynein motors positioned inside the sperm flagellum drive microtubule sliding giving rise to rhythmic beating of the flagellum. This force-generating action makes it possible for the sperm cell to move through viscous media. Here we report new nano-scale information on how the propulsive force is generated by the sperm flagellum and how this force varies over time. Single cell recordings reveal discretemore » {approx}50 ms pulses oscillating with amplitude 9.8 {+-} 2.6 nN independent of pulse frequency (3.5-19.5 Hz). The average work carried out by each cell is 4.6 x 10{sup -16} J per pulse, equivalent to the hydrolysis of {approx}5,500 ATP molecules. The mechanochemical coupling at each active dynein head is {approx}2.2 pN/ATP, and {approx}3.9 pN per dynein arm, in agreement with previously published values obtained using different methods.« less

Non-muscle myosin IIB is critical for nuclear translocation during 3D invasion

PubMed Central

Yenepalli, Aishwarya; Denais, Celine Marie; Rape, Andrew; Beach, Jordan R.; Wang, Yu-li; Schiemann, William P.; Baskaran, Harihara; Lammerding, Jan

2015-01-01

Non-muscle myosin II (NMII) is reported to play multiple roles during cell migration and invasion. However, the exact biophysical roles of different NMII isoforms during these processes remain poorly understood. We analyzed the contributions of NMIIA and NMIIB in three-dimensional (3D) migration and in generating the forces required for efficient invasion by mammary gland carcinoma cells. Using traction force microscopy and microfluidic invasion devices, we demonstrated that NMIIA is critical for generating force during active protrusion, and NMIIB plays a major role in applying force on the nucleus to facilitate nuclear translocation through tight spaces. We further demonstrate that the nuclear membrane protein nesprin-2 is a possible linker coupling NMIIB-based force generation to nuclear translocation. Together, these data reveal a central biophysical role for NMIIB in nuclear translocation during 3D invasive migration, a result with relevance not only to cancer metastasis but for 3D migration in other settings such as embryonic cell migration and wound healing. PMID:26261182

Do centrioles generate a polar ejection force?

PubMed

Wells, Jonathan

2005-01-01

A microtubule-dependent polar ejection force that pushes chromosomes away from spindle poles during prometaphase is observed in animal cells but not in the cells of higher plants. Elongating microtubules and kinesin-like motor molecules have been proposed as possible causes, but neither accounts for all the data. In the hypothesis proposed here a polar ejection force is generated by centrioles, which are found in animals but not in higher plants. Centrioles consist of nine microtubule triplets arranged like the blades of a tiny turbine. Instead of viewing centrioles through the spectacles of molecular reductionism and neo-Darwinism, this hypothesis assumes that they are holistically designed to be turbines. Orthogonally oriented centriolar turbines could generate oscillations in spindle microtubules that resemble the motion produced by a laboratory vortexer. The result would be a microtubule-mediated ejection force tending to move chromosomes away from the spindle axis and the poles. A rise in intracellular calcium at the onset of anaphase could regulate the polar ejection force by shutting down the centriolar turbines, but defective regulation could result in an excessive force that contributes to the chromosomal instability characteristic of most cancer cells.

Contact inhibition of locomotion determines cell-cell and cell-substrate forces in tissues.

PubMed

Zimmermann, Juliane; Camley, Brian A; Rappel, Wouter-Jan; Levine, Herbert

2016-03-08

Cells organized in tissues exert forces on their neighbors and their environment. Those cellular forces determine tissue homeostasis as well as reorganization during embryonic development and wound healing. To understand how cellular forces are generated and how they can influence the tissue state, we develop a particle-based simulation model for adhesive cell clusters and monolayers. Cells are contractile, exert forces on their substrate and on each other, and interact through contact inhibition of locomotion (CIL), meaning that cell-cell contacts suppress force transduction to the substrate and propulsion forces align away from neighbors. Our model captures the traction force patterns of small clusters of nonmotile cells and larger sheets of motile Madin-Darby canine kidney (MDCK) cells. In agreement with observations in a spreading MDCK colony, the cell density in the center increases as cells divide and the tissue grows. A feedback between cell density, CIL, and cell-cell adhesion gives rise to a linear relationship between cell density and intercellular tensile stress and forces the tissue into a nonmotile state characterized by a broad distribution of traction forces. Our model also captures the experimentally observed tissue flow around circular obstacles, and CIL accounts for traction forces at the edge.

Two-Layer Elastographic 3-D Traction Force Microscopy

PubMed Central

Álvarez-González, Begoña; Zhang, Shun; Gómez-González, Manuel; Meili, Ruedi; Firtel, Richard A.; Lasheras, Juan C.; del Álamo, Juan C.

2017-01-01

Cellular traction force microscopy (TFM) requires knowledge of the mechanical properties of the substratum where the cells adhere to calculate cell-generated forces from measurements of substratum deformation. Polymer-based hydrogels are broadly used for TFM due to their linearly elastic behavior in the range of measured deformations. However, the calculated stresses, particularly their spatial patterns, can be highly sensitive to the substratum’s Poisson’s ratio. We present two-layer elastographic TFM (2LETFM), a method that allows for simultaneously measuring the Poisson’s ratio of the substratum while also determining the cell-generated forces. The new method exploits the analytical solution of the elastostatic equation and deformation measurements from two layers of the substratum. We perform an in silico analysis of 2LETFM concluding that this technique is robust with respect to TFM experimental parameters, and remains accurate even for noisy measurement data. We also provide experimental proof of principle of 2LETFM by simultaneously measuring the stresses exerted by migrating Physarum amoeboae on the surface of polyacrylamide substrata, and the Poisson’s ratio of the substrata. The 2LETFM method could be generalized to concurrently determine the mechanical properties and cell-generated forces in more physiologically relevant extracellular environments, opening new possibilities to study cell-matrix interactions. PMID:28074837

Force-Mediating Magnetic Nanoparticles to Engineer Neuronal Cell Function

PubMed Central

Gahl, Trevor J.; Kunze, Anja

2018-01-01

Cellular processes like membrane deformation, cell migration, and transport of organelles are sensitive to mechanical forces. Technically, these cellular processes can be manipulated through operating forces at a spatial precision in the range of nanometers up to a few micrometers through chaperoning force-mediating nanoparticles in electrical, magnetic, or optical field gradients. But which force-mediating tool is more suitable to manipulate cell migration, and which, to manipulate cell signaling? We review here the differences in forces sensation to control and engineer cellular processes inside and outside the cell, with a special focus on neuronal cells. In addition, we discuss technical details and limitations of different force-mediating approaches and highlight recent advancements of nanomagnetics in cell organization, communication, signaling, and intracellular trafficking. Finally, we give suggestions about how force-mediating nanoparticles can be used to our advantage in next-generation neurotherapeutic devices. PMID:29867315

Force-Mediating Magnetic Nanoparticles to Engineer Neuronal Cell Function.

PubMed

Gahl, Trevor J; Kunze, Anja

2018-01-01

Cellular processes like membrane deformation, cell migration, and transport of organelles are sensitive to mechanical forces. Technically, these cellular processes can be manipulated through operating forces at a spatial precision in the range of nanometers up to a few micrometers through chaperoning force-mediating nanoparticles in electrical, magnetic, or optical field gradients. But which force-mediating tool is more suitable to manipulate cell migration, and which, to manipulate cell signaling? We review here the differences in forces sensation to control and engineer cellular processes inside and outside the cell, with a special focus on neuronal cells. In addition, we discuss technical details and limitations of different force-mediating approaches and highlight recent advancements of nanomagnetics in cell organization, communication, signaling, and intracellular trafficking. Finally, we give suggestions about how force-mediating nanoparticles can be used to our advantage in next-generation neurotherapeutic devices.

Effect of gel re-organization and tensional forces on alpha2beta1 integrin levels in dermal fibroblasts.

PubMed

Jenkins, G; Redwood, K L; Meadows, L; Green, M R

1999-07-01

Mechanical forces are known to play an important role in regulating cell function in a wide range of biological systems. This is of particular relevance to dermal fibroblast function, given that the skin is known to be held under an intrinsic natural tension. To understand more about the generation of force by dermal fibroblasts and their ability to respond to changes in it, we have studied the role of the beta1 integrin receptors expressed by dermal fibroblasts in their ability to generate tensional forces within a collagen type I matrix and the effect of altered tensional force on integrin expression by dermal fibroblasts. Using a purpose-built culture force monitor, function-blocking antibodies directed towards the beta1 receptors dramatically reduced the tensional forces generated by dermal fibroblasts in a 3D collagen I matrix. However, the specific involvement of alpha1 or alpha2 subunits could not be demonstrated. Analysis of cellular response demonstrated that cells isolated from contracting collagen gels expressed fourfold higher levels of alpha2 mRNA than cells isolated from fully restrained gels. The levels of beta1 messenger RNA were relatively unaffected by reductions in force. Cells exposed to single reductions in force, however, did not exhibit alterations in either alpha1 or beta1 mRNA levels. We propose, therefore that alpha2beta1 integrin receptor levels in dermal fibroblasts are not altered in response to single reductions of gel tension, but do change following a continual change in force and associated matrix re-organization

A numerical analysis of forces exerted by laminar flow on spreading cells in a parallel plate flow chamber assay.

PubMed

Olivier, L A; Truskey, G A

1993-10-01

Exposure of spreading anchorage-dependent cells to laminar flow is a common technique to measure the strength of cell adhesion. Since cells protrude into the flow stream, the force exerted by the fluid on the cells is a function of cell shape. To assess the relationship between cell shape and the hydrodynamic force on adherent cells, we obtained numerical solutions of the velocity and stress fields around bovine aortic endothelial cells during various stages of spreading and calculated the force required to detach the cells. Morphometric parameters were obtained from light and scanning electron microscopy measurements. Cells were assumed to have a constant volume, but the surface area increased during spreading until the membrane was stretched taut. Two-dimensional models of steady flow were generated using the software packages ANSYS (mesh generation) and FIDAP (problem solution). The validity of the numerical results was tested by comparison with published results for a semicircle in contact with the surface. The drag force and torque were greatest for round cells making initial contact with the surface. During spreading, the drag force and torque declined by factors of 2 and 20, respectively. The calculated forces and moments were used in adhesion models to predict the wall shear stress at which the cells detached. Based upon published values for the bond force and receptor number, round cells should detach at shear stresses between 2.5 and 6 dyn/cm(2), whereas substantially higher stresses are needed to detach spreading and fully spread cells. Results from the simulations indicate that (1) the drag force varies little with cell shape whereas the torque is very sensitive to cell shape, and (2) the increase in the strength of adhesion during spreading is due to increased contact area and receptor densities within the contact area. (c) 1993 John Wiley & Sons, Inc.

Force Generation upon T Cell Receptor Engagement

PubMed Central

Husson, Julien; Chemin, Karine; Bohineust, Armelle; Hivroz, Claire; Henry, Nelly

2011-01-01

T cells are major players of adaptive immune response in mammals. Recognition of an antigenic peptide in association with the major histocompatibility complex at the surface of an antigen presenting cell (APC) is a specific and sensitive process whose mechanism is not fully understood. The potential contribution of mechanical forces in the T cell activation process is increasingly debated, although these forces are scarcely defined and hold only limited experimental evidence. In this work, we have implemented a biomembrane force probe (BFP) setup and a model APC to explore the nature and the characteristics of the mechanical forces potentially generated upon engagement of the T cell receptor (TCR) and/or lymphocyte function-associated antigen-1 (LFA-1). We show that upon contact with a model APC coated with antibodies towards TCR-CD3, after a short latency, the T cell developed a timed sequence of pushing and pulling forces against its target. These processes were defined by their initial constant growth velocity and loading rate (force increase per unit of time). LFA-1 engagement together with TCR-CD3 reduced the growing speed during the pushing phase without triggering the same mechanical behavior when engaged alone. Intracellular Ca2+ concentration ([Ca2+]i) was monitored simultaneously to verify the cell commitment in the activation process. [Ca2+]i increased a few tens of seconds after the beginning of the pushing phase although no strong correlation appeared between the two events. The pushing phase was driven by actin polymerization. Tuning the BFP mechanical properties, we could show that the loading rate during the pulling phase increased with the target stiffness. This indicated that a mechanosensing mechanism is implemented in the early steps of the activation process. We provide here the first quantified description of force generation sequence upon local bidimensional engagement of TCR-CD3 and discuss its potential role in a T cell mechanically-regulated activation process. PMID:21572959

Force generation upon T cell receptor engagement.

PubMed

Husson, Julien; Chemin, Karine; Bohineust, Armelle; Hivroz, Claire; Henry, Nelly

2011-05-10

T cells are major players of adaptive immune response in mammals. Recognition of an antigenic peptide in association with the major histocompatibility complex at the surface of an antigen presenting cell (APC) is a specific and sensitive process whose mechanism is not fully understood. The potential contribution of mechanical forces in the T cell activation process is increasingly debated, although these forces are scarcely defined and hold only limited experimental evidence. In this work, we have implemented a biomembrane force probe (BFP) setup and a model APC to explore the nature and the characteristics of the mechanical forces potentially generated upon engagement of the T cell receptor (TCR) and/or lymphocyte function-associated antigen-1 (LFA-1). We show that upon contact with a model APC coated with antibodies towards TCR-CD3, after a short latency, the T cell developed a timed sequence of pushing and pulling forces against its target. These processes were defined by their initial constant growth velocity and loading rate (force increase per unit of time). LFA-1 engagement together with TCR-CD3 reduced the growing speed during the pushing phase without triggering the same mechanical behavior when engaged alone. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was monitored simultaneously to verify the cell commitment in the activation process. [Ca(2+)](i) increased a few tens of seconds after the beginning of the pushing phase although no strong correlation appeared between the two events. The pushing phase was driven by actin polymerization. Tuning the BFP mechanical properties, we could show that the loading rate during the pulling phase increased with the target stiffness. This indicated that a mechanosensing mechanism is implemented in the early steps of the activation process. We provide here the first quantified description of force generation sequence upon local bidimensional engagement of TCR-CD3 and discuss its potential role in a T cell mechanically-regulated activation process.

Finding the cell center by a balance of dynein and myosin pulling and microtubule pushing: a computational study.

PubMed

Zhu, Jie; Burakov, Anton; Rodionov, Vladimir; Mogilner, Alex

2010-12-01

The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces-dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles-is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility.

Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement

PubMed Central

Sorce, Barbara; Escobedo, Carlos; Toyoda, Yusuke; Stewart, Martin P.; Cattin, Cedric J.; Newton, Richard; Banerjee, Indranil; Stettler, Alexander; Roska, Botond; Eaton, Suzanne; Hyman, Anthony A.; Hierlemann, Andreas; Müller, Daniel J.

2015-01-01

Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells. PMID:26602832

Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement

NASA Astrophysics Data System (ADS)

Sorce, Barbara; Escobedo, Carlos; Toyoda, Yusuke; Stewart, Martin P.; Cattin, Cedric J.; Newton, Richard; Banerjee, Indranil; Stettler, Alexander; Roska, Botond; Eaton, Suzanne; Hyman, Anthony A.; Hierlemann, Andreas; Müller, Daniel J.

2015-11-01

Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells.

Mechanical stimulation improves tissue-engineered human skeletal muscle

NASA Technical Reports Server (NTRS)

Powell, Courtney A.; Smiley, Beth L.; Mills, John; Vandenburgh, Herman H.

2002-01-01

Human bioartificial muscles (HBAMs) are tissue engineered by suspending muscle cells in collagen/MATRIGEL, casting in a silicone mold containing end attachment sites, and allowing the cells to differentiate for 8 to 16 days. The resulting HBAMs are representative of skeletal muscle in that they contain parallel arrays of postmitotic myofibers; however, they differ in many other morphological characteristics. To engineer improved HBAMs, i.e., more in vivo-like, we developed Mechanical Cell Stimulator (MCS) hardware to apply in vivo-like forces directly to the engineered tissue. A sensitive force transducer attached to the HBAM measured real-time, internally generated, as well as externally applied, forces. The muscle cells generated increasing internal forces during formation which were inhibitable with a cytoskeleton depolymerizer. Repetitive stretch/relaxation for 8 days increased the HBAM elasticity two- to threefold, mean myofiber diameter 12%, and myofiber area percent 40%. This system allows engineering of improved skeletal muscle analogs as well as a nondestructive method to determine passive force and viscoelastic properties of the resulting tissue.

Interactions between the L1 cell adhesion molecule and ezrin support traction-force generation and can be regulated by tyrosine phosphorylation.

PubMed

Sakurai, Takeshi; Gil, Orlando D; Whittard, John D; Gazdoiu, Mihaela; Joseph, Todd; Wu, James; Waksman, Adam; Benson, Deanna L; Salton, Stephen R; Felsenfeld, Dan P

2008-09-01

An Ig superfamily cell-adhesion molecule, L1, forms an adhesion complex at the cell membrane containing both signaling molecules and cytoskeletal proteins. This complex mediates the transduction of extracellular signals and generates actin-mediated traction forces, both of which support axon outgrowth. The L1 cytoplasmic region binds ezrin, an adapter protein that interacts with the actin cytoskeleton. In this study, we analyzed L1-ezrin interactions in detail, assessed their role in generating traction forces by L1, and identified potential regulatory mechanisms controlling ezrin-L1 interactions. The FERM domain of ezrin binds to the juxtamembrane region of L1, demonstrated by yeast two-hybrid interaction traps and protein binding analyses in vitro. A lysine-to-leucine substitution in this domain of L1 (K1147L) shows reduced binding to the ezrin FERM domain. Additionally, in ND7 cells, the K1147L mutation inhibits retrograde movement of L1 on the cell surface that has been linked to the generation of the traction forces necessary for axon growth. A membrane-permeable peptide consisting of the juxtamembrane region of L1 that can disrupt endogenous L1-ezrin interactions inhibits neurite extension of cerebellar cells on L1 substrates. Moreover, the L1-ezrin interactions can be modulated by tyrosine phosphorylation of the L1 cytoplasmic region, namely, Y1151, possibly through Src-family kinases. Replacement of this tyrosine together with Y1176 with either aspartate or phenylalanine changes ezrin binding and alters colocalization with ezrin in ND7 cells. Collectively, these data suggest that L1-ezrin interactions mediated by the L1 juxtamembrane region are involved in traction-force generation and can be regulated by the phosphorylation of L1. (c) 2008 Wiley-Liss, Inc.

NuSAP governs chromosome oscillation by facilitating the Kid-generated polar ejection force

PubMed Central

Li, Chenyu; Xue, Chenyi; Yang, Qiaoyun; Low, Boon Chuan; Liou, Yih-Cherng

2016-01-01

In vertebrate cells, chromosomes oscillate to align precisely during metaphase. NuSAP, a microtubule-associated protein, plays a critical role in stabilizing spindle microtubules. In this study, we utilize 3D time-lapse live-cell imaging to monitor the role of NuSAP in chromosome oscillation and identify NuSAP as a novel regulator of the chromokinesin, Kid. Depletion of NuSAP significantly suppresses the amplitude and velocity of chromosome oscillation. We analyse the effects of NuSAP and Kid depletion in monopolar and bipolar cells with or without kinetochore microtubule depletion. Twelve postulated conditions are deciphered to reveal the contribution of NuSAP to the polar force generated at kinetochore microtubules and to the regulation of the polar ejection force generated by Kid, thus revealing a pivotal role of NuSAP in chromosome oscillation. PMID:26839278

NuSAP governs chromosome oscillation by facilitating the Kid-generated polar ejection force.

PubMed

Li, Chenyu; Xue, Chenyi; Yang, Qiaoyun; Low, Boon Chuan; Liou, Yih-Cherng

2016-02-03

In vertebrate cells, chromosomes oscillate to align precisely during metaphase. NuSAP, a microtubule-associated protein, plays a critical role in stabilizing spindle microtubules. In this study, we utilize 3D time-lapse live-cell imaging to monitor the role of NuSAP in chromosome oscillation and identify NuSAP as a novel regulator of the chromokinesin, Kid. Depletion of NuSAP significantly suppresses the amplitude and velocity of chromosome oscillation. We analyse the effects of NuSAP and Kid depletion in monopolar and bipolar cells with or without kinetochore microtubule depletion. Twelve postulated conditions are deciphered to reveal the contribution of NuSAP to the polar force generated at kinetochore microtubules and to the regulation of the polar ejection force generated by Kid, thus revealing a pivotal role of NuSAP in chromosome oscillation.

Flexible substrata for the detection of cellular traction forces

NASA Technical Reports Server (NTRS)

Beningo, Karen A.; Wang, Yu-Li

2002-01-01

By modulating adhesion signaling and cytoskeletal organization, mechanical forces play an important role in various cellular functions, from propelling cell migration to mediating communication between cells. Recent developments have resulted in several new approaches for the detection, analysis and visualization of mechanical forces generated by cultured cells. Combining these methods with other approaches, such as green-fluorescent protein (GFP) imaging and gene manipulation, proves to be particularly powerful for analyzing the interplay between extracellular physical forces and intracellular chemical events.

Finding the Cell Center by a Balance of Dynein and Myosin Pulling and Microtubule Pushing: A Computational Study

PubMed Central

Zhu, Jie; Burakov, Anton; Rodionov, Vladimir

2010-01-01

The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces—dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles—is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility. PMID:20980619

A micropatterning and image processing approach to simplify measurement of cellular traction forces

PubMed Central

Polio, Samuel R.; Rothenberg, Katheryn E.; Stamenović, Dimitrije; Smith, Michael L.

2012-01-01

Quantification of the traction forces that cells apply to their surroundings has been critical to the advancement of our understanding of cancer, development and basic cell biology. This field was made possible through the development of engineered cell culture systems that permit optical measurement of cell-mediated displacements and computational algorithms that allow conversion of these displacements into stresses and forces. Here, we present a novel advancement of traction force microscopy on polyacrylamide (PAA) gels that addresses limitations of existing technologies. Through an indirect patterning technique, we generated PAA gels with fluorescent 1 μm dot markers in a regularized array. This improves existing traction measurements since (i) multiple fields of view can be measured in one experiment without the need for cell removal; (ii) traction vectors are modeled as discrete point forces, and not as a continuous field, using an extremely simple computational algorithm that we have made available online; and (iii) the pattern transfer technique is amenable to any of the published techniques for producing patterns on glass. In the future, this technique will be used for measuring traction forces on complex patterns with multiple, spatially distinct ligands in systems for applying strain to the substrate, and in sandwich cultures that generate quasi-three-dimensional environments for cells. PMID:21884832

Maternal Forced Swimming Reduces Cell Proliferation in the Postnatal Dentate Gyrus of Mouse Offspring

PubMed Central

Wasinski, Frederick; Estrela, Gabriel R.; Arakaki, Aline M.; Bader, Michael; Alenina, Natalia; Klempin, Friederike; Araújo, Ronaldo C.

2016-01-01

Physical exercise positively affects the metabolism and induces proliferation of precursor cells in the adult brain. Maternal exercise likewise provokes adaptations early in the offspring. Using a high-intensity swimming protocol that comprises forced swim training before and during pregnancy, we determined the effect of maternal swimming on the mouse offspring's neurogenesis. Our data demonstrate decreased proliferation in sublayers of the postnatal dentate gyrus in offspring of swimming mother at postnatal day (P) 8 accompanied with decreased survival of newly generated cells 4 weeks later. The reduction in cell numbers was predominantly seen in the hilus and molecular layer. At P35, the reduced amount of cells was also reflected by a decrease in the population of newly generated immature and mature neurons of the granule cell layer. Our data suggest that forced maternal swimming at high-intensity has a negative effect on the neurogenic niche development in postnatal offspring. PMID:27621701

Cell migration through connective tissue in 3-D

NASA Astrophysics Data System (ADS)

Fabry, Ben

2008-03-01

A prerequisite for metastasis formation is the ability of tumor cells to invade and migrate through connective tissue. Four key components endow tumor cells with this ability: secretion of matrix-degrading enzymes, firm but temporary adhesion onto connective tissue fibers, contractile force generation, and rapid remodeling of cytoskeletal structures. Cell adhesion, contraction, and cytoskeletal remodeling are biomechanical parameter that can be measured on single cells using a panel of biophysical methods. We use 2-D and 3-D traction microscopy to measure contractile forces; magnetic tweezer microrheology to estimate adhesion strengths, cytoskeletal stiffness and molecular turn-over rates; and nanoscale particle tracking to measure cytoskeletal remodeling. On a wide range of tumor cell lines we could show that cell invasiveness correlates with increased expression of integrin adhesion receptors, increased contractile force generation, and increased speed of cytoskeletal reorganization. Each of those biomechanical parameters, however, varied considerably between cell lines of similar invasivity, suggesting that tumor cells employ multiple invasion strategies that cannot be unambiguously characterized using a single assay.

A positional code and anisotropic forces control tissue remodeling in Drosophila

NASA Astrophysics Data System (ADS)

Zallen, Jennifer

A major challenge in developmental biology is to understand how tissue-scale changes in organism structure arise from events that occur on a cellular and molecular level. We are using cell biological, biophysical, and quantitative live-embryo imaging approaches to understand how genes encode the forces that shape tissues, and to identify the mechanisms that modulate cell behavior in response to local forces. In many animals, the elongated head-to-tail body axis is achieved by rapid and coordinated movements of hundreds of cells. We found that in the fruit fly, these cell movements are regulated by subcellular asymmetries in the localization of proteins that generate contractile and adhesive forces between cells. Asymmetries in the force-generating machinery are in turn controlled by a positional code of spatial information provided by an ancient family of Toll-related receptors that are widely used for pathogen recognition by the innate immune system. I will describe how this spatial system systematically orients local cell movements and collective rosette-like clusters in the Drosophila embryo. Rosettes have now also been shown to shape the body axis in chicks, frogs, and mice, demonstrating that rosette behaviors are a general mechanism linking cellular asymmetry to tissue reorganization.

Contact inhibition of locomotion determines cell–cell and cell–substrate forces in tissues

PubMed Central

Zimmermann, Juliane; Camley, Brian A.; Rappel, Wouter-Jan; Levine, Herbert

2016-01-01

Cells organized in tissues exert forces on their neighbors and their environment. Those cellular forces determine tissue homeostasis as well as reorganization during embryonic development and wound healing. To understand how cellular forces are generated and how they can influence the tissue state, we develop a particle-based simulation model for adhesive cell clusters and monolayers. Cells are contractile, exert forces on their substrate and on each other, and interact through contact inhibition of locomotion (CIL), meaning that cell–cell contacts suppress force transduction to the substrate and propulsion forces align away from neighbors. Our model captures the traction force patterns of small clusters of nonmotile cells and larger sheets of motile Madin–Darby canine kidney (MDCK) cells. In agreement with observations in a spreading MDCK colony, the cell density in the center increases as cells divide and the tissue grows. A feedback between cell density, CIL, and cell–cell adhesion gives rise to a linear relationship between cell density and intercellular tensile stress and forces the tissue into a nonmotile state characterized by a broad distribution of traction forces. Our model also captures the experimentally observed tissue flow around circular obstacles, and CIL accounts for traction forces at the edge. PMID:26903658

Dynamics of Cell Area and Force during Spreading

PubMed Central

Brill-Karniely, Yifat; Nisenholz, Noam; Rajendran, Kavitha; Dang, Quynh; Krishnan, Ramaswamy; Zemel, Assaf

2014-01-01

Experiments on human pulmonary artery endothelial cells are presented to show that cell area and the force exerted on a substrate increase simultaneously, but with different rates during spreading; rapid-force increase systematically occurred several minutes past initial spreading. We examine this theoretically and present three complementary mechanisms that may accompany the development of lamellar stress during spreading and underlie the observed behavior. These include: 1), the dynamics of cytoskeleton assembly at the cell basis; 2), the strengthening of acto-myosin forces in response to the generated lamellar stresses; and 3), the passive strain-stiffening of the cytoskeleton. PMID:25517168

Cellular control lies in the balance of forces

NASA Technical Reports Server (NTRS)

Chicurel, M. E.; Chen, C. S.; Ingber, D. E.

1998-01-01

Mechanical tension generated within the cytoskeleton of living cells is emerging as a critical regulator of biological function in diverse situations ranging from the control of chromosome movement to the morphogenesis of the vertebrate brain. In this article, we review recent advances that have been made in terms of understanding how cells generate, transmit and sense mechanical tension, as well as how they use these forces to control their shape and behavior. An integrated view of cell regulation that incorporates mechanics and structure as well as chemistry is beginning to emerge.

Micropatterned Azopolymer Surfaces Modulate Cell Mechanics and Cytoskeleton Structure.

PubMed

Rianna, Carmela; Ventre, Maurizio; Cavalli, Silvia; Radmacher, Manfred; Netti, Paolo A

2015-09-30

Physical and chemical characteristics of materials are important regulators of cell behavior. In particular, cell elasticity is a fundamental parameter that reflects the state of a cell. Surface topography finely modulates cell fate and function via adhesion mediated signaling and cytoskeleton generated forces. However, how topographies alter cell mechanics is still unclear. In this work we have analyzed the mechanical properties of peripheral and nuclear regions of NIH-3T3 cells on azopolymer substrates with different topographic patterns. Micrometer scale patterns in the form of parallel ridges or square lattices of surface elevations were encoded on light responsive azopolymer films by means of contactless optical methods. Cell mechanics was investigated by atomic force microscopy (AFM). Cells and consequently the cell cytoskeleton were oriented along the linear patterns affecting cytoskeletal structures, e.g., formation of actin stress fibers. Our data demonstrate that topographic substrate patterns are recognized by cells and mechanical information is transferred by the cytoskeleton. Furthermore, cytoskeleton generated forces deform the nucleus, changing its morphology that appears to be related to different mechanical properties in the nuclear region.

Traction patterns of tumor cells.

PubMed

Ambrosi, D; Duperray, A; Peschetola, V; Verdier, C

2009-01-01

The traction exerted by a cell on a planar deformable substrate can be indirectly obtained on the basis of the displacement field of the underlying layer. The usual methodology used to address this inverse problem is based on the exploitation of the Green tensor of the linear elasticity problem in a half space (Boussinesq problem), coupled with a minimization algorithm under force penalization. A possible alternative strategy is to exploit an adjoint equation, obtained on the basis of a suitable minimization requirement. The resulting system of coupled elliptic partial differential equations is applied here to determine the force field per unit surface generated by T24 tumor cells on a polyacrylamide substrate. The shear stress obtained by numerical integration provides quantitative insight of the traction field and is a promising tool to investigate the spatial pattern of force per unit surface generated in cell motion, particularly in the case of such cancer cells.

Delivery of molecules into cells using carbon nanoparticles activated by femtosecond laser pulses

PubMed Central

Chakravarty, Prerona; Qian, Wei; El-Sayed, Mostafa A.; Prausnitz, Mark R.

2010-01-01

A major barrier to drug and gene delivery is crossing the cell's plasma membrane. Physical forces applied to cells via electroporation1, ultrasound2 and laser-irradiation3–6 generate nanoscale holes in the plasma membrane for direct delivery of drugs into the cytoplasm. Inspired by previous work showing that laser excitation of carbon nanoparticles can drive the carbon-steam reaction to generate highly controlled shock waves7–10, here we show carbon black (CB) nanoparticles activated by femtosecond laser pulses can facilitate the delivery of small molecules, proteins and DNA into two types of cells. Our initial results suggest that interaction between the laser energy and CB nanoparticles may generate photoacoustic forces by chemical reaction to create transient holes in the membrane for delivery. PMID:20639882

Fiber networks amplify active stress

NASA Astrophysics Data System (ADS)

Lenz, Martin; Ronceray, Pierre; Broedersz, Chase

Large-scale force generation is essential for biological functions such as cell motility, embryonic development, and muscle contraction. In these processes, forces generated at the molecular level by motor proteins are transmitted by disordered fiber networks, resulting in large-scale active stresses. While fiber networks are well characterized macroscopically, this stress generation by microscopic active units is not well understood. I will present a comprehensive theoretical study of force transmission in these networks. I will show that the linear, small-force response of the networks is remarkably simple, as the macroscopic active stress depends only on the geometry of the force-exerting unit. In contrast, as non-linear buckling occurs around these units, local active forces are rectified towards isotropic contraction and strongly amplified. This stress amplification is reinforced by the networks' disordered nature, but saturates for high densities of active units. I will show that our predictions are quantitatively consistent with experiments on reconstituted tissues and actomyosin networks, and that they shed light on the role of the network microstructure in shaping active stresses in cells and tissue.

Force dependence of phagosome trafficking in retinal pigment epithelial cells

NASA Astrophysics Data System (ADS)

Daniel, Rebekah; Koll, Andrew T.; Altman, David

2014-09-01

Retinal pigment epithelial (RPE) cells play an integral role in the renewal of photoreceptor disk membranes. As rod and cone cells shed their outer segments, they are phagocytosed and degraded by the RPE, and a failure in this process can result in retinal degeneration. We have studied the role of myosin VI in nonspecific phagocytosis in a human RPE primary cell line (ARPE-19), testing the hypothesis that this motor generates the forces required to traffic phagosomes in these cells. Experiments were conducted in the presence of forces through the use of in vivo optical trapping. Our results support a role for myosin VI in phagosome trafficking and demonstrate that applied forces modulate rates of phagosome trafficking.

Probing cooperative force generation in collective cancer invasion

NASA Astrophysics Data System (ADS)

Alobaidi, Amani A.; Xu, Yaopengxiao; Chen, Shaohua; Jiao, Yang; Sun, Bo

2017-08-01

Collective cellular dynamics in the three-dimensional extracellular matrix (ECM) plays a crucial role in many physiological processes such as cancer invasion. Both chemical and mechanical signaling support cell-cell communications on a variety of length scales, leading to collective migratory behaviors. Here we conduct experiments using 3D in vitro tumor models and develop a phenomenological model in order to probe the cooperativity of force generation in the collective invasion of breast cancer cells. In our model, cell-cell communication is characterized by a single parameter that quantifies the correlation length of cellular migration cycles. We devise a stochastic reconstruction method to generate realizations of cell colonies with specific contraction phase correlation functions and correlation length a. We find that as a increases, the characteristic size of regions containing cells with similar contraction phases grows. For small a values, the large fluctuations in individual cell contraction phases smooth out the temporal fluctuations in the time-dependent deformation field in the ECM. For large a values, the periodicity of an individual cell contraction cycle is clearly manifested in the temporal variation of the overall deformation field in the ECM. Through quantitative comparisons of the simulated and experimentally measured deformation fields, we find that the correlation length for collective force generation in the breast cancer diskoid in geometrically micropatterned ECM (DIGME) system is a≈ 25~μ \\text{m} , which is roughly twice the linear size of a single cell. One possible mechanism for this intermediate cell correlation length is the fiber-mediated stress propagation in the 3D ECM network in the DIGME system.

Multiphoton photochemical crosslinking-based fabrication of protein micropatterns with controllable mechanical properties for single cell traction force measurements

NASA Astrophysics Data System (ADS)

Tong, Ming Hui; Huang, Nan; Zhang, Wei; Zhou, Zhuo Long; Ngan, Alfonso Hing Wan; Du, Yanan; Chan, Barbara Pui

2016-01-01

Engineering 3D microstructures with predetermined properties is critical for stem cell niche studies. We have developed a multiphoton femtosecond laser-based 3D printing platform, which generates complex protein microstructures in minutes. Here, we used the platform to test a series of fabrication and reagent parameters in precisely controlling the mechanical properties of protein micropillars. Atomic force microscopy was utilized to measure the reduced elastic modulus of the micropillars, and transmission electron microscopy was used to visualize the porosity of the structures. The reduced elastic modulus of the micropillars associated positively and linearly with the scanning power. On the other hand, the porosity and pore size of the micropillars associated inversely and linearly with the scanning power and reagent concentrations. While keeping the elastic modulus constant, the stiffness of the micropillars was controlled by varying their height. Subsequently, the single cell traction forces of rabbit chondrocytes, human dermal fibroblasts, human mesenchymal stem cells, and bovine nucleus pulposus cells (bNPCs) were successfully measured by culturing the cells on micropillar arrays of different stiffness. Our results showed that the traction forces of all groups showed positive relationship with stiffness, and that the chondrocytes and bNPCs generated the highest and lowest traction forces, respectively.

Dynamics of cell area and force during spreading.

PubMed

Brill-Karniely, Yifat; Nisenholz, Noam; Rajendran, Kavitha; Dang, Quynh; Krishnan, Ramaswamy; Zemel, Assaf

2014-12-16

Experiments on human pulmonary artery endothelial cells are presented to show that cell area and the force exerted on a substrate increase simultaneously, but with different rates during spreading; rapid-force increase systematically occurred several minutes past initial spreading. We examine this theoretically and present three complementary mechanisms that may accompany the development of lamellar stress during spreading and underlie the observed behavior. These include: 1), the dynamics of cytoskeleton assembly at the cell basis; 2), the strengthening of acto-myosin forces in response to the generated lamellar stresses; and 3), the passive strain-stiffening of the cytoskeleton. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Accelerated generation of oligodendrocyte progenitor cells from human induced pluripotent stem cells by forced expression of Sox10 and Olig2.

PubMed

Li, Pengyan; Li, Mo; Tang, Xihe; Wang, Shuyan; Zhang, Y Alex; Chen, Zhiguo

2016-11-01

Oligodendrocyte progenitor cells (OPCs) hold great promise for treatment of dysmyelinating disorders, such as multiple sclerosis and cerebral palsy. Recent studies on generation of human OPCs mainly use human embryonic stem cells (hESCs) or neural stem cells (NSCs) as starter cell sources for the differentiation process. However, NSCs are restricted in availability and the present method for generation of oligodendrocytes (OLs) from ESCs often requires a lengthy period of time. Here, we demonstrated a protocol to efficiently derive OPCs from human induced pluripotent stem cells (hiPSCs) by forced expression of two transcription factors (2TFs), Sox10 and Olig2. With this method, PDGFRα + OPCs can be obtained in 14 days and O4 + OPCs in 56 days. Furthermore, OPCs may be able to differentiate to mature OLs that could ensheath axons when co-cultured with rat cortical neurons. The results have implications in the development of autologous cell therapies.

Mechanical regulation of T-cell functions

PubMed Central

Chen, Wei; Zhu, Cheng

2013-01-01

Summary T cells are key players of the mammalian adaptive immune system. They experience different mechanical microenvironments during their life cycles, from the thymus, secondary lymph organs, and peripheral tissues that are free of externally applied force but display variable substrate rigidities, to the blood and lymphatic circulation systems where complicated hydrodynamic forces are present. Regardless of whether T cells are subject to external forces or generate their own internal forces, they response and adapt to different biomechanical cues to modulate their adhesion, migration, trafficking, and triggering of immune functions through mechanical regulation of various molecules that bear force. These include adhesive receptors, immunoreceptors, motor proteins, cytoskeletal proteins, and their associated molecules. Here we discuss the forces acting on various surface and cytoplasmic proteins of a T cell in different mechanical milieus. We review existing data on how force regulates protein conformational changes and interactions with counter molecules, including integrins, actin, and the T-cell receptor, and how each relates to T-cell functions. PMID:24117820

Cell-populated collagen lattice contraction model for the investigation of fibroblast collagen interactions.

PubMed

Ehrlich, H Paul; Moyer, Kurtis E

2013-01-01

The fibroblast-populated collagen lattice (FPCL) was intended to act as the dermal component for "skin-equivalent" or artificial skin developed for skin grafting burn patients. The "skin-equivalent" was clinically unsuccessful as a skin graft, but today it is successfully used as a dressing for the management of chronic wounds. The FPCL has, however, become an instrument for investigating cell-connective tissue interactions within a three-dimensional matrix. Through the capacity of cell compaction of collagen fibrils, the FPCL undergoes a reduction in volume referred to as lattice contraction. Lattice contraction proceeds by cell-generated forces that reduce the water mass between collagen fibers, generating a closer relationship between collagen fibers. The compaction of collagen fibers is responsible for the reduction in the FPCL volume. Cell-generated forces through the linkage of collagen fibers with fibroblast's cytoskeletal actin-rich microfilament structures are responsible for the completion of the collagen matrix compaction. The type of culture dish used to cast FPCL as well as the cell number will dictate the mechanism for compacting collagen matrices. Fibroblasts, at moderate density, cast as an FPCL within a petri dish and released from the surface of the dish soon after casting compact collagen fibers through cell tractional forces. Fibroblasts at moderate density cast as an FPCL within a tissue culture dish and not released for 4 days upon release show rapid lattice contraction through a mechanism of cell contraction forces. Fibroblasts at high density cast in an FPCL within a petri dish, released from the surface of the dish soon after casting, compact a collagen lattice very rapidly through forces related to cell elongation. The advantage of the FPCL contraction model is the study of cells in the three-dimensional environment, which is similar to the environment from which these cells were isolated. In this chapter methods are described for manufacturing collagen lattices, which assess the three forces involved in compacting and/or organizing collagen fibrils into thicker collagen fibers. The clinical relevance of the FPCL contraction model is related to advancing our understanding of wound contraction and scar contracture.

Fluid flows and forces in development: functions, features and biophysical principles

PubMed Central

Freund, Jonathan B.; Goetz, Jacky G.; Hill, Kent L.; Vermot, Julien

2012-01-01

Throughout morphogenesis, cells experience intracellular tensile and contractile forces on microscopic scales. Cells also experience extracellular forces, such as static forces mediated by the extracellular matrix and forces resulting from microscopic fluid flow. Although the biological ramifications of static forces have received much attention, little is known about the roles of fluid flows and forces during embryogenesis. Here, we focus on the microfluidic forces generated by cilia-driven fluid flow and heart-driven hemodynamics, as well as on the signaling pathways involved in flow sensing. We discuss recent studies that describe the functions and the biomechanical features of these fluid flows. These insights suggest that biological flow determines many aspects of cell behavior and identity through a specific set of physical stimuli and signaling pathways. PMID:22395739

Polarization of cells and soft objects driven by mechanical interactions: consequences for migration and chemotaxis.

PubMed

Leoni, M; Sens, P

2015-02-01

We study a generic model for the polarization and motility of self-propelled soft objects, biological cells, or biomimetic systems, interacting with a viscous substrate. The active forces generated by the cell on the substrate are modeled by means of oscillating force multipoles at the cell-substrate interface. Symmetry breaking and cell polarization for a range of cell sizes naturally "emerge" from long range mechanical interactions between oscillating units, mediated both by the intracellular medium and the substrate. However, the harnessing of cell polarization for motility requires substrate-mediated interactions. Motility can be optimized by adapting the oscillation frequency to the relaxation time of the system or when the substrate and cell viscosities match. Cellular noise can destroy mechanical coordination between force-generating elements within the cell, resulting in sudden changes of polarization. The persistence of the cell's motion is found to depend on the cell size and the substrate viscosity. Within such a model, chemotactic guidance of cell motion is obtained by directionally modulating the persistence of motion, rather than by modulating the instantaneous cell velocity, in a way that resembles the run and tumble chemotaxis of bacteria.

Physical Limits on the Precision of Mitotic Spindle Positioning by Microtubule Pushing forces: Mechanics of mitotic spindle positioning.

PubMed

Howard, Jonathon; Garzon-Coral, Carlos

2017-11-01

Tissues are shaped and patterned by mechanical and chemical processes. A key mechanical process is the positioning of the mitotic spindle, which determines the size and location of the daughter cells within the tissue. Recent force and position-fluctuation measurements indicate that pushing forces, mediated by the polymerization of astral microtubules against- the cell cortex, maintain the mitotic spindle at the cell center in Caenorhabditis elegans embryos. The magnitude of the centering forces suggests that the physical limit on the accuracy and precision of this centering mechanism is determined by the number of pushing microtubules rather than by thermally driven fluctuations. In cells that divide asymmetrically, anti-centering, pulling forces generated by cortically located dyneins, in conjunction with microtubule depolymerization, oppose the pushing forces to drive spindle displacements away from the center. Thus, a balance of centering pushing forces and anti-centering pulling forces localize the mitotic spindles within dividing C. elegans cells. © 2017 The Authors. BioEssays published by Wiley Periodicals, Inc.

Hindrances to precise recovery of cellular forces in fibrous biopolymer networks.

PubMed

Zhang, Yunsong; Feng, Jingchen; Heizler, Shay I; Levine, Herbert

2018-01-11

How cells move through the three-dimensional extracellular matrix (ECM) is of increasing interest in attempts to understand important biological processes such as cancer metastasis. Just as in motion on flat surfaces, it is expected that experimental measurements of cell-generated forces will provide valuable information for uncovering the mechanisms of cell migration. However, the recovery of forces in fibrous biopolymer networks may suffer from large errors. Here, within the framework of lattice-based models, we explore possible issues in force recovery by solving the inverse problem: how can one determine the forces cells exert to their surroundings from the deformation of the ECM? Our results indicate that irregular cell traction patterns, the uncertainty of local fiber stiffness, the non-affine nature of ECM deformations and inadequate knowledge of network topology will all prevent the precise force determination. At the end, we discuss possible ways of overcoming these difficulties.

Hindrances to precise recovery of cellular forces in fibrous biopolymer networks

NASA Astrophysics Data System (ADS)

Zhang, Yunsong; Feng, Jingchen; Heizler, Shay I.; Levine, Herbert

2018-03-01

How cells move through the three-dimensional extracellular matrix (ECM) is of increasing interest in attempts to understand important biological processes such as cancer metastasis. Just as in motion on flat surfaces, it is expected that experimental measurements of cell-generated forces will provide valuable information for uncovering the mechanisms of cell migration. However, the recovery of forces in fibrous biopolymer networks may suffer from large errors. Here, within the framework of lattice-based models, we explore possible issues in force recovery by solving the inverse problem: how can one determine the forces cells exert to their surroundings from the deformation of the ECM? Our results indicate that irregular cell traction patterns, the uncertainty of local fiber stiffness, the non-affine nature of ECM deformations and inadequate knowledge of network topology will all prevent the precise force determination. At the end, we discuss possible ways of overcoming these difficulties.

Elongated Nanoparticle Aggregates in Cancer Cells for Mechanical Destruction with Low Frequency Rotating Magnetic Field.

PubMed

Shen, Yajing; Wu, Congyu; Uyeda, Taro Q P; Plaza, Gustavo R; Liu, Bin; Han, Yu; Lesniak, Maciej S; Cheng, Yu

2017-01-01

Magnetic nanoparticles (MNPs) functionalized with targeting moieties can recognize specific cell components and induce mechanical actuation under magnetic field. Their size is adequate for reaching tumors and targeting cancer cells. However, due to the nanometric size, the force generated by MNPs is smaller than the force required for largely disrupting key components of cells. Here, we show the magnetic assembly process of the nanoparticles inside the cells, to form elongated aggregates with the size required to produce elevated mechanical forces. We synthesized iron oxide nanoparticles doped with zinc, to obtain high magnetization, and functionalized with the epidermal growth factor (EGF) peptide for targeting cancer cells. Under a low frequency rotating magnetic field at 15 Hz and 40 mT, the internalized EGF-MNPs formed elongated aggregates and generated hundreds of pN to dramatically damage the plasma and lysosomal membranes. The physical disruption, including leakage of lysosomal hydrolases into the cytosol, led to programmed cell death and necrosis. Our work provides a novel strategy of designing magnetic nanomedicines for mechanical destruction of cancer cells.

Elongated Nanoparticle Aggregates in Cancer Cells for Mechanical Destruction with Low Frequency Rotating Magnetic Field

PubMed Central

Shen, Yajing; Wu, Congyu; Uyeda, Taro Q. P.; Plaza, Gustavo R.; Liu, Bin; Han, Yu; Lesniak, Maciej S.; Cheng, Yu

2017-01-01

Magnetic nanoparticles (MNPs) functionalized with targeting moieties can recognize specific cell components and induce mechanical actuation under magnetic field. Their size is adequate for reaching tumors and targeting cancer cells. However, due to the nanometric size, the force generated by MNPs is smaller than the force required for largely disrupting key components of cells. Here, we show the magnetic assembly process of the nanoparticles inside the cells, to form elongated aggregates with the size required to produce elevated mechanical forces. We synthesized iron oxide nanoparticles doped with zinc, to obtain high magnetization, and functionalized with the epidermal growth factor (EGF) peptide for targeting cancer cells. Under a low frequency rotating magnetic field at 15 Hz and 40 mT, the internalized EGF-MNPs formed elongated aggregates and generated hundreds of pN to dramatically damage the plasma and lysosomal membranes. The physical disruption, including leakage of lysosomal hydrolases into the cytosol, led to programmed cell death and necrosis. Our work provides a novel strategy of designing magnetic nanomedicines for mechanical destruction of cancer cells. PMID:28529648

Mechanical Control of Myotendinous Junction Formation and Tendon Differentiation during Development.

PubMed

Valdivia, Mauricio; Vega-Macaya, Franco; Olguín, Patricio

2017-01-01

The development of the musculoskeletal system is a great model to study the interplay between chemical and mechanical inter-tissue signaling in cell adhesion, tissue morphogenesis and differentiation. In both vertebrates and invertebrates (e.g., Drosophila melanogaster ) the formation of muscle-tendon interaction generates mechanical forces which are required for myotendinous junction maturation and tissue differentiation. In addition, these forces must be withstood by muscles and tendons in order to prevent detachment from each other, deformation or even losing their integrity. Extracellular matrix remodeling at the myotendinous junction is key to resist mechanical load generated by muscle contraction. Recent evidences in vertebrates indicate that mechanical forces generated during junction formation regulate chemical signaling leading to extracellular matrix remodeling, however, the mechanotransduction mechanisms associated to this response remains elusive. In addition to extracellular matrix remodeling, the ability of Drosophila tendon-cells to bear mechanical load depends on rearrangement of tendon cell cytoskeleton, thus studying the molecular mechanisms involved in this process is critical to understand the contribution of mechanical forces to the development of the musculoskeletal system. Here, we review recent findings regarding the role of chemical and mechanical signaling in myotendinous junction formation and tendon differentiation, and discuss molecular mechanisms of mechanotransduction that may allow tendon cells to withstand mechanical load during development of the musculoskeletal system.

Molecular analysis of antigen-independent adhesion forces between T and B lymphocytes.

PubMed Central

Amblard, F; Auffray, C; Sekaly, R; Fischer, A

1994-01-01

The low-affinity interactions underlying antigen recognition by T-cell receptors (TCRs) are thought to involve antigen-independent adhesion mechanisms. Using a hydrodynamic approach, we found that antigen-independent adhesion occurred between human B cells and resting T cells in a transient and temperature-dependent fashion. The mean cell-cell adhesion force was 0.32 x 10(-9) N and was generated by similar contributions (0.16 x 10(-9) N) of the LFA-1- and CD2-dependent adhesion pathways. After T-cell stimulation with a phorbol ester, the force contributed by LFA-1 was drastically increased, while that of CD2 was unaffected. We propose that weak receptor-mediated adhesion initiates antigen-independent intercellular contacts required for antigen recognition by the TCR and is upregulated following TCR engagement. The method used permits adhesion forces between living cells to be resolved at the molecular level and should prove valuable for the rapid assessment of interaction forces between various types of cells and cell-sized particles. Images PMID:7909604

Microfabricated tissues for investigating traction forces involved in cell migration and tissue morphogenesis

PubMed Central

Nerger, Bryan A.; Siedlik, Michael J.; Nelson, Celeste M.

2016-01-01

Cell-generated forces drive an array of biological processes ranging from wound healing to tumor metastasis. Whereas experimental techniques such as traction force microscopy are capable of quantifying traction forces in multidimensional systems, the physical mechanisms by which these forces induce changes in tissue form remain to be elucidated. Understanding these mechanisms will ultimately require techniques that are capable of quantifying traction forces with high precision and accuracy in vivo or in systems that recapitulate in vivo conditions, such as microfabricated tissues and engineered substrata. To that end, here we review the fundamentals of traction forces, their quantification, and the use of microfabricated tissues designed to study these forces during cell migration and tissue morphogenesis. We emphasize the differences between traction forces in two- and three-dimensional systems, and highlight recently developed techniques for quantifying traction forces. PMID:28008471

Measuring traction forces of motile dendritic cells on micropost arrays.

PubMed

Ricart, Brendon G; Yang, Michael T; Hunter, Christopher A; Chen, Christopher S; Hammer, Daniel A

2011-12-07

Dendritic cells (DCs) migrate from sites of inflammation to secondary lymphoid organs where they initiate the adaptive immune response. Although motility is essential to DC function, the mechanisms by which they migrate are not fully understood. We incorporated micropost array detectors into a microfluidic gradient generator to develop what we consider to be a novel method for probing low magnitude traction forces during directional migration. We found migration of primary murine DCs is driven by short-lived traction stresses at the leading edge or filopodia. The traction forces generated by DCs are smaller in magnitude than found in neutrophils, and of similar magnitude during chemotaxis and chemokinesis, at 18 ± 1.4 and 16 ± 1.3 nN/cell, respectively. The characteristic duration of local DC traction forces was 3 min. The maximum principal stress in the cell occurred in the plane perpendicular to the axis of motion, forward of the centroid. We illustrate that the spatiotemporal pattern of traction stresses can be used to predict the direction of future DC motion. Overall, DCs show a mode of migration distinct from both mesenchymal cells and neutrophils, characterized by rapid turnover of traction forces in leading filopodia. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Traction force microscopy in rapidly moving cells reveals separate roles for ROCK and MLCK in the mechanics of retraction.

PubMed

Morin, Timothy R; Ghassem-Zadeh, Sean A; Lee, Juliet

2014-08-15

Retraction is a major rate-limiting step in cell motility, particularly in slow moving cell types that form large stable adhesions. Myosin II dependent contractile forces are thought to facilitate detachment by physically pulling up the rear edge. However, retraction can occur in the absence of myosin II activity in cell types that form small labile adhesions. To investigate the role of contractile force generation in retraction, we performed traction force microscopy during the movement of fish epithelial keratocytes. By correlating changes in local traction stress at the rear with the area retracted, we identified four distinct modes of retraction. "Recoil" retractions are preceded by a rise in local traction stress, while rear edge is temporarily stuck, followed by a sharp drop in traction stress upon detachment. This retraction type was most common in cells generating high average traction stress. In "pull" type retractions local traction stress and area retracted increase concomitantly. This was the predominant type of retraction in keratocytes and was observed mostly in cells generating low average traction stress. "Continuous" type retractions occur without any detectable change in traction stress, and are seen in cells generating low average traction stress. In contrast, to many other cell types, "release" type retractions occur in keratocytes following a decrease in local traction stress. Our identification of distinct modes of retraction suggests that contractile forces may play different roles in detachment that are related to rear adhesion strength. To determine how the regulation of contractility via MLCK or Rho kinase contributes to the mechanics of detachment, inhibitors were used to block or augment these pathways. Modulation of MLCK activity led to the most rapid change in local traction stress suggesting its importance in regulating attachment strength. Surprisingly, Rho kinase was not required for detachment, but was essential for localizing retraction to the rear. We suggest that in keratocytes MLCK and Rho kinase play distinct, complementary roles in the respective temporal and spatial control of rear detachment that is essential for maintaining rapid motility. Copyright © 2014 Elsevier Inc. All rights reserved.

An Improved Optical Tweezers Assay for Measuring the Force Generation of Single Kinesin Molecules

PubMed Central

Nicholas, Matthew P.; Rao, Lu; Gennerich, Arne

2014-01-01

Numerous microtubule-associated molecular motors, including several kinesins and cytoplasmic dynein, produce opposing forces that regulate spindle and chromosome positioning during mitosis. The motility and force generation of these motors are therefore critical to normal cell division, and dysfunction of these processes may contribute to human disease. Optical tweezers provide a powerful method for studying the nanometer motility and piconewton force generation of single motor proteins in vitro. Using kinesin-1 as a prototype, we present a set of step-by-step, optimized protocols for expressing a kinesin construct (K560-GFP) in Escherichia coli, purifying it, and studying its force generation in an optical tweezers microscope. We also provide detailed instructions on proper alignment and calibration of an optical trapping microscope. These methods provide a foundation for a variety of similar experiments. PMID:24633799

Mechanics of composite actin networks: in vitro and cellular perspectives

NASA Astrophysics Data System (ADS)

Upadhyaya, Arpita

2014-03-01

Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not well understood. The ABP, palladin, is essential for the integrity of cell morphology and movement during development. Palladin coexists with alpha-actinin in stress fibers and focal adhesions and binds to both actin and alpha-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we have characterized the micro-structure and mechanics of actin networks crosslinked with palladin and alpha-actinin. Our studies on composite networks of alpha-actinin/palladin/actin show that palladin and alpha-actinin synergistically determine network viscoelasticity. We have further examined the role of palladin in cellular force generation and mechanosensing. Traction force microscopy revealed that TAFs are sensitive to substrate stiffness as they generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells, and also inhibited the ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in the actin organization and adhesion dynamics of palladin knock down cells. Perturbation experiments also suggest altered myosin activity in palladin KD cells. Our results suggest that the actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis.

Fiber networks amplify active stress

PubMed Central

Ronceray, Pierre; Broedersz, Chase P.

2016-01-01

Large-scale force generation is essential for biological functions such as cell motility, embryonic development, and muscle contraction. In these processes, forces generated at the molecular level by motor proteins are transmitted by disordered fiber networks, resulting in large-scale active stresses. Although these fiber networks are well characterized macroscopically, this stress generation by microscopic active units is not well understood. Here we theoretically study force transmission in these networks. We find that collective fiber buckling in the vicinity of a local active unit results in a rectification of stress towards strongly amplified isotropic contraction. This stress amplification is reinforced by the networks’ disordered nature, but saturates for high densities of active units. Our predictions are quantitatively consistent with experiments on reconstituted tissues and actomyosin networks and shed light on the role of the network microstructure in shaping active stresses in cells and tissue. PMID:26921325

Directional migration of leading-edge mesoderm generates physical forces: Implication in Xenopus notochord formation during gastrulation.

PubMed

Hara, Yusuke; Nagayama, Kazuaki; Yamamoto, Takamasa S; Matsumoto, Takeo; Suzuki, Makoto; Ueno, Naoto

2013-10-15

Gastrulation is a dynamic tissue-remodeling process occurring during early development and fundamental to the later organogenesis. It involves both chemical signals and physical factors. Although much is known about the molecular pathways involved, the roles of physical forces in regulating cellular behavior and tissue remodeling during gastrulation have just begun to be explored. Here, we characterized the force generated by the leading edge mesoderm (LEM) that migrates preceding axial mesoderm (AM), and investigated the contribution of LEM during Xenopus gastrulation. First, we constructed an assay system using micro-needle which could measure physical forces generated by the anterior migration of LEM, and estimated the absolute magnitude of the force to be 20-80nN. Second, laser ablation experiments showed that LEM could affect the force distribution in the AM (i.e. LEM adds stretch force on axial mesoderm along anterior-posterior axis). Third, migrating LEM was found to be necessary for the proper gastrulation cell movements and the establishment of organized notochord structure; a reduction of LEM migratory activity resulted in the disruption of mediolateral cell orientation and convergence in AM. Finally, we found that LEM migration cooperates with Wnt/PCP to form proper notochord. These results suggest that the force generated by the directional migration of LEM is transmitted to AM and assists the tissue organization of notochord in vivo independently of the regulation by Wnt/PCP. We propose that the LEM may have a mechanical role in aiding the AM elongation through the rearrangement of force distribution in the dorsal marginal zone. © 2013 Elsevier Inc. All rights reserved.

Actin Filament Elasticity and Retrograde Flow Shape the Force-Velocity Relation of Motile Cells

PubMed Central

Zimmermann, Juliane; Brunner, Claudia; Enculescu, Mihaela; Goegler, Michael; Ehrlicher, Allen; Käs, Josef; Falcke, Martin

2012-01-01

Cells migrate through a crowded environment during processes such as metastasis or wound healing, and must generate and withstand substantial forces. The cellular motility responses to environmental forces are represented by their force-velocity relation, which has been measured for fish keratocytes but remains unexplained. Even pN opposing forces slow down lamellipodium motion by three orders of magnitude. At larger opposing forces, the retrograde flow of the actin network accelerates until it compensates for polymerization, and cell motion stalls. Subsequently, the lamellipodium adapts to the stalled state. We present a mechanism quantitatively explaining the cell's force-velocity relation and its changes upon application of drugs that hinder actin polymerization or actomyosin-based contractility. Elastic properties of filaments, close to the lamellipodium leading edge, and retrograde flow shape the force-velocity relation. To our knowledge, our results shed new light on how these migratory responses are regulated, and on the mechanics and structure of the lamellipodium. PMID:22339865

Reverse transduction measured in the living cochlea by low-coherence heterodyne interferometry.

PubMed

Ren, Tianying; He, Wenxuan; Barr-Gillespie, Peter G

2016-01-06

It is generally believed that the remarkable sensitivity and frequency selectivity of mammalian hearing depend on outer hair cell-generated force, which amplifies sound-induced vibrations inside the cochlea. This 'reverse transduction' force production has never been demonstrated experimentally, however, in the living ear. Here by directly measuring microstructure vibrations inside the cochlear partition using a custom-built interferometer, we demonstrate that electrical stimulation can evoke both fast broadband and slow sharply tuned responses of the reticular lamina, but only a slow tuned response of the basilar membrane. Our results indicate that outer hair cells can generate sufficient force to drive the reticular lamina over all audible frequencies in living cochleae. Contrary to expectations, the cellular force causes a travelling wave rather than an immediate local vibration of the basilar membrane; this travelling wave vibrates in phase with the reticular lamina at the best frequency, and results in maximal vibration at the apical ends of outer hair cells.

Traction force dynamics predict gap formation in activated endothelium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valent, Erik T.; Nieuw Amerongen, Geerten P. van; Hinsbergh, Victor W.M. van

In many pathological conditions the endothelium becomes activated and dysfunctional, resulting in hyperpermeability and plasma leakage. No specific therapies are available yet to control endothelial barrier function, which is regulated by inter-endothelial junctions and the generation of acto-myosin-based contractile forces in the context of cell-cell and cell-matrix interactions. However, the spatiotemporal distribution and stimulus-induced reorganization of these integral forces remain largely unknown. Traction force microscopy of human endothelial monolayers was used to visualize contractile forces in resting cells and during thrombin-induced hyperpermeability. Simultaneously, information about endothelial monolayer integrity, adherens junctions and cytoskeletal proteins (F-actin) were captured. This revealed a heterogeneousmore » distribution of traction forces, with nuclear areas showing lower and cell-cell junctions higher traction forces than the whole-monolayer average. Moreover, junctional forces were asymmetrically distributed among neighboring cells. Force vector orientation analysis showed a good correlation with the alignment of F-actin and revealed contractile forces in newly formed filopodia and lamellipodia-like protrusions within the monolayer. Finally, unstable areas, showing high force fluctuations within the monolayer were prone to form inter-endothelial gaps upon stimulation with thrombin. To conclude, contractile traction forces are heterogeneously distributed within endothelial monolayers and force instability, rather than force magnitude, predicts the stimulus-induced formation of intercellular gaps. - Highlights: • Endothelial monolayers exert dynamic- and heterogeneous traction forces. • High traction forces correlate with junctional areas and the F-actin cytoskeleton. • Newly formed inter-endothelial gaps are characterized by opposing traction forces. • Force stability is a key feature controlling endothelial permeability.« less

Single Vector Calibration System for Multi-Axis Load Cells and Method for Calibrating a Multi-Axis Load Cell

NASA Technical Reports Server (NTRS)

Parker, Peter A. (Inventor)

2003-01-01

A single vector calibration system is provided which facilitates the calibration of multi-axis load cells, including wind tunnel force balances. The single vector system provides the capability to calibrate a multi-axis load cell using a single directional load, for example loading solely in the gravitational direction. The system manipulates the load cell in three-dimensional space, while keeping the uni-directional calibration load aligned. The use of a single vector calibration load reduces the set-up time for the multi-axis load combinations needed to generate a complete calibration mathematical model. The system also reduces load application inaccuracies caused by the conventional requirement to generate multiple force vectors. The simplicity of the system reduces calibration time and cost, while simultaneously increasing calibration accuracy.

Viability evaluation of culture cells patterned by femtosecond laser-induced impulsive force

NASA Astrophysics Data System (ADS)

Takizawa, Noriko; Okano, Kazunori; Uwada, Takayuki; Hosokawa, Yoichiroh; Masuhara, Hiroshi

2008-02-01

PC12 cells, which are derived from a rat pheochromocytoma, were independently patterned utilizing an impulsive force resulting in impulsive shockwave and cavitation bubble generation by focused femtosecond laser irradiation. Since the PC12 cells respond reversibly to nerve growth factor by induction of the neuronal phenotype, we can assess an influence that the impulsive force gives to the bioactivity in term of the cell differentiation. The patterned cells were accumulated on an intact dish and cultured for 3 days. The behavior of appearance and cell differentiation was observed by multipoint time-lapse system. On bases of these results, it was proved that the biological activity of the cell is unaffected by the femtosecond laser patterning.

FRET measurements of cell-traction forces and nano-scale clustering of adhesion ligands varied by substrate stiffness.

PubMed

Kong, Hyun Joon; Polte, Thomas R; Alsberg, Eben; Mooney, David J

2005-03-22

The mechanical properties of cell adhesion substrates regulate cell phenotype, but the mechanism of this relation is currently unclear. It may involve the magnitude of traction force applied by the cell, and/or the ability of the cells to rearrange the cell adhesion molecules presented from the material. In this study, we describe a FRET technique that can be used to evaluate the mechanics of cell-material interactions at the molecular level and simultaneously quantify the cell-based nanoscale rearrangement of the material itself. We found that these events depended on the mechanical rigidity of the adhesion substrate. Furthermore, both the proliferation and differentiation of preosteoblasts (MC3T3-E1) correlated to the magnitude of force that cells generate to cluster the cell adhesion ligands, but not the extent of ligand clustering. Together, these data demonstrate the utility of FRET in analyzing cell-material interactions, and suggest that regulation of phenotype with substrate stiffness is related to alterations in cellular traction forces.

Myosin II Dynamics during Embryo Morphogenesis

NASA Astrophysics Data System (ADS)

Kasza, Karen

2013-03-01

During embryonic morphogenesis, the myosin II motor protein generates forces that help to shape tissues, organs, and the overall body form. In one dramatic example in the Drosophila melanogaster embryo, the epithelial tissue that will give rise to the body of the adult animal elongates more than two-fold along the head-to-tail axis in less than an hour. This elongation is accomplished primarily through directional rearrangements of cells within the plane of the tissue. Just prior to elongation, polarized assemblies of myosin II accumulate perpendicular to the elongation axis. The contractile forces generated by myosin activity orient cell movements along a common axis, promoting local cell rearrangements that contribute to global tissue elongation. The molecular and mechanical mechanisms by which myosin drives this massive change in embryo shape are poorly understood. To investigate these mechanisms, we generated a collection of transgenic flies expressing variants of myosin II with altered motor function and regulation. We found that variants that are predicted to have increased myosin activity cause defects in tissue elongation. Using biophysical approaches, we found that these myosin variants also have decreased turnover dynamics within cells. To explore the mechanisms by which molecular-level myosin dynamics are translated into tissue-level elongation, we are using time-lapse confocal imaging to observe cell movements in embryos with altered myosin activity. We are utilizing computational approaches to quantify the dynamics and directionality of myosin localization and cell rearrangements. These studies will help elucidate how myosin-generated forces control cell movements within tissues. This work is in collaboration with J. Zallen at the Sloan-Kettering Institute.

Mechanical dynamics in live cells and fluorescence-based force/tension sensors

PubMed Central

Yang, Chao; Zhang, Xiaohan; Guo, Yichen; Meng, Fanjie; Sachs, Frederick; Guo, Jun

2016-01-01

Three signaling systems play the fundamental roles in modulating cell activities: chemical, electrical, and mechanical. While the former two are well studied, the mechanical signaling system is still elusive because of the lack of methods to measure structural forces in real time at cellular and subcellular levels. Indeed, almost all biological processes are responsive to modulation by mechanical forces that trigger dispersive downstream electrical and biochemical pathways. Communication among the three systems is essential to make cells and tissues receptive to environmental changes. Cells have evolved many sophisticated mechanisms for the generation, perception and transduction of mechanical forces, including motor proteins and mechanosensors. In this review, we introduce some background information about mechanical dynamics in live cells, including the ubiquitous mechanical activity, various types of mechanical stimuli exerted on cells and the different mechanosensors. We also summarize recent results obtained using genetically encoded FRET (fluorescence resonance energy transfer)-based force/tension sensors; a new technique used to measure mechanical forces in structural proteins. The sensors have been incorporated into many specific structural proteins and have measured the force gradients in real time within live cells, tissues, and animals. PMID:25958335

Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels

PubMed Central

Delorme-Walker, Violaine; Seo, Ji-Yeon; Gohla, Antje; Fowler, Bruce; Bohl, Ben; DerMardirossian, Céline

2015-01-01

Cofilin, a critical player of actin dynamics, is spatially and temporally regulated to control the direction and force of membrane extension required for cell locomotion. In carcinoma cells, although the signaling pathways regulating cofilin activity to control cell direction have been established, the molecular machinery required to generate the force of the protrusion remains unclear. We show that the cofilin phosphatase chronophin (CIN) spatiotemporally regulates cofilin activity at the cell edge to generate persistent membrane extension. We show that CIN translocates to the leading edge in a PI3-kinase–, Rac1-, and cofilin-dependent manner after EGF stimulation to activate cofilin, promotes actin free barbed end formation, accelerates actin turnover, and enhances membrane protrusion. In addition, we establish that CIN is crucial for the balance of protrusion/retraction events during cell migration. Thus, CIN coordinates the leading edge dynamics by controlling active cofilin levels to promote MTLn3 cell protrusion. PMID:26324884

Force-activatable coating enables high-resolution cellular force imaging directly on regular cell culture surfaces.

PubMed

Sarkar, Anwesha; Zhao, Yuanchang; Wang, Yongliang; Wang, Xuefeng

2018-06-25

Integrin-transmitted cellular forces are crucial mechanical signals regulating a vast range of cell functions. Although various methods have been developed to visualize and quantify cellular forces at the cell-matrix interface, a method with high performance and low technical barrier is still in demand. Here we developed a force-activatable coating (FAC), which can be simply coated on regular cell culture apparatus' surfaces by physical adsorption, and turn these surfaces to force reporting platforms that enable cellular force mapping directly by fluorescence imaging. The FAC molecule consists of an adhesive domain for surface coating and a force-reporting domain which can be activated to fluoresce by integrin molecular tension. The tension threshold required for FAC activation is tunable in 10-60 piconewton (pN), allowing the selective imaging of cellular force contributed by integrin tension at different force levels. We tested the performance of two FACs with tension thresholds of 12 and 54 pN (nominal values), respectively, on both glass and polystyrene surfaces. Cellular forces were successfully mapped by fluorescence imaging on all the surfaces. FAC-coated surfaces also enable co-imaging of cellular forces and cell structures in both live cells and immunostained cells, therefore opening a new avenue for the study of the interplay of force and structure. We demonstrated the co-imaging of integrin tension and talin clustering in live cells, and concluded that talin clustering always occurs before the generation of integrin tension above 54 pN, reinforcing the notion that talin is an important adaptor protein for integrin tension transmission. Overall, FAC provides a highly convenient approach that is accessible to general biological laboratories for the study of cellular forces with high sensitivity and resolution, thus holding the potential to greatly boost the research of cell mechanobiology.

Cellular dynamics of bovine aortic smooth muscle cells measured using MEMS force sensors

NASA Astrophysics Data System (ADS)

Tsukagoshi, Takuya; Nguyen, Thanh-Vinh; Hirayama Shoji, Kayoko; Takahashi, Hidetoshi; Matsumoto, Kiyoshi; Shimoyama, Isao

2018-04-01

Adhesive cells perceive the mechanical properties of the substrates to which they adhere, adjusting their cellular mechanical forces according to their biological characteristics. This mechanical interaction subsequently affects the growth, locomotion, and differentiation of the cell. However, little is known about the detailed mechanism that underlies this interaction between adherent cells and substrates because dynamically measuring mechanical phenomena is difficult. Here, we utilize microelectromechamical systems force sensors that can measure cellular traction forces with high temporal resolution (~2.5 µs) over long periods (~3 h). We found that the cellular dynamics reflected physical phenomena with time scales from milliseconds to hours, which contradicts the idea that cellular motion is slow. A single focal adhesion (FA) generates an average force of 7 nN, which disappears in ms via the action of trypsin-ethylenediaminetetraacetic acid. The force-changing rate obtained from our measurements suggests that the time required for an FA to decompose was nearly proportional to the force acting on the FA.

Wounded cells drive rapid epidermal repair in the early Drosophila embryo

PubMed Central

Fernandez-Gonzalez, Rodrigo; Zallen, Jennifer A.

2013-01-01

Epithelial tissues are protective barriers that display a remarkable ability to repair wounds. Wound repair is often associated with an accumulation of actin and nonmuscle myosin II around the wound, forming a purse string. The role of actomyosin networks in generating mechanical force during wound repair is not well understood. Here we investigate the mechanisms of force generation during wound repair in the epidermis of early and late Drosophila embryos. We find that wound closure is faster in early embryos, where, in addition to a purse string around the wound, actomyosin networks at the medial cortex of the wounded cells contribute to rapid wound repair. Laser ablation demonstrates that both medial and purse-string actomyosin networks generate contractile force. Quantitative analysis of protein localization dynamics during wound closure indicates that the rapid contraction of medial actomyosin structures during wound repair in early embryos involves disassembly of the actomyosin network. By contrast, actomyosin purse strings in late embryos contract more slowly in a mechanism that involves network condensation. We propose that the combined action of two force-generating structures—a medial actomyosin network and an actomyosin purse string—contributes to the increased efficiency of wound repair in the early embryo. PMID:23985320

An Analytical Model for Determining Two-Dimensional Receptor-Ligand Kinetics

PubMed Central

Cheung, Luthur Siu-Lun; Konstantopoulos, Konstantinos

2011-01-01

Cell-cell adhesive interactions play a pivotal role in major pathophysiological vascular processes, such as inflammation, infection, thrombosis, and cancer metastasis, and are regulated by hemodynamic forces generated by blood flow. Cell adhesion is mediated by the binding of receptors to ligands, which are both anchored on two-dimensional (2-D) membranes of apposing cells. Biophysical assays have been developed to determine the unstressed (no-force) 2-D affinity but fail to disclose its dependence on force. Here we develop an analytical model to estimate the 2-D kinetics of diverse receptor-ligand pairs as a function of force, including antibody-antigen, vascular selectin-ligand, and bacterial adhesin-ligand interactions. The model can account for multiple bond interactions necessary to mediate adhesion and resist detachment amid high hemodynamic forces. Using this model, we provide a generalized biophysical interpretation of the counterintuitive force-induced stabilization of cell rolling observed by a select subset of receptor-ligand pairs with specific intrinsic kinetic properties. This study enables us to understand how single-molecule and multibond biophysics modulate the macroscopic cell behavior in diverse pathophysiological processes. PMID:21575567

PIEZO channel protein naturally expressed in human breast cancer cell MDA-MB-231 as probed by atomic force microscopy

NASA Astrophysics Data System (ADS)

Weng, Yuanqi; Yan, Fei; Chen, Runkang; Qian, Ming; Ou, Yun; Xie, Shuhong; Zheng, Hairong; Li, Jiangyu

2018-05-01

Mechanical stimuli drives many physiological processes through mechanically activated channels, and the recent discovery of PIEZO channel has generated great interests in its mechanotransduction. Many previous researches investigated PIEZO proteins by transcribing them in cells that originally have no response to mechanical stimulation, or by forming PIEZO-combined complexes in vitro, and few studied PIEZO protein's natural characteristics in cells. In this study we show that MDA-MB-231, a malignant cell in human breast cancer cell line, expresses the mechanosensitive behavior of PIEZO in nature without extra treatment, and we report its characteristics in response to localized mechanical stimulation under an atomic force microscope, wherein a correlation between the force magnitude applied and the channel opening probability is observed. The results on PIEZO of MDA-MB-231 can help establish a basis of preventing and controlling of human breast cancer cell via mechanical forces.

Cell and molecular mechanics of biological materials

NASA Astrophysics Data System (ADS)

Bao, G.; Suresh, S.

2003-11-01

Living cells can sense mechanical forces and convert them into biological responses. Similarly, biological and biochemical signals are known to influence the abilities of cells to sense, generate and bear mechanical forces. Studies into the mechanics of single cells, subcellular components and biological molecules have rapidly evolved during the past decade with significant implications for biotechnology and human health. This progress has been facilitated by new capabilities for measuring forces and displacements with piconewton and nanometre resolutions, respectively, and by improvements in bio-imaging. Details of mechanical, chemical and biological interactions in cells remain elusive. However, the mechanical deformation of proteins and nucleic acids may provide key insights for understanding the changes in cellular structure, response and function under force, and offer new opportunities for the diagnosis and treatment of disease. This review discusses some basic features of the deformation of single cells and biomolecules, and examines opportunities for further research.

Apical constriction drives tissue-scale hydrodynamic flow to mediate cell elongation

PubMed Central

He, Bing; Doubrovinski, Konstantin; Polyakov, Oleg; Wieschaus, Eric

2014-01-01

Epithelial folding mediated by apical constriction converts flat epithelial sheets into multilayered, complex tissue structures and is employed throughout the development in most animals1. Little is known, however, how forces produced near the apical surface of the tissue are transmitted within individual cells to generate the global changes in cell shape that characterize tissue deformation. Here we apply particle tracking velocimetry in gastrulating Drosophila embryos to measure the movement of cytoplasm and plasma membrane during ventral furrow (VF) formation2, 3. We find that cytoplasmic redistribution during the lengthening phase of VF formation can be precisely described by viscous flows that quantitatively match the predictions of hydrodynamics. Cell membranes move with the ambient cytoplasm, with little resistance to or driving force on the flow. Strikingly, apical constriction produces similar flow patterns in mutant embryos that fail to form cells prior to gastrulation (“acellular” embryos), such that the global redistribution of cytoplasm mirrors the summed redistribution occurring in individual cells of wild type embryos. Our results suggest that during the lengthening phase of VF formation, hydrodynamic behavior of the cytoplasm provides the predominant mechanism transmitting apically generated forces deep into the tissue and that cell individualization is dispensable. PMID:24590071

Capillary photoelectrode structures for photoelectrochemical and photocatalytic cells

DOEpatents

Wang, Xudong; Li, Zhaodong; Cai, Zhiyong; Yao, Chunhua

2017-05-02

Photocatalytic structures having a capillary-force based electrolyte delivery system are provided. Also provided are photoelectrochemical and photocatalytic cells incorporating the structures and methods for using the cells to generate hydrogen and/or oxygen from water. The photocatalytic structures use an electrolyte-transporting strip comprising a porous network of cellulose nanofibers to transport electrolyte from a body of the electrolyte to a porous photoelectrode or a porous photocatalytic substrate via capillary force.

Generation and Long-term Maintenance of Nerve-free Hydra.

PubMed

Tran, Cassidy M; Fu, Sharon; Rowe, Trevor; Collins, Eva-Maria S

2017-07-07

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Development of living cell force sensors for the interrogation of cell surface interactions

NASA Astrophysics Data System (ADS)

Brown, Scott Chang

The measurement of cell surface interactions, or cell interaction forces, are critical for the early diagnosis and prevention of disease, the design of targeted drug and gene delivery vehicles, the development of next-generation implant materials, and much more. However, the technologies and devices that are currently available are highly limited with respect to the dynamic force range over which they can measure cell-cell or cell-substratum interactions, and with their ability to adequately mimic biologically relevant systems. Consequently, research efforts that involve cell surface interactions have been limited. In this dissertation, existing tools for research at the nanoscale (i.e., atomic force microscopy microcantilevers) are modified to develop living cell force sensors that allow for the highly sensitive measurement of cell-mediated interactions over the entire range of forces expected in biotechnology (and nano-biotechnology) research (from a single to millions of receptor-ligand bonds). Several force sensor motifs have been developed that can be used to measure interactions using single adherent cells, single suspension culture cell, and cell monolayers (tissues) over a wide range of interaction conditions (e.g., approach velocity, shear rate, contact time) using a conventional atomic force microscope. This new tool has been applied to study the pathogenesis of spontaneous pneumothorax and the interaction of cells with 14 man-made interfaces. Consequently, a new hypothesis of the interactions that manifest spontaneous pneumothorax has been developed. Additionally, these findings have the potential to lead to the development of tools for data mining materials and surfaces for unique cell interactions that could have an immense societal impact.

Shortening actin filaments cause force generation in actomyosin network to change from contractile to extensile

NASA Astrophysics Data System (ADS)

Kumar, Nitin; Gardel, Margaret

Motor proteins in conjunction with filamentous proteins convert biochemical energy into mechanical energy which serves a number of cellular processes including cell motility, force generation and intracellular cargo transport. In-vitro experiments suggest that the forces generated by kinesin motors on microtubule bundles are extensile in nature whereas myosin motors on actin filaments are contractile. It is not clear how qualitatively similar systems can show completely different behaviors in terms of the nature of force generation. In order to answer this question, we carry out in vitro experiments where we form quasi 2D filamentous actomyosin networks and vary the length of actin filaments by adding capping protein. We show that when filaments are much shorter than their typical persistence length (approximately 10 microns), the forces generated are extensile and we see active nematic defect propagation, as seen in the microtubule-kinesin system. Based on this observation, we claim that the rigidity of rods plays an important role in dictating the nature of force generation in such systems. In order to understand this transition, we selectively label individual filaments and find that longer filaments show considerable bending and buckling, making them difficult to slide and extend along their length.

Prestin modulates mechanics and electromechanical force of the plasma membrane.

PubMed

Zhang, Rui; Qian, Feng; Rajagopalan, Lavanya; Pereira, Fred A; Brownell, William E; Anvari, Bahman

2007-07-01

The voltage-dependent movement, or electromotility, of cochlear outer hair cells contributes to cochlear amplification in mammalian hearing. Outer hair-cell electromotility involves a membrane-based motor in which the membrane protein prestin plays a central role. We have investigated the contribution of prestin to the mechanics and electromechanical force (EMF) generation of the membrane using membrane tethers formed from human embryonic kidney (HEK) cells. Several measures of membrane tether mechanics are greater in tethers pulled from HEK cells transfected with prestin when compared to control untransfected HEK cells. A single point mutation of alanine to tryptophan (A100W) in prestin eliminates prestin-associated charge movement and diminishes EMF but does not alter passive membrane mechanics. These results suggest that prestin-associated charge transfer is necessary for maximal EMF generation by the membrane.

Prestin Modulates Mechanics and Electromechanical Force of the Plasma Membrane

PubMed Central

Zhang, Rui; Qian, Feng; Rajagopalan, Lavanya; Pereira, Fred A.; Brownell, William E.; Anvari, Bahman

2007-01-01

The voltage-dependent movement, or electromotility, of cochlear outer hair cells contributes to cochlear amplification in mammalian hearing. Outer hair-cell electromotility involves a membrane-based motor in which the membrane protein prestin plays a central role. We have investigated the contribution of prestin to the mechanics and electromechanical force (EMF) generation of the membrane using membrane tethers formed from human embryonic kidney (HEK) cells. Several measures of membrane tether mechanics are greater in tethers pulled from HEK cells transfected with prestin when compared to control untransfected HEK cells. A single point mutation of alanine to tryptophan (A100W) in prestin eliminates prestin-associated charge movement and diminishes EMF but does not alter passive membrane mechanics. These results suggest that prestin-associated charge transfer is necessary for maximal EMF generation by the membrane. PMID:17468166

Modeling and predictions of biphasic mechanosensitive cell migration altered by cell-intrinsic properties and matrix confinement.

PubMed

Pathak, Amit

2018-04-12

Motile cells sense the stiffness of their extracellular matrix (ECM) through adhesions and respond by modulating the generated forces, which in turn lead to varying mechanosensitive migration phenotypes. Through modeling and experiments, cell migration speed is known to vary with matrix stiffness in a biphasic manner, with optimal motility at an intermediate stiffness. Here, we present a two-dimensional cell model defined by nodes and elements, integrated with subcellular modeling components corresponding to mechanotransductive adhesion formation, force generation, protrusions and node displacement. On 2D matrices, our calculations reproduce the classic biphasic dependence of migration speed on matrix stiffness and predict that cell types with higher force-generating ability do not slow down on very stiff matrices, thus disabling the biphasic response. We also predict that cell types defined by lower number of total receptors require stiffer matrices for optimal motility, which also limits the biphasic response. For a cell type with robust biphasic migration on 2D surface, simulations in channel-like confined environments of varying width and height predict faster migration in more confined matrices. Simulations performed in shallower channels predict that the biphasic mechanosensitive cell migration response is more robust on 2D micro-patterns as compared to the channel-like 3D confinement. Thus, variations in the dimensionality of matrix confinement alters the way migratory cells sense and respond to the matrix stiffness. Our calculations reveal new phenotypes of stiffness- and topography-sensitive cell migration that critically depend on both cell-intrinsic and matrix properties. These predictions may inform our understanding of various mechanosensitive modes of cell motility that could enable tumor invasion through topographically heterogeneous microenvironments. © 2018 IOP Publishing Ltd.

Emergence of an apical epithelial cell surface in vivo

PubMed Central

Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté; Wallingford, John B.

2016-01-01

Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, we combine molecular, pharmacological and laser-dissection experiments with theoretical modelling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium in vivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging cell and mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis. PMID:26766441

Stiffness nanotomography of human epithelial cancer cells

NASA Astrophysics Data System (ADS)

Staunton, Jack R.; Doss, Bryant L.; Gilbert, C. Michael; Kasas, Sandor; Ros, Robert

2012-02-01

The mechanical stiffness of individual cells is important in both cancer initiation and metastasis. We present atomic force microscopy (AFM) based nanoindentation experiments on various human mammary and esophagus cell lines covering the spectrum from normal immortalized cells to highly metastatic ones. The combination of an AFM with a confocal fluorescence lifetime imaging microscope (FLIM) in conjunction with the ability to move the sample and objective independently allow for precise alignment of AFM probe and laser focus with an accuracy down to a few nanometers. This enables us to correlate the mechanical properties with the point of indentation in the FLIM image. We are using force-volume measurements as well as force indentation curves on distinct points on the cells to compare the elastic moduli of the nuclei, nucleoli, and the cytoplasm, and how they vary within and between individual cells and cell lines. Further, a detailed analysis of the force-indentation curves allows study of the cells' mechanical properties at different indentation depths and to generate 3D elasticity maps.

Filopodial retraction force is generated by cortical actin dynamics and controlled by reversible tethering at the tip

PubMed Central

Bornschlögl, Thomas; Romero, Stéphane; Vestergaard, Christian L.; Joanny, Jean-François; Van Nhieu, Guy Tran; Bassereau, Patricia

2013-01-01

Filopodia are dynamic, finger-like plasma membrane protrusions that sense the mechanical and chemical surroundings of the cell. Here, we show in epithelial cells that the dynamics of filopodial extension and retraction are determined by the difference between the actin polymerization rate at the tip and the retrograde flow at the base of the filopodium. Adhesion of a bead to the filopodial tip locally reduces actin polymerization and leads to retraction via retrograde flow, reminiscent of a process used by pathogens to invade cells. Using optical tweezers, we show that filopodial retraction occurs at a constant speed against counteracting forces up to 50 pN. Our measurements point toward retrograde flow in the cortex together with frictional coupling between the filopodial and cortical actin networks as the main retraction-force generator for filopodia. The force exerted by filopodial retraction, however, is limited by the connection between filopodial actin filaments and the membrane at the tip. Upon mechanical rupture of the tip connection, filopodia exert a passive retraction force of 15 pN via their plasma membrane. Transient reconnection at the tip allows filopodia to continuously probe their surroundings in a load-and-fail manner within a well-defined force range. PMID:24198333

Traction Stresses Exerted by Adherent Cells: From Angiogenesis to Metastasis

NASA Astrophysics Data System (ADS)

Reinhart-King, Cynthia

2010-03-01

Cells exert traction stresses against their substrate that mediate their ability to sense the mechanical properties of their microenvironment. These same forces mediate cell adhesion, migration and the formation of stable cell-cell contacts during tissue formation. In this talk, I will present our data on the traction stresses generated by endothelial cells and metastatic breast cancer cells focused on understanding the processes of angiogenesis and metastasis, respectively. In the context of capillary formation, our data indicate that the mechanics of the substrate play a critical role in establishing endothelial cell-cell contacts. On more compliant substrates, endothelial cell shape and traction stresses polarize and promote the formation of stable cell-cell contacts. On stiffer substrates, traction stresses are less polarized and cell connectivity is disrupted. These data indicate that the mechanical properties of the microenvironment may drive cell connectivity and the formation of stable cell-cell contacts through the reorientation of traction stresses. In our studies of metastatic cell migration, we have found that traction stresses increase with increasing metastatic potential. We investigated three lines of varying metastatic potential (MCF10A, MCF7 and MDAMB231). MDAMB231, which are the most invasive, exert the most significant forces as measured by Traction Force Microscopy. These data present the possibility that cellular traction stress generation aids in the ability of metastatic cells to migrate through the matrix-dense tumor microenvironment. Such measurements are integral to link the mechanical and chemical microenvironment with the resulting response of the cell in health and disease.

Using cell deformation and motion to predict forces and collective behavior in morphogenesis.

PubMed

Merkel, Matthias; Manning, M Lisa

2017-07-01

In multi-cellular organisms, morphogenesis translates processes at the cellular scale into tissue deformation at the scale of organs and organisms. To understand how biochemical signaling regulates tissue form and function, we must understand the mechanical forces that shape cells and tissues. Recent progress in developing mechanical models for tissues has led to quantitative predictions for how cell shape changes and polarized cell motility generate forces and collective behavior on the tissue scale. In particular, much insight has been gained by thinking about biological tissues as physical materials composed of cells. Here we review these advances and discuss how they might help shape future experiments in developmental biology. Copyright © 2016 Elsevier Ltd. All rights reserved.

Active mechanical stabilization of the viscoplastic intracellular space of Dictyostelia cells by microtubule-actin crosstalk.

PubMed

Heinrich, Doris; Sackmann, Erich

2006-11-01

The micro-viscoelasticity of the intracellular space of Dictyostelium discoideum cells is studied by evaluating the intracellular transport of magnetic force probes and their viscoelastic responses to force pulses of 20-700 pN. The role of the actin cortex, the microtubule (MT) aster and their crosstalk is explored by comparing the behaviour of wild-type cells, myosin II null mutants, latrunculin A and benomyl treated cells. The MT coupled beads perform irregular local and long range directed motions which are characterized by measuring their velocity distributions (P(v)). The correlated motion of the MT and the centrosome are evaluated by microfluorescence of GFP-labelled MTs. P(v) can be represented by log-normal distributions with long tails and it is determined by random sweeping motions (v approximately 0.5 microm/s) of the MTs (caused by tangential forces on the filament ends coupled to the actin cortex) and by intermittent bead transports parallel to the MTs (v(max) approximately 1.5 microm/s). The tails are due to spontaneous filament deflections (with speeds up to 10 microm/s) attributed to pre-stressing of the MT by local cortical tensions, generated by dynactin motors generating plus-end directed forces in the MTs. The viscoelastic responses are strongly non-linear and are mostly directed opposite or perpendicular to the force, showing that the cytoplasm behaves as an active viscoplastic body with time and force dependent drag coefficients. Nano-Newton loads exerted on the soft MT are balanced by traction forces arising at the MT ends coupled to the actin cortex and the centrosome, respectively. The mechanical coupling between the soft microtubules and the viscoelastic actin cortex provides cells with high mechanical stability despite the softness of the cytoplasm.

Alpha-actinin binding kinetics modulate cellular dynamics and force generation

PubMed Central

Ehrlicher, Allen J.; Krishnan, Ramaswamy; Guo, Ming; Bidan, Cécile M.; Weitz, David A.; Pollak, Martin R.

2015-01-01

The actin cytoskeleton is a key element of cell structure and movement whose properties are determined by a host of accessory proteins. Actin cross-linking proteins create a connected network from individual actin filaments, and though the mechanical effects of cross-linker binding affinity on actin networks have been investigated in reconstituted systems, their impact on cellular forces is unknown. Here we show that the binding affinity of the actin cross-linker α-actinin 4 (ACTN4) in cells modulates cytoplasmic mobility, cellular movement, and traction forces. Using fluorescence recovery after photobleaching, we show that an ACTN4 mutation that causes human kidney disease roughly triples the wild-type binding affinity of ACTN4 to F-actin in cells, increasing the dissociation time from 29 ± 13 to 86 ± 29 s. This increased affinity creates a less dynamic cytoplasm, as demonstrated by reduced intracellular microsphere movement, and an approximate halving of cell speed. Surprisingly, these less motile cells generate larger forces. Using traction force microscopy, we show that increased binding affinity of ACTN4 increases the average contractile stress (from 1.8 ± 0.7 to 4.7 ± 0.5 kPa), and the average strain energy (0.4 ± 0.2 to 2.1 ± 0.4 pJ). We speculate that these changes may be explained by an increased solid-like nature of the cytoskeleton, where myosin activity is more partitioned into tension and less is dissipated through filament sliding. These findings demonstrate the impact of cross-linker point mutations on cell dynamics and forces, and suggest mechanisms by which such physical defects lead to human disease. PMID:25918384

Quantifying the effect of electric current on cell adhesion studied by single-cell force spectroscopy.

PubMed

Jaatinen, Leena; Young, Eleanore; Hyttinen, Jari; Vörös, János; Zambelli, Tomaso; Demkó, László

2016-03-20

This study presents the effect of external electric current on the cell adhesive and mechanical properties of the C2C12 mouse myoblast cell line. Changes in cell morphology, viability, cytoskeleton, and focal adhesion structure were studied by standard staining protocols, while single-cell force spectroscopy based on the fluidic force microscopy technology provided a rapid, serial quantification and detailed analysis of cell adhesion and its dynamics. The setup allowed measurements of adhesion forces up to the μN range, and total detachment distances over 40 μm. Force-distance curves have been fitted with a simple elastic model including a cell detachment protocol in order to estimate the Young's modulus of the cells, as well as to reveal changes in the dynamic properties as functions of the applied current dose. While the cell spreading area decreased monotonously with increasing current doses, small current doses resulted only in differences related to cell elasticity. Current doses above 11 As/m(2), however, initiated more drastic changes in cell morphology, viability, cellular structure, as well as in properties related to cell adhesion. The observed differences, eventually leading to cell death toward higher doses, might originate from both the decrease in pH and the generation of reactive oxygen species.

Acoustic manipulation of bacteria cells suspensions

NASA Astrophysics Data System (ADS)

GutiéRrez-Ramos, Salomé; Hoyos, Mauricio; Aider, Jean Luc; Ruiz, Carlos; Acoustofluidics Team Team; Soft; Bio Group Collaboration

An acoustic contacless manipulation gives advantages in the exploration of the complex dynamics enviroment that active matter exhibits. Our works reports the control confinement and dispersion of Escherichia coliRP437-pZA3R-YFP suspensions (M9Glu-Ca) via acoustic levitation.The manipulation of the bacteria bath in a parallel plate resonator is achieved using the acoustic radiation force and the secondary radiation force. The primary radiation force generates levitation of the bacteria cells at the nodal plane of the ultrasonic standing wave generated inside the resonator. On the other side, secondary forces leads to the consolidation of stable aggregates. All the experiments were performed in the acoustic trap described, where we excite the emission plate with a continuous sinusoidal signal at a frequency in the order of MHz and a quartz slide as the reflector plate. In a typical experiment we observed that, before the input of the signal, the bacteria cells exhibit their typical run and tumble behavior and after the sound is turned on all of them displace towards the nodal plane, and instantaneously the aggregation begins in this region. CNRS French National Space Studies, CONACYT Mexico.

Dissipation of contractile forces: the missing piece in cell mechanics.

PubMed

Kurzawa, Laetitia; Vianay, Benoit; Senger, Fabrice; Vignaud, Timothée; Blanchoin, Laurent; Théry, Manuel

2017-07-07

Mechanical forces are key regulators of cell and tissue physiology. The basic molecular mechanism of fiber contraction by the sliding of actin filament upon myosin leading to conformational change has been known for decades. The regulation of force generation at the level of the cell, however, is still far from elucidated. Indeed, the magnitude of cell traction forces on the underlying extracellular matrix in culture is almost impossible to predict or experimentally control. The considerable variability in measurements of cell-traction forces indicates that they may not be the optimal readout to properly characterize cell contractile state and that a significant part of the contractile energy is not transferred to cell anchorage but instead is involved in actin network dynamics. Here we discuss the experimental, numerical, and biological parameters that may be responsible for the variability in traction force production. We argue that limiting these sources of variability and investigating the dissipation of mechanical work that occurs with structural rearrangements and the disengagement of force transmission is key for further understanding of cell mechanics. © 2017 Kurzawa et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Transient ectopic expression of the histone demethylase JMJD3 accelerates the differentiation of human pluripotent stem cells

PubMed Central

Wakabayashi, Shunichi; Soma, Atsumi; Sato, Saeko; Nakatake, Yuhki; Oda, Mayumi; Murakami, Miyako; Sakota, Miki; Chikazawa-Nohtomi, Nana

2016-01-01

Harnessing epigenetic regulation is crucial for the efficient and proper differentiation of pluripotent stem cells (PSCs) into desired cell types. Histone H3 lysine 27 trimethylation (H3K27me3) functions as a barrier against cell differentiation through the suppression of developmental gene expression in PSCs. Here, we have generated human PSC (hPSC) lines in which genome-wide reduction of H3K27me3 can be induced by ectopic expression of the catalytic domain of the histone demethylase JMJD3 (called JMJD3c). We found that transient, forced demethylation of H3K27me3 alone triggers the upregulation of mesoendodermal genes, even when the culture conditions for the hPSCs are not changed. Furthermore, transient and forced expression of JMJD3c followed by the forced expression of lineage-defining transcription factors enabled the hPSCs to activate tissue-specific genes directly. We have also shown that the introduction of JMJD3c facilitates the differentiation of hPSCs into functional hepatic cells and skeletal muscle cells. These results suggest the utility of the direct manipulation of epigenomes for generating desired cell types from hPSCs for cell transplantation therapy and platforms for drug screenings. PMID:27802135

How well can an amoeba climb?

PubMed Central

Fukui, Yoshio; Uyeda, Taro Q. P.; Kitayama, Chikako; Inoué, Shinya

2000-01-01

We report here our efforts to measure the crawling force generated by cells undergoing amoeboid locomotion. In a centrifuge microscope, acceleration was increased until amoebae of Dictyostelium discoideum were “stalled” or no longer able to “climb up.” The “apparent weight” of the amoebae at stalling rpm in myosin mutants depended on the presence of myosin II (but not myosins IA and IB) and paralleled the cortical strength of the cells. Surprisingly, however, the cell stalled not only in low-density media as expected but also in media with densities greater than the cell density where the buoyant force should push the amoeba upward. We find that the leading pseudopod is bent under centrifugal force in all stalled amoebae, suggesting that this pseudopod is very dense indeed. This finding also suggests that directional cell locomotion against resistive forces requires a turgid forward-pointing pseudopod, most likely sustained by cortical actomyosin II. PMID:10963666

MRCK-1 drives apical constriction in C. elegans by linking developmental patterning to force generation

PubMed Central

Marston, Daniel J.; Higgins, Christopher D.; Peters, Kimberly A.; Cupp, Timothy D.; Dickinson, Daniel J.; Pani, Ariel M.; Moore, Regan P.; Cox, Amanda H.; Kiehart, Daniel P.; Goldstein, Bob

2016-01-01

Summary Apical constriction is a change in cell shape that drives key morphogenetic events including gastrulation and neural tube formation. Apical force-producing actomyosin networks drive apical constriction by contracting while connected to cell-cell junctions. The mechanisms by which developmental patterning regulates these actomyosin networks and associated junctions with spatial precision are not fully understood. Here, we identify a myosin light chain kinase MRCK-1 as a key regulator of C. elegans gastrulation that integrates spatial and developmental patterning information. We show that MRCK-1 is required for activation of contractile actomyosin dynamics and elevated cortical tension in the apical cell cortex of endodermal precursor cells. MRCK-1 is apically localized by active Cdc42 at the external, cell-cell contact-free surfaces of apically constricting cells, downstream of cell fate determination mechanisms. We establish that the junctional components α-catenin, β-catenin, and cadherin become highly enriched at the apical junctions of apically-constricting cells, and that MRCK-1 and myosin activity are required in vivo for this enrichment. Taken together, our results define mechanisms that position a myosin activator to a specific cell surface where it both locally increases cortical tension and locally enriches junctional components to facilitate apical constriction. These results reveal crucial links that can tie spatial information to local force generation to drive morphogenesis. PMID:27451898

Engineering the Intracellular Micro- and Nano-environment via Magnetic Nanoparticles

NASA Astrophysics Data System (ADS)

Tseng, Peter

Single cells, despite being the base unit of living organisms, possess a high degree of hierarchical structure and functional compartmentalization. This complexity exists for good reason: cells must respond efficiently and effectively to its surrounding environment by differentiating, moving, interacting, and more in order to survive or inhabit its role in the larger biological system. At the core of these responses is cellular decision-making. Cells process cues internally and externally from the environment and effect intracellular asymmetry in biochemistry and structure in order to carry out the proper biological responses. Functionalized magnetic particles have shown to be a powerful tool in interacting with biological matter, through either cell or biomolecule sorting, and the activation of biological processes. This dissertation reports on techniques utilizing manipulated magnetic nanoparticles (internalized by cells) to spatially and temporally localize intracellular cues, and examines the resulting asymmetry in biological processes generated by our methods. We first examine patterned micromagnetic elements as a simple strategy of rapidly manipulating magnetic nanoparticles throughout the intracellular space. Silicon or silicon dioxide substrates form the base for electroplated NiFe rods, which are repeated at varying size and pitch. A planarizing resin, initially SU-8, is used as the substrate layer for cellular adhesion. We demonstrate that through the manipulations of a simple external magnet, these micro-fabricated substrates can mediate rapid (under 2 s) and precise (submicron), reversible translation of magnetic nanoparticles through cellular space. Seeding cells on substrates composed of these elements allows simultaneous control of ensembles of nanoparticles over thousands of cells at a time. We believe such substrates could form the basis of magnetically based tools for the activation of biological matter. We further utilize these strategies to generate user-controllable (time-varying and localizable), massively parallel forces on arrays of cells mediated by coalesced ensembles of magnetic nanoparticles. The above process is simplified and adapted for single cell analysis by precisely aligning fibronectin patterned cells to a single flanking micromagnet. The cells are loaded with magnetic-fluorescent nanoparticles, which are then localized to uniform positions at the internal edge of the cell membrane over huge arrays of cells using large external fields, allowing us to conduct composed studies on cellular response to force. By applying forces approaching the yield tension (5 nN / mum) of single cells, we are able to generate highly coordinated responses in cellular behavior. We discover that increasing tension generates highly directed, PAK-dependent leading-edge type filopodia that increase in intensity with rising tension. In addition, we find that our generated forces can simulate cues created during cellular mitosis, as we are consistently able to generate significant (45 to 90 degree) biasing of the metaphase plate during cell division. Large sample size and rapid sample generation also allow us to analyze cells at an unprecedented rate---a single sample can simultaneously stimulate thousands of cells for high statistical accuracy in measurements. We believe these approaches have potential not just as a tool to study single-cell response, but as a means of cell control, potentially through modifying cell movement, division, or differentiation. More generally, once approaches to release nanoparticles from endosomes are implemented, the technique provides a platform to dynamically apply a range of localized stimuli arbitrarily within cells. Through the bioconjugation of proteins, nucleic acids, small molecules, or whole organelles a broad range of questions should be accessible concerning molecular localization and its importance in cell function.

Imaging viscoelastic properties of live cells by AFM: power-law rheology on the nanoscale.

PubMed

Hecht, Fabian M; Rheinlaender, Johannes; Schierbaum, Nicolas; Goldmann, Wolfgang H; Fabry, Ben; Schäffer, Tilman E

2015-06-21

We developed force clamp force mapping (FCFM), an atomic force microscopy (AFM) technique for measuring the viscoelastic creep behavior of live cells with sub-micrometer spatial resolution. FCFM combines force-distance curves with an added force clamp phase during tip-sample contact. From the creep behavior measured during the force clamp phase, quantitative viscoelastic sample properties are extracted. We validate FCFM on soft polyacrylamide gels. We find that the creep behavior of living cells conforms to a power-law material model. By recording short (50-60 ms) force clamp measurements in rapid succession, we generate, for the first time, two-dimensional maps of power-law exponent and modulus scaling parameter. Although these maps reveal large spatial variations of both parameters across the cell surface, we obtain robust mean values from the several hundreds of measurements performed on each cell. Measurements on mouse embryonic fibroblasts show that the mean power-law exponents and the mean modulus scaling parameters differ greatly among individual cells, but both parameters are highly correlated: stiffer cells consistently show a smaller power-law exponent. This correlation allows us to distinguish between wild-type cells and cells that lack vinculin, a dominant protein of the focal adhesion complex, even though the mean values of viscoelastic properties between wildtype and knockout cells did not differ significantly. Therefore, FCFM spatially resolves viscoelastic sample properties and can uncover subtle mechanical signatures of proteins in living cells.

Feedback-tracking microrheology in living cells

PubMed Central

Nishizawa, Kenji; Bremerich, Marcel; Ayade, Heev; Schmidt, Christoph F.; Ariga, Takayuki; Mizuno, Daisuke

2017-01-01

Living cells are composed of active materials, in which forces are generated by the energy derived from metabolism. Forces and structures self-organize to shape the cell and drive its dynamic functions. Understanding the out-of-equilibrium mechanics is challenging because constituent materials, the cytoskeleton and the cytosol, are extraordinarily heterogeneous, and their physical properties are strongly affected by the internally generated forces. We have analyzed dynamics inside two types of eukaryotic cells, fibroblasts and epithelial-like HeLa cells, with simultaneous active and passive microrheology using laser interferometry and optical trapping technology. We developed a method to track microscopic probes stably in cells in the presence of vigorous cytoplasmic fluctuations, by using smooth three-dimensional (3D) feedback of a piezo-actuated sample stage. To interpret the data, we present a theory that adapts the fluctuation-dissipation theorem (FDT) to out-of-equilibrium systems that are subjected to positional feedback, which introduces an additional nonequilibrium effect. We discuss the interplay between material properties and nonthermal force fluctuations in the living cells that we quantify through the violations of the FDT. In adherent fibroblasts, we observed a well-known polymer network viscoelastic response where the complex shear modulus scales as G* ∝ (−iω)3/4. In the more 3D confluent epithelial cells, we found glassy mechanics with G* ∝ (−iω)1/2 that we attribute to glassy dynamics in the cytosol. The glassy state in living cells shows characteristics that appear distinct from classical glasses and unique to nonequilibrium materials that are activated by molecular motors. PMID:28975148

Emergent behavior of cells on microfabricated soft polymeric substrates

NASA Astrophysics Data System (ADS)

Anand, Sandeep Venkit

In recent years, cell based bio-actuators like cardiomyocytes and skeletal muscle cells have emerged as popular choices for powering biological machines consisting of soft polymeric scaffolds at the micro and macro scales. This is owing to their unique ability to generate spontaneous, synchronous contractions either autonomously or under externally applied fields. Most of the biological machine designs reported in literature use single cells or cell clusters conjugated with biocompatible soft polymers like polydimethylsiloxane (PDMS) and hydrogels to produce some form of locomotion by converting chemical energy of the cells to mechanical energy. The mode of locomotion may vary, but the fundamental mechanism that these biological machines exploit to achieve locomotion stems from cell substrate interactions leading to large deformations of the substrates (relative to the cell size). However, the effect of such large scale, dynamic deformation of the substrates on the cellular and cluster level organization of the cells remains elusive. This dissertation tries to explore the emergent behavior of cells on different types of micro-scale deformable, soft polymeric substrates. In the first part of the dissertation, contractile dynamics of primary cardiomyocyte clusters is studied by culturing them on deformable thin polymeric films. The cell clusters beat and generate sufficient forces to deform the substrates out of plane. Over time, the clusters reorient their force dipoles along the direction of maximum compliance. This suggests that the cells are capable of sensing substrate deformations through a mechanosensitive feedback mechanism and dynamically reorganizing themselves. Results are further validated through finite element analysis. The development, characterization and quantification of a novel 1D/2D like polymeric platform for cell culture is presented in the second part. The platform consists of a 2D surface anchoring a long (few millimeters) narrow filament (1D) with a single cell scale (micro scale) cross section. We plate C2C12 cells on the platform and characterize their migration, proliferation, and differentiation patterns in contrast to 2D culture. We find that the cells land on the 2D surface, and then migrate to the filament only when the 2D surface has become nearly confluent. Individual and isolated cells randomly approaching the filament always retract away towards the 2D surface. Once on the filament, their differentiation to myotubes is expedited compared to that on 2D substrate. The myotubes generate periodic twitching forces that deform the filament producing more than 17 um displacement at the tip. Such flagellar motion can be used to develop autonomous micro scale bio-bots. Finally, the design and fabrication of a polymeric micro-pillar based force sensor capable of measuring cellular focal-adhesion forces under externally applied stretch is discussed. The force sensor consists of arrays of uniformly spaced PDMS micro-pillars of 1-2 um diameter and 2-3 um spacing on a macroscale PDMS substrate. The tips of the micro-pillars are selectively patterned with fluorescently labeled ECM proteins using micro-contact printing to promote cell adhesion while simultaneously acting as markers for strain measurements. Cells adhere and spread on top of the pillars causing them to deform. When stretched, the cells reorganize their internal structure and modulate their traction forces in response to the applied stretch. The dynamically varying cellular forces in response to the stretch are computed by measuring the cell induced displacements estimated by isolating the displacements caused by the applied stretch from the net displacements of the tips.

Contractile and mechanical properties of epithelia with perturbed actomyosin dynamics.

PubMed

Fischer, Sabine C; Blanchard, Guy B; Duque, Julia; Adams, Richard J; Arias, Alfonso Martinez; Guest, Simon D; Gorfinkiel, Nicole

2014-01-01

Mechanics has an important role during morphogenesis, both in the generation of forces driving cell shape changes and in determining the effective material properties of cells and tissues. Drosophila dorsal closure has emerged as a reference model system for investigating the interplay between tissue mechanics and cellular activity. During dorsal closure, the amnioserosa generates one of the major forces that drive closure through the apical contraction of its constituent cells. We combined quantitation of live data, genetic and mechanical perturbation and cell biology, to investigate how mechanical properties and contraction rate emerge from cytoskeletal activity. We found that a decrease in Myosin phosphorylation induces a fluidization of amnioserosa cells which become more compliant. Conversely, an increase in Myosin phosphorylation and an increase in actin linear polymerization induce a solidification of cells. Contrary to expectation, these two perturbations have an opposite effect on the strain rate of cells during DC. While an increase in actin polymerization increases the contraction rate of amnioserosa cells, an increase in Myosin phosphorylation gives rise to cells that contract very slowly. The quantification of how the perturbation induced by laser ablation decays throughout the tissue revealed that the tissue in these two mutant backgrounds reacts very differently. We suggest that the differences in the strain rate of cells in situations where Myosin activity or actin polymerization is increased arise from changes in how the contractile forces are transmitted and coordinated across the tissue through ECadherin-mediated adhesion. Altogether, our results show that there is an optimal level of Myosin activity to generate efficient contraction and suggest that the architecture of the actin cytoskeleton and the dynamics of adhesion complexes are important parameters for the emergence of coordinated activity throughout the tissue.

Contractile and Mechanical Properties of Epithelia with Perturbed Actomyosin Dynamics

PubMed Central

Fischer, Sabine C.; Blanchard, Guy B.; Duque, Julia; Adams, Richard J.; Arias, Alfonso Martinez; Guest, Simon D.; Gorfinkiel, Nicole

2014-01-01

Mechanics has an important role during morphogenesis, both in the generation of forces driving cell shape changes and in determining the effective material properties of cells and tissues. Drosophila dorsal closure has emerged as a reference model system for investigating the interplay between tissue mechanics and cellular activity. During dorsal closure, the amnioserosa generates one of the major forces that drive closure through the apical contraction of its constituent cells. We combined quantitation of live data, genetic and mechanical perturbation and cell biology, to investigate how mechanical properties and contraction rate emerge from cytoskeletal activity. We found that a decrease in Myosin phosphorylation induces a fluidization of amnioserosa cells which become more compliant. Conversely, an increase in Myosin phosphorylation and an increase in actin linear polymerization induce a solidification of cells. Contrary to expectation, these two perturbations have an opposite effect on the strain rate of cells during DC. While an increase in actin polymerization increases the contraction rate of amnioserosa cells, an increase in Myosin phosphorylation gives rise to cells that contract very slowly. The quantification of how the perturbation induced by laser ablation decays throughout the tissue revealed that the tissue in these two mutant backgrounds reacts very differently. We suggest that the differences in the strain rate of cells in situations where Myosin activity or actin polymerization is increased arise from changes in how the contractile forces are transmitted and coordinated across the tissue through ECadherin-mediated adhesion. Altogether, our results show that there is an optimal level of Myosin activity to generate efficient contraction and suggest that the architecture of the actin cytoskeleton and the dynamics of adhesion complexes are important parameters for the emergence of coordinated activity throughout the tissue. PMID:24759936

Generation of hair cells in neonatal mice by β-catenin overexpression in Lgr5-positive cochlear progenitors

PubMed Central

Shi, Fuxin; Hu, Lingxiang; Edge, Albert S. B.

2013-01-01

Mammalian hair cells do not regenerate, and their loss is a major cause of deafness. We recently identified leucine-rich repeat containing, G-protein-coupled receptor 5 (Lgr5)-expressing cochlear supporting cells with the capacity for self-renewal and hair cell differentiation in vitro. We found that these cells, a subset of cochlear supporting cells, were responsive to Wnt signaling. Here we asked whether these Lgr5-positive cells, despite their lack of contribution to hair cell replacement after degenerative loss, could be driven by forced expression of β-catenin to act as hair cell progenitors in vivo. We showed that forced stabilization of β-catenin in supporting cells in neonatal animals resulted in proliferation of supporting cells and generation of hair cells. Although β-catenin expression was increased by genetic means in all supporting cells, entry to the cell cycle and differentiation to hair cells of the normally postmitotic cells was restricted to the Lgr5-positive population. Our finding suggests that Wnt/β-catenin can drive Lgr5-positive cells to act as hair cell progenitors, even after their exit from the cell cycle and apparent establishment of cell fate. PMID:23918377

Understanding force-generating microtubule systems through in vitro reconstitution

PubMed Central

Kok, Maurits; Dogterom, Marileen

2016-01-01

ABSTRACT Microtubules switch between growing and shrinking states, a feature known as dynamic instability. The biochemical parameters underlying dynamic instability are modulated by a wide variety of microtubule-associated proteins that enable the strict control of microtubule dynamics in cells. The forces generated by controlled growth and shrinkage of microtubules drive a large range of processes, including organelle positioning, mitotic spindle assembly, and chromosome segregation. In the past decade, our understanding of microtubule dynamics and microtubule force generation has progressed significantly. Here, we review the microtubule-intrinsic process of dynamic instability, the effect of external factors on this process, and how the resulting forces act on various biological systems. Recently, reconstitution-based approaches have strongly benefited from extensive biochemical and biophysical characterization of individual components that are involved in regulating or transmitting microtubule-driven forces. We will focus on the current state of reconstituting increasingly complex biological systems and provide new directions for future developments. PMID:27715396

Mechanical Coordination of Single-Cell and Collective-Cell Amoeboid Migration

NASA Astrophysics Data System (ADS)

Del Alamo, Juan Carlos

Amoeboid migration consists of the sequential repetition of pseudopod extensions and retractions driven by actin polymerization and actomyosin contraction, and requires cells to apply mechanical forces on their surroundings. We measure the three-dimensional forces exerted by chemotaxing Dictyostelium cells, and examine wild-type cells as well as mutants with defects in contractility, F-actin polymerization, internal F-actin crosslinking, and cortical integrity. We find that cells pull on their substrate adhesions using two distinct, yet interconnected mechanisms: axial actomyosin contractility and cortical tension. The 3D pulling forces generated by both mechanisms are internally balanced by an increase in cytoplasmic pressure that allows cells to push on their substrate, and we show that these pushing forces are relevant for cell invasion and migration in three-dimensional environments. We observe that cells migrate mainly by forming two stationary adhesion sites at the front and back of the cell, over which the cell body moves forward in a step-wise fashion. During this process, the traction forces at each adhesion site are switched off and subsequently their direction is reversed. The cell migration speed is found to be proportional to the rate at which cells are able regulate these forces to produce the cell shape changes needed for locomotion, which is increased when axial contractility overcomes the stabilizing effect of cortical tension. This spatiotemporal coordination is conserved in streams of multiple migratory cells connected head to tail, which also migrate by exerting traction forces on stationary sites. Furthermore, we observe that trailing cells reuse the adhesion sites of the leading cells. Finally, we provide evidence that the above modes of migration may be conserved in a range of other amoeboid-type moving cells such as neutrophils.

Motility, Force Generation, and Energy Consumption of Unicellular Parasites.

PubMed

Hochstetter, Axel; Pfohl, Thomas

2016-07-01

Motility is a key factor for pathogenicity of unicellular parasites, enabling them to infiltrate and evade host cells, and perform several of their life-cycle events. State-of-the-art methods of motility analysis rely on a combination of optical tweezers with high-resolution microscopy and microfluidics. With this technology, propulsion forces, energies, and power generation can be determined so as to shed light on the motion mechanisms, chemotactic behavior, and specific survival strategies of unicellular parasites. With these new tools in hand, we can elucidate the mechanisms of motility and force generation of unicellular parasites, and identify ways to manipulate and eventually inhibit them. Copyright © 2016 Elsevier Ltd. All rights reserved.

Friction forces position the neural anlage

PubMed Central

Smutny, Michael; Ákos, Zsuzsa; Grigolon, Silvia; Shamipour, Shayan; Ruprecht, Verena; Čapek, Daniel; Behrndt, Martin; Papusheva, Ekaterina; Tada, Masazumi; Hof, Björn; Vicsek, Tamás; Salbreux, Guillaume; Heisenberg, Carl-Philipp

2017-01-01

During embryonic development, mechanical forces are essential for cellular rearrangements driving tissue morphogenesis. Here, we show that in the early zebrafish embryo, friction forces are generated at the interface between anterior axial mesoderm (prechordal plate, ppl) progenitors migrating towards the animal pole and neurectoderm progenitors moving in the opposite direction towards the vegetal pole of the embryo. These friction forces lead to global rearrangement of cells within the neurectoderm and determine the position of the neural anlage. Using a combination of experiments and simulations, we show that this process depends on hydrodynamic coupling between neurectoderm and ppl as a result of E-cadherin-mediated adhesion between those tissues. Our data thus establish the emergence of friction forces at the interface between moving tissues as a critical force-generating process shaping the embryo. PMID:28346437

Friction forces position the neural anlage.

PubMed

Smutny, Michael; Ákos, Zsuzsa; Grigolon, Silvia; Shamipour, Shayan; Ruprecht, Verena; Čapek, Daniel; Behrndt, Martin; Papusheva, Ekaterina; Tada, Masazumi; Hof, Björn; Vicsek, Tamás; Salbreux, Guillaume; Heisenberg, Carl-Philipp

2017-04-01

During embryonic development, mechanical forces are essential for cellular rearrangements driving tissue morphogenesis. Here, we show that in the early zebrafish embryo, friction forces are generated at the interface between anterior axial mesoderm (prechordal plate, ppl) progenitors migrating towards the animal pole and neurectoderm progenitors moving in the opposite direction towards the vegetal pole of the embryo. These friction forces lead to global rearrangement of cells within the neurectoderm and determine the position of the neural anlage. Using a combination of experiments and simulations, we show that this process depends on hydrodynamic coupling between neurectoderm and ppl as a result of E-cadherin-mediated adhesion between those tissues. Our data thus establish the emergence of friction forces at the interface between moving tissues as a critical force-generating process shaping the embryo.

A new technique for calculating individual dermal fibroblast contractile forces generated within collagen-GAG scaffolds.

PubMed

Harley, Brendan A; Freyman, Toby M; Wong, Matthew Q; Gibson, Lorna J

2007-10-15

Cell-mediated contraction plays a critical role in many physiological and pathological processes, notably organized contraction during wound healing. Implantation of an appropriately formulated (i.e., mean pore size, chemical composition, degradation rate) three-dimensional scaffold into an in vivo wound site effectively blocks the majority of organized wound contraction and results in induced regeneration rather than scar formation. Improved understanding of cell contraction within three-dimensional constructs therefore represents an important area of study in tissue engineering. Studies of cell contraction within three-dimensional constructs typically calculate an average contractile force from the gross deformation of a macroscopic substrate by a large cell population. In this study, cellular solids theory has been applied to conventional column buckling relationships to quantify the magnitude of individual cell contraction events within a three-dimensional, collagen-glycosaminoglycan scaffold. This new technique can be used for studying cell mechanics with a wide variety of porous scaffolds that resemble low-density, open-cell foams. It extends previous methods for analyzing cell buckling of two-dimensional substrates to three-dimensional constructs. From data available in the literature, the mean contractile force (Fc) generated by individual dermal fibroblasts within the collagen-glycosaminoglycan scaffold was calculated to range between 11 and 41 nN (Fc=26+/-13 nN, mean+/-SD), with an upper bound of cell contractility estimated at 450 nN.

Study of Chemotaxis and Cell–Cell Interactions in Cancer with Microfluidic Devices

PubMed Central

Sai, Jiqing; Rogers, Matthew; Hockemeyer, Kathryn; Wikswo, John P.; Richmond, Ann

2017-01-01

Microfluidic devices have very broad applications in biological assays from simple chemotaxis assays to much more complicated 3D bioreactors. In this chapter, we describe the design and methods for performing chemotaxis assays using simple microfluidic chemotaxis chambers. With these devices, using real-time video microscopy we can examine the chemotactic responses of neutrophil-like cells under conditions of varying gradient steepness or flow rate and then utilize software programs to calculate the speed and angles of cell migration as gradient steepness and flow are varied. Considering the shearing force generated on the cells by the constant flow that is required to produce and maintain a stable gradient, the trajectories of the cell migration will reflect the net result of both shear force generated by flow and the chemotactic force resulting from the chemokine gradient. Moreover, the effects of mutations in chemokine receptors or the presence of inhibitors of intracellular signals required for gradient sensing can be evaluated in real time. We also describe a method to monitor intracellular signals required for cells to alter cell polarity in response to an abrupt switch in gradient direction. Lastly, we demonstrate an in vitro method for studying the interactions of human cancer cells with human endothelial cells, fibroblasts, and leukocytes, as well as environmental chemokines and cytokines, using 3D microbioreactors that mimic the in vivo microenvironment. PMID:26921940

Cell origami: self-folding of three-dimensional cell-laden microstructures driven by cell traction force.

PubMed

Kuribayashi-Shigetomi, Kaori; Onoe, Hiroaki; Takeuchi, Shoji

2012-01-01

This paper describes a method of generating three-dimensional (3D) cell-laden microstructures by applying the principle of origami folding technique and cell traction force (CTF). We harness the CTF as a biological driving force to fold the microstructures. Cells stretch and adhere across multiple microplates. Upon detaching the microplates from a substrate, CTF causes the plates to lift and fold according to a prescribed pattern. This self-folding technique using cells is highly biocompatible and does not involve special material requirements for the microplates and hinges to induce folding. We successfully produced various 3D cell-laden microstructures by just changing the geometry of the patterned 2D plates. We also achieved mass-production of the 3D cell-laden microstructures without causing damage to the cells. We believe that our methods will be useful for biotechnology applications that require analysis of cells in 3D configurations and for self-assembly of cell-based micro-medical devices.

A theoretical and computational framework for mechanics of the cortex

NASA Astrophysics Data System (ADS)

Torres-SáNchez, Alejandro; Arroyo, Marino

The cell cortex is a thin network of actin filaments lying beneath the cell surface of animal cells. Myosin motors exert contractile forces in this network leading to active stresses, which play a key role in processes such as cytokinesis or cell migration. Thus, understanding the mechanics of the cortex is fundamental to understand the mechanics of animal cells. Due to the dynamic remodeling of the actin network, the cortex behaves as a viscoelastic fluid. Furthermore, due to the difference between its thickness (tens of nanometers) and its dimensions (tens of microns), the cortex can be regarded a surface. Thus, we can model the cortex as a viscoelastic fluid, confined to a surface, that generates active stresses. Interestingly, geometric confinement results in the coupling between shape generation and material flows. In this work we present a theoretical framework to model the mechanics of the cortex that couples elasticity, hydrodynamics and force generation. We complement our theoretical description with a computational setting to simulate the resulting non-linear equations. We use this methodology to understand different processes such as asymmetric cell division or experimental probing of the rheology of the cortex We acknowledge the support of the Europen Research Council through Grant ERC CoG-681434.

Fibrous nonlinear elasticity enables positive mechanical feedback between cells and ECMs

PubMed Central

Hall, Matthew S.; Alisafaei, Farid; Ban, Ehsan; Feng, Xinzeng; Hui, Chung-Yuen; Shenoy, Vivek B.; Wu, Mingming

2016-01-01

In native states, animal cells of many types are supported by a fibrous network that forms the main structural component of the ECM. Mechanical interactions between cells and the 3D ECM critically regulate cell function, including growth and migration. However, the physical mechanism that governs the cell interaction with fibrous 3D ECM is still not known. In this article, we present single-cell traction force measurements using breast tumor cells embedded within 3D collagen matrices. We recreate the breast tumor mechanical environment by controlling the microstructure and density of type I collagen matrices. Our results reveal a positive mechanical feedback loop: cells pulling on collagen locally align and stiffen the matrix, and stiffer matrices, in return, promote greater cell force generation and a stiffer cell body. Furthermore, cell force transmission distance increases with the degree of strain-induced fiber alignment and stiffening of the collagen matrices. These findings highlight the importance of the nonlinear elasticity of fibrous matrices in regulating cell–ECM interactions within a 3D context, and the cell force regulation principle that we uncover may contribute to the rapid mechanical tissue stiffening occurring in many diseases, including cancer and fibrosis. PMID:27872289

Spatial differences of cellular origins and in vivo hypoxia modify contractile properties of pulmonary artery smooth muscle cells: lessons for arterial tissue engineering.

PubMed

Hall, S M; Soueid, A; Smith, T; Brown, R A; Haworth, S G; Mudera, V

2007-01-01

Tissue engineering of functional arteries is challenging. Within the pulmonary artery wall, smooth muscle cells (PASMCs) have site-specific developmental and functional phenotypes, reflecting differing contractile roles. The force generated by PASMCs isolated from the inner 25% and outer 50% of the media of intrapulmonary elastic arteries from five normal and eight chronically hypoxic (hypertensive) 14 day-old piglets was quantified in a three-dimensional (3D) collagen construct, using a culture force monitor. Outer medial PASMCs from normal piglets exerted more force (528 +/- 50 dynes) than those of hypoxic piglets (177 +/- 42 dynes; p < 0.01). Force generation by inner medial PASMCs from normal and hypoxic piglets was similar (349 +/- 35 and 239 +/- 60 dynes). In response to agonist (thromboxane) stimulation, all PASMCs from normal and hypoxic piglets contracted, but the increase in force generated by outer and inner hypoxic PASMCs (ranges 13-72 and 14-56 dynes) was less than by normal PASMCs (ranges 27-154 and 34-159 dynes, respectively; p < 0.05 for both). All hypoxic PASMCs were unresponsive to antagonist (sodium nitroprusside) stimulation, all normal PASMCs relaxed (range - 87 to - 494 dynes). Myosin heavy chain expression by both hypoxic PASMC phenotypes was less than normal (p < 0.05 for both), as was the activity of focal adhesion kinase, regulating contraction, in hypoxic inner PASMCs (p < 0.01). Chronic hypoxia resulted in the development of abnormal PASMC phenotypes, which in collagen constructs exhibited a reduction in contractile force and reactivity to agonists. Characterization of the mechanical response of spatially distinct cells and modification of their behaviour by hypoxia is critical for successful tissue engineering of major blood vessels.

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix.

PubMed

Stout, David A; Toyjanova, Jennet; Franck, Christian

2015-06-12

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Characterizing the mechanics of cultured cell monolayers

PubMed Central

Peter, Loic; Bellis, Julien; Baum, Buzz; Kabla, Alexandre J.; Charras, Guillaume T.

2012-01-01

One-cell-thick monolayers are the simplest tissues in multicellular organisms, yet they fulfill critical roles in development and normal physiology. In early development, embryonic morphogenesis results largely from monolayer rearrangement and deformation due to internally generated forces. Later, monolayers act as physical barriers separating the internal environment from the exterior and must withstand externally applied forces. Though resisting and generating mechanical forces is an essential part of monolayer function, simple experimental methods to characterize monolayer mechanical properties are lacking. Here, we describe a system for tensile testing of freely suspended cultured monolayers that enables the examination of their mechanical behavior at multi-, uni-, and subcellular scales. Using this system, we provide measurements of monolayer elasticity and show that this is two orders of magnitude larger than the elasticity of their isolated cellular components. Monolayers could withstand more than a doubling in length before failing through rupture of intercellular junctions. Measurement of stress at fracture enabled a first estimation of the average force needed to separate cells within truly mature monolayers, approximately ninefold larger than measured in pairs of isolated cells. As in single cells, monolayer mechanical properties were strongly dependent on the integrity of the actin cytoskeleton, myosin, and intercellular adhesions interfacing adjacent cells. High magnification imaging revealed that keratin filaments became progressively stretched during extension, suggesting they participate in monolayer mechanics. This multiscale study of monolayer response to deformation enabled by our device provides the first quantitative investigation of the link between monolayer biology and mechanics. PMID:22991459

Shaping tissues by balancing active forces and geometric constraints

NASA Astrophysics Data System (ADS)

Foolen, Jasper; Yamashita, Tadahiro; Kollmannsberger, Philip

2016-02-01

The self-organization of cells into complex tissues during growth and regeneration is a combination of physical-mechanical events and biochemical signal processing. Cells actively generate forces at all stages in this process, and according to the laws of mechanics, these forces result in stress fields defined by the geometric boundary conditions of the cell and tissue. The unique ability of cells to translate such force patterns into biochemical information and vice versa sets biological tissues apart from any other material. In this topical review, we summarize the current knowledge and open questions of how forces and geometry act together on scales from the single cell to tissues and organisms, and how their interaction determines biological shape and structure. Starting with a planar surface as the simplest type of geometric constraint, we review literature on how forces during cell spreading and adhesion together with geometric constraints impact cell shape, stress patterns, and the resulting biological response. We then move on to include cell-cell interactions and the role of forces in monolayers and in collective cell migration, and introduce curvature at the transition from flat cell sheets to three-dimensional (3D) tissues. Fibrous 3D environments, as cells experience them in the body, introduce new mechanical boundary conditions and change cell behaviour compared to flat surfaces. Starting from early work on force transmission and collagen remodelling, we discuss recent discoveries on the interaction with geometric constraints and the resulting structure formation and network organization in 3D. Recent literature on two physiological scenarios—embryonic development and bone—is reviewed to demonstrate the role of the force-geometry balance in living organisms. Furthermore, the role of mechanics in pathological scenarios such as cancer is discussed. We conclude by highlighting common physical principles guiding cell mechanics, tissue patterning and matrix organization under geometric constraints across multiple length and time scales.

Biophysical Aspects of T Lymphocyte Activation at the Immune Synapse

PubMed Central

Hivroz, Claire; Saitakis, Michael

2016-01-01

T lymphocyte activation is a pivotal step of the adaptive immune response. It requires the recognition by T-cell receptors (TCR) of peptides presented in the context of major histocompatibility complex molecules (pMHC) present at the surface of antigen-presenting cells (APCs). T lymphocyte activation also involves engagement of costimulatory receptors and adhesion molecules recognizing ligands on the APC. Integration of these different signals requires the formation of a specialized dynamic structure: the immune synapse. While the biochemical and molecular aspects of this cell–cell communication have been extensively studied, its mechanical features have only recently been addressed. Yet, the immune synapse is also the place of exchange of mechanical signals. Receptors engaged on the T lymphocyte surface are submitted to many tensile and traction forces. These forces are generated by various phenomena: membrane undulation/protrusion/retraction, cell mobility or spreading, and dynamic remodeling of the actomyosin cytoskeleton inside the T lymphocyte. Moreover, the TCR can both induce force development, following triggering, and sense and convert forces into biochemical signals, as a bona fide mechanotransducer. Other costimulatory molecules, such as LFA-1, engaged during immune synapse formation, also display these features. Moreover, T lymphocytes themselves are mechanosensitive, since substrate stiffness can modulate their response. In this review, we will summarize recent studies from a biophysical perspective to explain how mechanical cues can affect T lymphocyte activation. We will particularly discuss how forces are generated during immune synapse formation; how these forces affect various aspects of T lymphocyte biology; and what are the key features of T lymphocyte response to stiffness. PMID:26913033

Coiled fiber scaffolds embedded with gold nanoparticles improve the performance of engineered cardiac tissues

NASA Astrophysics Data System (ADS)

Fleischer, Sharon; Shevach, Michal; Feiner, Ron; Dvir, Tal

2014-07-01

Coiled perimysial fibers within the heart muscle provide it with the ability to contract and relax efficiently. Here, we report on a new nanocomposite scaffold for cardiac tissue engineering, integrating coiled electrospun fibers with gold nanoparticles. Cultivation of cardiac cells within the hybrid scaffolds promoted cell organization into elongated and aligned tissues generating a strong contraction force, high contraction rate and low excitation threshold.Coiled perimysial fibers within the heart muscle provide it with the ability to contract and relax efficiently. Here, we report on a new nanocomposite scaffold for cardiac tissue engineering, integrating coiled electrospun fibers with gold nanoparticles. Cultivation of cardiac cells within the hybrid scaffolds promoted cell organization into elongated and aligned tissues generating a strong contraction force, high contraction rate and low excitation threshold. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr00300d

Internal Stresses Lead to Net Forces and Torques on Extended Elastic Bodies

NASA Astrophysics Data System (ADS)

Aharoni, Hillel; Kolinski, John M.; Moshe, Michael; Meirzada, Idan; Sharon, Eran

2016-09-01

A geometrically frustrated elastic body will develop residual stresses arising from the mismatch between the intrinsic geometry of the body and the geometry of the ambient space. We analyze these stresses for an ambient space with gradients in its intrinsic curvature, and show that residual stresses generate effective forces and torques on the center of mass of the body. We analytically calculate these forces in two dimensions, and experimentally demonstrate their action by the migration of a non-Euclidean gel disc in a curved Hele-Shaw cell. An extension of our analysis to higher dimensions shows that these forces are also generated in three dimensions, but are negligible compared to gravity.

Active turnover regulates pattern formation and stress transmission in disordered acto-myosin networks

NASA Astrophysics Data System (ADS)

McCall, Patrick; Stam, Samantha; Kovar, David; Gardel, Margaret

The shape and mechanics of animal cells are controlled by a dynamic, thin network of semiflexible actin filaments and myosin-II motor proteins called the actomyosin cortex. Motor-generated stresses in the cortex drive changes in cell shape during cell division and morphogenesis, while dynamic turnover of actin filaments dissipates stress. The relative effects that force generation, force dissipation, and disassembly and reassembly of material have on motion in these networks are unknown. We find that cross-linked actin networks in vitro contract under myosin-generated stresses, resulting in partial filament disassembly, the formation of asters, and clustering of myosin motors. We observe a rapid restoration of uniform polymer density in the presence of the assembly factors which catalyze network turnover through elongation of severed actin filaments. When severing is accelerated further by the addition of a severing protein, network contraction and motor clustering are dramatically suppressed. We test the relative effects of material regeneration and force transmission using image analysis, and conclude that the dominant mechanism for this effect is relatively short-lived stresses that do not propagate over considerable distance or push network deformation into the nonlinear contractile regime we have previously characterized. Our results present a framework to understand cytoskeletal active matter that are influenced by a complex interplay between stress generation, network reorganization, and polymer turnover.

Distinct Motion of GFP-Tagged Histone Expressing Cells Under AC Electrokinetics in Electrode-Multilayered Microfluidic Device.

PubMed

Yao, Jiafeng; Sugawara, Michiko; Obara, Hiromichi; Mizutani, Takeomi; Takei, Masahiro

2017-12-01

The distinct motion of GFP-tagged histone expressing cells (Histone-GFP type cells) has been investigated under ac electrokinetics in an electrode-multilayered microfluidic device as compared with Wild type cells and GFP type cells in terms of different intracellular components. The Histone-GFP type cells were modified by the transfection of green fluorescent protein-fused histone from the human lung fibroblast cell line. The velocity of the Histone-GFP type cells obtained by particle tracking velocimetry technique is faster than Wild type cells by 24.9% and GFP type cells by 57.1%. This phenomenon is caused by the more amount of proteins in the intracellular of single Histone-GFP type cell than that of the Wild type and GFP type cells. The more amount of proteins in the Histone-GFP type cells corresponds to a lower electric permittivity ϵ c of the cells, which generates a lower dielectrophoretic force exerting on the cells. The velocity of Histone-GFP type cells is well agreed with Eulerian-Lagrangian two-phase flow simulation by 4.2% mean error, which proves that the fluid motion driven by thermal buoyancy and electrothermal force dominates the direction of cells motion, while the distinct motion of Histone-GFP type cells is caused by dielectrophoretic force. The fluid motion does not generate a distinct drag motion for Histone-GFP type cells because the Histone-GFP type cells have the same size to the Wild type and GFP type cells. These results clarified the mechanism of cells motion in terms of intracellular components, which helps to improve the cell manipulation efficiency with electrokinetics.

Theory of Force Regulation by Nascent Adhesion Sites

PubMed Central

Bruinsma, Robijn

2005-01-01

The mechanical coupling of a cell with the extracellular matrix relies on adhesion sites, clusters of membrane-associated proteins that communicate forces generated along the F-Actin filaments of the cytoskeleton to connecting tissue. Nascent adhesion sites have been shown to regulate these forces in response to tissue rigidity. Force-regulation by substrate rigidity of adhesion sites with fixed area is not possible for stationary adhesion sites, according to elasticity theory. A simple model is presented to describe force regulation by dynamical adhesion sites. PMID:15849245

YAP is essential for mechanical force production and epithelial cell proliferation during lung branching morphogenesis

PubMed Central

Lin, Chuwen; Yao, Erica; Zhang, Kuan; Jiang, Xuan; Croll, Stacey; Thompson-Peer, Katherine; Chuang, Pao-Tien

2017-01-01

Branching morphogenesis is a fundamental program for tissue patterning. We show that active YAP, a key mediator of Hippo signaling, is distributed throughout the murine lung epithelium and loss of epithelial YAP severely disrupts branching. Failure to branch is restricted to regions where YAP activity is removed. This suggests that YAP controls local epithelial cell properties. In support of this model, mechanical force production is compromised and cell proliferation is reduced in Yap mutant lungs. We propose that defective force generation and insufficient epithelial cell number underlie the branching defects. Through genomic analysis, we also uncovered a feedback control of pMLC levels, which is critical for mechanical force production, likely through the direct induction of multiple regulators by YAP. Our work provides a molecular pathway that could control epithelial cell properties required for proper morphogenetic movement and pattern formation. DOI: http://dx.doi.org/10.7554/eLife.21130.001 PMID:28323616

Physical probing of cells

NASA Astrophysics Data System (ADS)

Rehfeldt, Florian; Schmidt, Christoph F.

2017-11-01

In the last two decades, it has become evident that the mechanical properties of the microenvironment of biological cells are as important as traditional biochemical cues for the control of cellular behavior and fate. The field of cell and matrix mechanics is quickly growing and so is the development of the experimental approaches used to study active and passive mechanical properties of cells and their surroundings. Within this topical review we will provide a brief overview, on the one hand, over how cellular mechanics can be probed physically, how different geometries allow access to different cellular properties, and, on the other hand, how forces are generated in cells and transmitted to the extracellular environment. We will describe the following experimental techniques: atomic force microscopy, traction force microscopy, magnetic tweezers, optical stretcher and optical tweezers pointing out both their advantages and limitations. Finally, we give an outlook on the future of the physical probing of cells.

Up-regulation of MHC class I in transgenic mice results in reduced force-generating capacity in slow-twitch muscle

PubMed Central

Salomonsson, Stina; Grundtman, Cecilia; Zhang, Shi-Jin; Lanner, Johanna T.; Li, Charles; Katz, Abram; Wedderburn, Lucy R.; Nagaraju, Kanneboyina; Lundberg, Ingrid E.; Westerblad, Håkan

2008-01-01

Expression of major histocompatibility complex (MHC) class I in skeletal muscle fibers is an early and consistent finding in inflammatory myopathies. To test if MHC class I has a primary role in muscle impairment; we used transgenic mice with inducible over-expression of MHC class I in their skeletal muscle cells. Contractile function was studied in isolated extensor digitorum longus (EDL, fast-twitch) and soleus (slow-twitch) muscles. We found that EDL was smaller, whereas soleus muscle was slightly larger. Both muscles generated less absolute force in myopathic compared to control mice, however when force was expressed per cross-sectional area, only soleus muscle generated less force. Inflammation was markedly increased, but no changes were found in the activities of key mitochondrial and glycogenolytic enzymes in myopathic mice. The induction of MHC class I results in muscle atrophy and an intrinsic decrease in force-generation capacity. These observations may have important implications for our understanding of the pathophysiological processes of muscle weakness seen in inflammatory myopathies. PMID:19229963

Mechanical forces in plant growth and development

NASA Technical Reports Server (NTRS)

Fisher, D. D.; Cyr, R. J.

2000-01-01

Plant cells perceive forces that arise from the environment and from the biophysics of plant growth. These forces provide meaningful cues that can affect the development of the plant. Seedlings of Arabidopsis thaliana were used to examine the cytoplasmic tensile character of cells that have been implicated in the gravitropic response. Laser-trapping technology revealed that the starch-containing statoliths of the central columella cells in root caps are held loosely within the cytoplasm. In contrast, the peripheral cells have starch granules that are relatively resistant to movement. The role of the actin cytoskeleton in affecting the tensile character of these cells is discussed. To explore the role that biophysical forces might play in generating developmental cues, we have developed an experimental model system in which protoplasts, embedded in a synthetic agarose matrix, are subjected to stretching or compression. We have found that protoplasts subjected to these forces from five minutes to two hours will subsequently elongate either at right angles or parallel to the tensive or compressive force vector. Moreover, the cortical microtubules are found to be organized either at right angles or parallel to the tensive or compressive force vector. We discuss these results in terms of an interplay of information between the extracellular matrix and the underlying cytoskeleton.

Tension and Elasticity Contribute to Fibroblast Cell Shape in Three Dimensions.

PubMed

Brand, Christoph A; Linke, Marco; Weißenbruch, Kai; Richter, Benjamin; Bastmeyer, Martin; Schwarz, Ulrich S

2017-08-22

The shape of animal cells is an important regulator for many essential processes such as cell migration or division. It is strongly determined by the organization of the actin cytoskeleton, which is also the main regulator of cell forces. Quantitative analysis of cell shape helps to reveal the physical processes underlying cell shape and forces, but it is notoriously difficult to conduct it in three dimensions. Here we use direct laser writing to create 3D open scaffolds for adhesion of connective tissue cells through well-defined adhesion platforms. Due to actomyosin contractility in the cell contour, characteristic invaginations lined by actin bundles form between adjacent adhesion sites. Using quantitative image processing and mathematical modeling, we demonstrate that the resulting shapes are determined not only by contractility, but also by elastic stress in the peripheral actin bundles. In this way, cells can generate higher forces than through contractility alone. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The microtubule-binding and coiled-coil domains of Kid are required to turn off the polar ejection force at anaphase.

PubMed

Soeda, Shou; Yamada-Nomoto, Kaori; Ohsugi, Miho

2016-10-01

Mitotic chromosomes move dynamically along the spindle microtubules using the forces generated by motor proteins such as chromokinesin Kid (also known as KIF22). Kid generates a polar ejection force and contributes to alignment of the chromosome arms during prometaphase and metaphase, whereas during anaphase, Kid contributes to chromosome compaction. How Kid is regulated and how this regulation is important for chromosome dynamics remains unclear. Here, we address these questions by expressing mutant forms of Kid in Kid-deficient cells. We demonstrate that Cdk1-mediated phosphorylation of Thr463 is required to generate the polar ejection force on Kid-binding chromosomes, whereas dephosphorylation of Thr463 prevents generation of the ejection force on such chromosomes. In addition to activation of the second microtubule-binding domain through dephosphorylation of Thr463, the coiled-coil domain is essential in suspending generation of the polar ejection force, preventing separated chromosomes from becoming recongressed during anaphase. We propose that phosphorylation of Thr463 switches the mitotic chromosome movement from an anti-poleward direction to a poleward direction by converting the Kid functional mode from polar-ejection-force-ON to -OFF during the metaphase-anaphase transition, and that both the second microtubule-binding domain and the coiled-coil domain are involved in this switching process. © 2016. Published by The Company of Biologists Ltd.

Realizations of highly heterogeneous collagen networks via stochastic reconstruction for micromechanical analysis of tumor cell invasion

NASA Astrophysics Data System (ADS)

Nan, Hanqing; Liang, Long; Chen, Guo; Liu, Liyu; Liu, Ruchuan; Jiao, Yang

2018-03-01

Three-dimensional (3D) collective cell migration in a collagen-based extracellular matrix (ECM) is among one of the most significant topics in developmental biology, cancer progression, tissue regeneration, and immune response. Recent studies have suggested that collagen-fiber mediated force transmission in cellularized ECM plays an important role in stress homeostasis and regulation of collective cellular behaviors. Motivated by the recent in vitro observation that oriented collagen can significantly enhance the penetration of migrating breast cancer cells into dense Matrigel which mimics the intravasation process in vivo [Han et al. Proc. Natl. Acad. Sci. USA 113, 11208 (2016), 10.1073/pnas.1610347113], we devise a procedure for generating realizations of highly heterogeneous 3D collagen networks with prescribed microstructural statistics via stochastic optimization. Specifically, a collagen network is represented via the graph (node-bond) model and the microstructural statistics considered include the cross-link (node) density, valence distribution, fiber (bond) length distribution, as well as fiber orientation distribution. An optimization problem is formulated in which the objective function is defined as the squared difference between a set of target microstructural statistics and the corresponding statistics for the simulated network. Simulated annealing is employed to solve the optimization problem by evolving an initial network via random perturbations to generate realizations of homogeneous networks with randomly oriented fibers, homogeneous networks with aligned fibers, heterogeneous networks with a continuous variation of fiber orientation along a prescribed direction, as well as a binary system containing a collagen region with aligned fibers and a dense Matrigel region with randomly oriented fibers. The generation and propagation of active forces in the simulated networks due to polarized contraction of an embedded ellipsoidal cell and a small group of cells are analyzed by considering a nonlinear fiber model incorporating strain hardening upon large stretching and buckling upon compression. Our analysis shows that oriented fibers can significantly enhance long-range force transmission in the network. Moreover, in the oriented-collagen-Matrigel system, the forces generated by a polarized cell in collagen can penetrate deeply into the Matrigel region. The stressed Matrigel fibers could provide contact guidance for the migrating cell cells, and thus enhance their penetration into Matrigel. This suggests a possible mechanism for the observed enhanced intravasation by oriented collagen.

A force sensor using nanowire arrays to understand biofilm formation (Conference Presentation)

NASA Astrophysics Data System (ADS)

Sahoo, Prasana K.; Cavalli, Alessandro; Pelegati, Vitor B.; Murillo, Duber M.; Souza, Alessandra A.; Cesar, Carlos L.; Bakkers, Erik P. A. M.; Cotta, Monica A.

2016-03-01

Understanding the cellular signaling and function at the nano-bio interface can pave the way towards developing next-generation smart diagnostic tools. From this perspective, limited reports detail so far the cellular and subcellular forces exerted by bacterial cells during the interaction with abiotic materials. Nanowire arrays with high aspect ratio have been used to detect such small forces. In this regard, live force measurements were performed ex-vivo during the interaction of Xylella fastidiosa bacterial cells with InP nanowire arrays. The influence of nanowire array topography and surface chemistry on the response and motion of bacterial cells was studied in detail. The nanowire arrays were also functionalized with different cell adhesive promoters, such as amines and XadA1, an afimbrial protein of X.fastidiosa. By employing the well-defined InP nanowire arrays platform, and single cell confocal imaging system, we were able to trace the bacterial growth pattern, and show that their initial attachment locations are strongly influenced by the surface chemistry and nanoscale surface topography. In addition, we measure the cellular forces down to few nanonewton range using these nanowire arrays. In case of nanowire functionalized with XadA1, the force exerted by vertically and horizontally attached single bacteria on the nanowire is in average 14% and 26% higher than for the pristine array, respectively. These results provide an excellent basis for live-cell force measurements as well as unravel the range of forces involved during the early stages of bacterial adhesion and biofilm formation.

Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening.

PubMed

Amaral, Robson L F; Miranda, Mariza; Marcato, Priscyla D; Swiech, Kamilla

2017-01-01

Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better. Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 10 4 cells/mL), and then, subjected to drug resistance evaluation. Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300-500 μm) was obtained using cultures initiated with 0.5 and 1.25 × 10 4 cells/mL for the ULA method and 2.5 and 3.75 × 10 4 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC 50 of 1.00 and 0.83 μg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC 50 ranging from 0.39 to 0.43). Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application.

Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening

PubMed Central

Amaral, Robson L. F.; Miranda, Mariza; Marcato, Priscyla D.; Swiech, Kamilla

2017-01-01

Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better. Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 104 cells/mL), and then, subjected to drug resistance evaluation. Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300–500 μm) was obtained using cultures initiated with 0.5 and 1.25 × 104 cells/mL for the ULA method and 2.5 and 3.75 × 104 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC50 of 1.00 and 0.83 μg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC50 ranging from 0.39 to 0.43). Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application. PMID:28878686

The push for a place in the crowd

NASA Astrophysics Data System (ADS)

Notbohm, Jacob; Burkel, Brian

2018-06-01

Cells change shape and volume when they divide — not a simple task, especially when they are confined by surrounding tissue. Experiments now reveal that hydrostatic pressure changes generate the pushing forces that cells need to create space for division.

The extracellular matrix remodeled

PubMed Central

Kirmse, Robert; Otto, Hannes

2012-01-01

Membrane Type-1 Matrix Metalloproteinase (MT1-MMP, MMP-14) is regarded as the prototype of a membrane- tethered protease. It drives fundamental biological processes ranging from embryogenesis to cancer metastasis. The proteolytic cleavage of proteins by MT1-MMP can rapidly alter the biophysical properties of a cell’s microenvironment. Cell’s must thus be able to sense and react to these alterations and transduce these effectively in biochemical signals and cell responses. Although many cells react as acutely to such physical stimuli as they do to chemical ones, the regulatory effects of these have been less extensively explored. In order to investigate a possible interdependency of proteolytic matrix cleavage by MT1-MMP and the generation and sensing of force by cells, a model system was established which exploits the properties of a matrix array of parallel collagen-I fibers. The resulting an-isotropy of the matrix with high tensile strength along the fibers and high mobility perpendicular to it allows the convenient detection of bundling and cleavage of the collagen fibers, as well as spreading and durotaxis of the cells. In summary, we have demonstrated that cell adhesion, force generation, and force sensing are vital for the regulation of MT1-MMP for efficient cleavage of collagen-I. PMID:22482015

Roughness-dependent friction force of the tarsal claw system in the beetle Pachnoda marginata (Coleoptera, Scarabaeidae).

PubMed

Dai, Zhendong; Gorb, Stanislav N; Schwarz, Uli

2002-08-01

This paper studies slide-resisting forces generated by claws in the free-walking beetle Pachnoda marginata (Coleoptera, Scarabaeoidea) with emphasis on the relationship between the dimension of the claw tip and the substrate texture. To evaluate the force range by which the claw can interact with a substrate, forces generated by the freely moving legs were measured using a load cell force transducer. To obtain information about material properties of the claw, its mechanical strength was tested in a fracture experiment, and the internal structure of the fractured claw material was studied by scanning electron microscopy. The bending stress of the claw was evaluated as 143.4-684.2 MPa, depending on the cross-section model selected. Data from these different approaches led us to propose a model explaining the saturation of friction force with increased texture roughness. The forces are determined by the relative size of the surface roughness R(a) (or an average particle diameter) and the diameter of the claw tip. When surface roughness is much bigger than the claw tip diameter, the beetle can grasp surface irregularities and generate a high degree of attachment due to mechanical interlocking with substrate texture. When R(a) is lower than or comparable to the claw tip diameter, the frictional properties of the contact between claw and substrate particles play a key role in the generation of the friction force.

Computer-aided mechanogenesis of skeletal muscle organs from single cells in vitro

NASA Technical Reports Server (NTRS)

Vanderburgh, Herman H.; Swasdison, Somporn; Karlisch, Patricia

1991-01-01

Complex mechanical forces generated in the growing embryo play an important role in organogenesis. Computerized application of similar forces to differentiating skeletal muscle myoblasts in vitro generate three dimensional artificial muscle organs. These organs contain parallel networks of long unbranched myofibers organized into fascicle-like structures. Tendon development is initiated and the muscles are capable of performing directed, functional work. Kinetically engineered organs provide a new method for studying the growth and development of normal and diseased skeletal muscle.

Computer aided mechanogenesis of skeletal muscle organs from single cells in vitro

NASA Technical Reports Server (NTRS)

Vandenburgh, Herman H.; Swasdison, Somporn; Karlisch, Patricia

1990-01-01

Complex mechanical forces generated in the growing embryo play an important role in organogenesis. Computerized application of similar forces to differentiating skeletal muscle myoblasts in vitro generate three dimensional artificial muscle organs. These organs contain parallel networks of long unbranched myofibers organized into fascicle-like structures. Tendon development is initiated and the muscles are capable of performing directed, functional work. Kinetically engineered organs provide a new method for studying the growth and development of normal and diseased skeletal muscle.

Bistable front dynamics in a contractile medium: travelling wave and cortical advection define stable zones of RhoA signaling at epithelial adherens junctions

NASA Astrophysics Data System (ADS)

Neufeld, Zoltan

Recent studies have demonstrated that mechanical forces can lead to novel mechanisms of pattern formation such as clustering and oscillations in contractile systems. We investigate how contractile forces in mechanically active media can affect bistable front propagation. We found that contraction regulates the front speed or can fully suppress its propagation in space to create a static localized zone. We demonstrate how the interplay between biochemical signaling through positive feedback, combined with diffusion on the cell membrane and mechanical forces generated in the actomyosin cortex, can determine the spatial distribution of RhoA signaling at cell-cell junctions. The dynamical mechanism relies on the balance between a propagating bistable signal that is opposed by an advective flow generated by an actomyosin stress gradient. Experimental observations on the behaviour of the system when contractility is inhibited are in qualitative agreement with the predictions of the model. In collaboration with: Zoltan Neufeld, Guillermo A. Gomez, and Alpha S. Yap, University of Queensland, Brisbane, Australia

Living matter—nexus of physics and biology in the 21st century

PubMed Central

Gardel, Margaret L.

2012-01-01

Cells are made up of complex assemblies of cytoskeletal proteins that facilitate force transmission from the molecular to cellular scale to regulate cell shape and force generation. The “living matter” formed by the cytoskeleton facilitates versatile and robust behaviors of cells, including their migration, adhesion, division, and morphology, that ultimately determine tissue architecture and mechanics. Elucidating the underlying physical principles of such living matter provides great opportunities in both biology and physics. For physicists, the cytoskeleton provides an exceptional toolbox to study materials far from equilibrium. For biologists, these studies will provide new understanding of how molecular-scale processes determine cell morphological changes. PMID:23112229

Mechanosensing drives acuity of αβ T-cell recognition

PubMed Central

Feng, Yinnian; Brazin, Kristine N.; Kobayashi, Eiji; Mallis, Robert J.; Reinherz, Ellis L.; Lang, Matthew J.

2017-01-01

T lymphocytes use surface αβ T-cell receptors (TCRs) to recognize peptides bound to MHC molecules (pMHCs) on antigen-presenting cells (APCs). How the exquisite specificity of high-avidity T cells is achieved is unknown but essential, given the paucity of foreign pMHC ligands relative to the ubiquitous self-pMHC array on an APC. Using optical traps, we determine physicochemical triggering thresholds based on load and force direction. Strikingly, chemical thresholds in the absence of external load require orders of magnitude higher pMHC numbers than observed physiologically. In contrast, force applied in the shear direction (∼10 pN per TCR molecule) triggers T-cell Ca2+ flux with as few as two pMHC molecules at the interacting surface interface with rapid positional relaxation associated with similarly directed motor-dependent transport via ∼8-nm steps, behaviors inconsistent with serial engagement during initial TCR triggering. These synergistic directional forces generated during cell motility are essential for adaptive T-cell immunity against infectious pathogens and cancers. PMID:28811364

The potential of prolonged tissue culture to reduce stress generation and retraction in engineered heart valve tissues.

PubMed

van Vlimmeren, Marijke A A; Driessen-Mol, Anita; Oomens, Cees W J; Baaijens, Frank P T

2013-03-01

In tissue-engineered (TE) heart valves, cell-mediated processes cause tissue compaction during culture and leaflet retraction at time of implantation. We have quantified and correlated stress generation, compaction, retraction, and tissue quality during a prolonged culture period of 8 weeks. Polyglycolic acid/poly-4-hydroxybutyrate strips were seeded with vascular-derived cells and cultured for 4-8 weeks. Compaction in width, generated force, and stress was measured during culture. Retraction in length, generated force, and stress was measured after release of constraints at weeks 4, 6, and 8. Further, the amount of DNA, glycosaminoglycans (GAGs), collagen, and collagen cross-links was assessed. During culture, compaction and force generation increased to, respectively, 63.9% ± 0.8% and 43.7 ± 4.3 mN at week 4, after which they remained stable. Stress generation reached 27.7 ± 3.2 kPa at week 4, after which it decreased to ∼8.5 kPa. At release of constraints, tissue retraction was 44.0% ± 3.7% at week 4 and decreased to 29.2% ± 2.8% and 26.1% ± 2.2% at, respectively, 6 and 8 weeks. Generated force (8-16 mN) was lower at week 6 than at weeks 4 and 8. Generated stress decreased from 11.8 ± 0.9 kPa at week 4 to 1.4 ± 0.3 and 2.4 ± 0.4 kPa at, respectively, weeks 6 and 8. The amount of GAGs increased at weeks 6 and 8 compared to week 4 and correlated to the reduced stress and retraction. In summary, prolonged culture resulted in decreased stress generation and retraction, likely as a result of the increased amount of GAGs. These results demonstrate the potential of prolonged tissue culture in developing functional, nonretracting, TE heart valves.

The nucleus is an intracellular propagator of tensile forces in NIH 3T3 fibroblasts

PubMed Central

Alam, Samer G.; Lovett, David; Kim, Dae In; Roux, Kyle J.; Dickinson, Richard B.; Lele, Tanmay P.

2015-01-01

ABSTRACT Nuclear positioning is a crucial cell function, but how a migrating cell positions its nucleus is not understood. Using traction-force microscopy, we found that the position of the nucleus in migrating fibroblasts closely coincided with the center point of the traction-force balance, called the point of maximum tension (PMT). Positioning of the nucleus close to the PMT required nucleus–cytoskeleton connections through linker of nucleoskeleton-to-cytoskeleton (LINC) complexes. Although the nucleus briefly lagged behind the PMT following spontaneous detachment of the uropod during migration, the nucleus quickly repositioned to the PMT within a few minutes. Moreover, traction-generating spontaneous protrusions deformed the nearby nucleus surface to pull the nuclear centroid toward the new PMT, and subsequent retraction of these protrusions relaxed the nuclear deformation and restored the nucleus to its original position. We propose that the protruding or retracting cell boundary transmits a force to the surface of the nucleus through the intervening cytoskeletal network connected by the LINC complexes, and that these forces help to position the nucleus centrally and allow the nucleus to efficiently propagate traction forces across the length of the cell during migration. PMID:25908852

Computational Model of Secondary Palate Fusion and Disruption

EPA Science Inventory

Morphogenetic events are driven by cell-generated physical forces and complex cellular dynamics. To improve our capacity to predict developmental effects from cellular alterations, we built a multi-cellular agent-based model in CompuCell3D that recapitulates the cellular networks...

Concise Review: Plasma and Nuclear Membranes Convey Mechanical Information to Regulate Mesenchymal Stem Cell Lineage.

PubMed

Uzer, Gunes; Fuchs, Robyn K; Rubin, Janet; Thompson, William R

2016-06-01

Numerous factors including chemical, hormonal, spatial, and physical cues determine stem cell fate. While the regulation of stem cell differentiation by soluble factors is well-characterized, the role of mechanical force in the determination of lineage fate is just beginning to be understood. Investigation of the role of force on cell function has largely focused on "outside-in" signaling, initiated at the plasma membrane. When interfaced with the extracellular matrix, the cell uses integral membrane proteins, such as those found in focal adhesion complexes to translate force into biochemical signals. Akin to these outside-in connections, the internal cytoskeleton is physically linked to the nucleus, via proteins that span the nuclear membrane. Although structurally and biochemically distinct, these two forms of mechanical coupling influence stem cell lineage fate and, when disrupted, often lead to disease. Here we provide an overview of how mechanical coupling occurs at the plasma and nuclear membranes. We also discuss the role of force on stem cell differentiation, with focus on the biochemical signals generated at the cell membrane and the nucleus, and how those signals influence various diseases. While the interaction of stem cells with their physical environment and how they respond to force is complex, an understanding of the mechanical regulation of these cells is critical in the design of novel therapeutics to combat diseases associated with aging, cancer, and osteoporosis. Stem Cells 2016;34:1455-1463. © 2016 AlphaMed Press.

A model of cell-wall dynamics during sporulation in Bacillus subtilis

NASA Astrophysics Data System (ADS)

Yap, Li-Wei; Endres, Robert G.

To survive starvation, Bacillus subtilis forms durable spores. After asymmetric cell division, the septum grows around the forespore in a process called engulfment, but the mechanism of force generation is unknown. Here, we derived a novel biophysical model for the dynamics of cell-wall remodeling during engulfment based on a balancing of dissipative, active, and mechanical forces. By plotting phase diagrams, we predict that sporulation is promoted by a line tension from the attachment of the septum to the outer cell wall, as well as by an imbalance in turgor pressures in the mother-cell and forespore compartments. We also predict that significant mother-cell growth hinders engulfment. Hence, relatively simple physical principles may guide this complex biological process.

Optical Tweezers-Based Measurements of Forces and Dynamics at Microtubule Ends.

PubMed

Baclayon, Marian; Kalisch, Svenja-Marei; Hendel, Ed; Laan, Liedewij; Husson, Julien; Munteanu, E Laura; Dogterom, Marileen

2017-01-01

Microtubules are dynamic cytoskeletal polymers that polymerize and depolymerize while interacting with different proteins and structures within the cell. The highly regulated dynamic properties as well as the pushing and pulling forces generated by dynamic microtubule ends play important roles in processes such as in cell division. For instance, microtubule end-binding proteins are known to affect dramatically the dynamic properties of microtubules, and cortical dyneins are known to mediate pulling forces on microtubule ends. We discuss in this chapter our efforts to reconstitute these systems in vitro and mimic their interactions with structures within the cell using micro-fabricated barriers. Using an optical tweezers setup, we investigate the dynamics and forces of microtubules growing against functionalized barriers in the absence and presence of end-binding proteins and barrier-attached motor proteins. This setup allows high-speed as well as nanometer and piconewton resolution measurements on dynamic microtubules.

Branched actin networks push against each other at adherens junctions to maintain cell-cell adhesion.

PubMed

Efimova, Nadia; Svitkina, Tatyana M

2018-05-07

Adherens junctions (AJs) are mechanosensitive cadherin-based intercellular adhesions that interact with the actin cytoskeleton and carry most of the mechanical load at cell-cell junctions. Both Arp2/3 complex-dependent actin polymerization generating pushing force and nonmuscle myosin II (NMII)-dependent contraction producing pulling force are necessary for AJ morphogenesis. Which actin system directly interacts with AJs is unknown. Using platinum replica electron microscopy of endothelial cells, we show that vascular endothelial (VE)-cadherin colocalizes with Arp2/3 complex-positive actin networks at different AJ types and is positioned at the interface between two oppositely oriented branched networks from adjacent cells. In contrast, actin-NMII bundles are located more distally from the VE-cadherin-rich zone. After Arp2/3 complex inhibition, linear AJs split, leaving gaps between cells with detergent-insoluble VE-cadherin transiently associated with the gap edges. After NMII inhibition, VE-cadherin is lost from gap edges. We propose that the actin cytoskeleton at AJs acts as a dynamic push-pull system, wherein pushing forces maintain extracellular VE-cadherin transinteraction and pulling forces stabilize intracellular adhesion complexes. © 2018 Efimova and Svitkina.

Review of cellular mechanotransduction

NASA Astrophysics Data System (ADS)

Wang, Ning

2017-06-01

Living cells and tissues experience physical forces and chemical stimuli in the human body. The process of converting mechanical forces into biochemical activities and gene expression is mechanochemical transduction or mechanotransduction. Significant advances have been made in understanding mechanotransduction at the cellular and molecular levels over the last two decades. However, major challenges remain in elucidating how a living cell integrates signals from mechanotransduction with chemical signals to regulate gene expression and to generate coherent biological responses in living tissues in physiological conditions and diseases.

Mechanosensitivity in axon growth and guidance

NASA Astrophysics Data System (ADS)

Urbach, Jeff

2013-03-01

In the developing nervous system, axons respond to a diverse array of cues to generate the intricate connection network required for proper function. The growth cone, a highly motile structure at the tip of a growing axon, integrates information about the local environment and modulates outgrowth and guidance, but little is known about effects of external mechanical cues and internal mechanical forces on growth cone behavior. We have investigated axon outgrowth and force generation on soft elastic substrates for dorsal root ganglion (DRG) neurons (from the peripheral nervous system) and hippocampal neurons (from the central) to see how the mechanics of the microenvironment affect different populations. We find that force generation and stiffness-dependent outgrowth are strongly dependent on cell type. We also observe very different internal dynamics and substrate coupling in the two populations, suggesting that the difference in force generation is due to stronger adhesions and therefore stronger substrate engagement in the peripheral nervous system neurons. We will discuss the biological origins of these differences, and recent analyses of the dynamic aspects of growth cone force generation and the implications for the role of mechanosensitivity in axon guidance. In collaboration with D. Koch, W. Rosoff, and H. M. Geller. Supported by NINDS grant 1R01NS064250-01 (J.S.U.) and the NHLBI Intramural Research Program (H.M.G.).

The Effects of the Interplay between Motor and Brownian Forces on the Rheology of Active Gels.

PubMed

Córdoba, Andrés

2018-04-19

Active gels perform key mechanical roles inside the cell, such as cell division, motion, and force sensing. The unique mechanical properties required to perform such functions arise from the interactions between molecular motors and semiflexible polymeric filaments. Molecular motors can convert the energy released in the hydrolysis of ATP into forces of up to piconewton magnitudes. Moreover, the polymeric filaments that form active gels are flexible enough to respond to Brownian forces but also stiff enough to support the large tensions induced by the motor-generated forces. Brownian forces are expected to have a significant effect especially at motor activities at which stable noncontractile in vitro active gels are prepared for rheological measurements. Here, a microscopic mean-field theory of active gels originally formulated in the limit of motor-dominated dynamics is extended to include Brownian forces. In the model presented here, Brownian forces are included accurately, at real room temperature, even in systems with high motor activity. It is shown that a subtle interplay, or competition, between motor-generated forces and Brownian forces has an important impact on the mass transport and rheological properties of active gels. The model predictions show that at low frequencies the dynamic modulus of active gels is determined mostly by motor protein dynamics. However, Brownian forces significantly increase the breadth of the relaxation spectrum and can affect the shape of the dynamic modulus over a wide frequency range even for ratios of motor to Brownian forces of more than a hundred. Since the ratio between motor and Brownian forces is sensitive to ATP concentration, the results presented here shed some light on how the transient mechanical response of active gels changes with varying ATP concentration.

Dynamics of Individual cilia to external loading- A simple one dimensional picture

NASA Astrophysics Data System (ADS)

Swaminathan, Vinay; Hill, David; Superfine, R.

2008-10-01

From being called the cellular janitors to swinging debauchers, cilia have captured the fascinations of researchers for over 200 years. In cystic fibrosis and chronic obstructive pulmonary disease where the cilia loses it's function, the protective mucus layer in the lung thickens and mucociliary clearance breaks down, leading to inflammation along the airways and an increased rate of infection. The mechanistic understanding of mucus clearance depends on a quantitative assessment of the axoneme dynamics and the maximum force the cilia are capable of generating and imparting to the mucus layer. Similar to the situation in molecular motors, detailed quantitative measurements of dynamics under applied load conditions are expected to be essential in developing predictive models. Based on our measurements of the dynamics of individual ciliary motion in the human bronchial epithelial cell under the application of an applied load, we present a simple one dimensional model for the axoneme dynamics and quantify the axoneme stiffness, the internal force generated by the axoneme, the stall force and show how the dynamics sheds insight on the time dependence of the internal force generation. The internal force generated by the axoneme is related to the ability of cilia to propel fluids and to their potential role in force sensing.

Single-Cell Functional Analysis of Stem-Cell Derived Cardiomyocytes on Micropatterned Flexible Substrates.

PubMed

Kijlstra, Jan David; Hu, Dongjian; van der Meer, Peter; Domian, Ibrahim J

2017-11-15

Human pluripotent stem-cell derived cardiomyocytes (hPSC-CMs) hold great promise for applications in human disease modeling, drug discovery, cardiotoxicity screening, and, ultimately, regenerative medicine. The ability to study multiple parameters of hPSC-CM function, such as contractile and electrical activity, calcium cycling, and force generation, is therefore of paramount importance. hPSC-CMs cultured on stiff substrates like glass or polystyrene do not have the ability to shorten during contraction, making them less suitable for the study of hPSC-CM contractile function. Other approaches require highly specialized hardware and are difficult to reproduce. Here we describe a protocol for the preparation of hPSC-CMs on soft substrates that enable shortening, and subsequently the simultaneous quantitative analysis of their contractile and electrical activity, calcium cycling, and force generation at single-cell resolution. This protocol requires only affordable and readily available materials and works with standard imaging hardware. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

From Red Cells to Soft Porous Lubrication

NASA Astrophysics Data System (ADS)

Wu, Qianhong; Gacka, Thomas; Nathan, Rungun; Crawford, Robert; Vucbmss Team

2014-11-01

Biological scientists have wondered, since the motion of red cells was first observed in capillaries, how the highly flexible red cell can move with so little friction in tightly fitting microvessels without being damaged or undergoing hemolysis. Theoretical studies (Feng and Weinbaum, 2000, JFM; Wu et al., 2004, PRL) attributed this frictionless motion to the dramatically enhanced hydrodynamic lifting force generated inside the soft, porous, endothelial surface layer (ESL) covering the inner surfaces of our capillaries, as a red blood cell glides over it. Herein we report the first experimental examination of this concept. The results conclusively demonstrate that significant fraction of the overall lifting force generated in a soft porous layer as a planing surface glides over it, is contributed by the pore fluid pressure, and thus frictional loss is reduced significantly. Moreover, the experimental predictions showed excellent agreement with the experimental data. This finding has the potential of dramatically changing existing lubrication approaches, and can result in substantial savings in energy consumption and thus reduction in greenhouse gas emissions.

Forces Generated by Cell Intercalation Tow Epidermal Sheets in Mammalian Tissue Morphogenesis

PubMed Central

Heller, Evan; Kumar, K. Vijay; Grill, Stephan W.; Fuchs, Elaine

2014-01-01

Summary While gastrulation movements offer mechanistic paradigms for how collective cellular movements shape developing embryos, far less is known about coordinated cellular movements that occur later in development. Studying eyelid closure, we explore a case where an epithelium locally reshapes, expands, and moves over another epithelium. Live imaging, gene targeting and cell cycle inhibitors reveal that closure does not require overlying periderm, proliferation or supracellular actin cable assembly. Laser ablation and quantitative analyses of tissue deformations further distinguish the mechanism from wound-repair and dorsal closure. Rather, cell intercalations parallel to the tissue front locally compress it perpendicularly, pulling the surrounding epidermis along the closure axis. Functional analyses in vivo show that the mechanism requires localized myosin-IIA and α5β1-fibronectin-mediated migration, and E-cadherin downregulation likely stimulated by Wnt signaling. These studies uncover a mode of epithelial closure in which forces generated by cell intercalation are leveraged to tow the surrounding tissue. PMID:24697897

Brushes, cables, and anchors: recent insights into multiscale assembly and mechanics of cellular structural networks.

PubMed

Lele, Tanmay P; Kumar, Sanjay

2007-01-01

The remarkable ability of living cells to sense, process, and respond to mechanical stimuli in their environment depends on the rapid and efficient interconversion of mechanical and chemical energy at specific times and places within the cell. For example, application of force to cells leads to conformational changes in specific mechanosensitive molecules which then trigger cellular signaling cascades that may alter cellular structure, mechanics, and migration and profoundly influence gene expression. Similarly, the sensitivity of cells to mechanical stresses is governed by the composition, architecture, and mechanics of the cellular cytoskeleton and extracellular matrix (ECM), which are in turn driven by molecular-scale forces between the constituent biopolymers. Understanding how these mechanochemical systems coordinate over multiple length and time scales to produce orchestrated cell behaviors represents a fundamental challenge in cell biology. Here, we review recent advances in our understanding of these complex processes in three experimental systems: the assembly of axonal neurofilaments, generation of tensile forces by actomyosin stress fiber bundles, and mechanical control of adhesion assembly.

Modeling human development in 3D culture.

PubMed

Ader, Marius; Tanaka, Elly M

2014-12-01

Recently human embryonic stem cell research has taken on a new dimension - the third dimension. Capitalizing on increasing knowledge on directing pluripotent cells along different lineages, combined with ECM supported three-dimensional culture conditions, it has become possible to generate highly organized tissues of the central nervous system, gut, liver and kidney. Each system has been used to study different aspects of organogenesis and function including physical forces underlying optic cup morphogenesis, the function of disease related genes in progenitor cell control, as well as interaction of the generated tissues with host tissue upon transplantation. Pluripotent stem cell derived organoids represent powerful systems for the study of how cells self-organize to generate tissues with a given shape, pattern and form. Copyright © 2014 Elsevier Ltd. All rights reserved.

High-resolution imaging of cellular processes across textured surfaces using an indexed-matched elastomer.

PubMed

Ravasio, Andrea; Vaishnavi, Sree; Ladoux, Benoit; Viasnoff, Virgile

2015-03-01

Understanding and controlling how cells interact with the microenvironment has emerged as a prominent field in bioengineering, stem cell research and in the development of the next generation of in vitro assays as well as organs on a chip. Changing the local rheology or the nanotextured surface of substrates has proved an efficient approach to improve cell lineage differentiation, to control cell migration properties and to understand environmental sensing processes. However, introducing substrate surface textures often alters the ability to image cells with high precision, compromising our understanding of molecular mechanisms at stake in environmental sensing. In this paper, we demonstrate how nano/microstructured surfaces can be molded from an elastomeric material with a refractive index matched to the cell culture medium. Once made biocompatible, contrast imaging (differential interference contrast, phase contrast) and high-resolution fluorescence imaging of subcellular structures can be implemented through the textured surface using an inverted microscope. Simultaneous traction force measurements by micropost deflection were also performed, demonstrating the potential of our approach to study cell-environment interactions, sensing processes and cellular force generation with unprecedented resolution. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

On the origins of the universal dynamics of endogenous granules in mammalian cells.

PubMed

Vanapalli, Siva A; Li, Yixuan; Mugele, Frieder; Duits, Michel H G

2009-12-01

Endogenous granules (EGs) that consist of lipid droplets and mitochondria have been commonly used to assess intracellular mechanical properties via multiple particle tracking microrheology (MPTM). Despite their widespread use, the nature of interaction of EGs with the cytoskeletal network and the type of forces driving their dynamics--both of which are crucial for the interpretation of the results from MPTM technique--are yet to be resolved. In this report, we study the dynamics of endogenous granules in mammalian cells using particle tracking methods. We find that the ensemble dynamics of EGs is diffusive in three types of mammalian cells (endothelial cells, smooth muscle cells and fibroblasts), thereby suggesting an apparent universality in their dynamical behavior. Moreover, in a given cell, the amplitude of the mean-squared displacement for EGs is an order of magnitude larger than that of injected particles. This observation along with results from ATP depletion and temperature intervention studies suggests that cytoskeletal active forces drive the dynamics of EGs. To elucidate the dynamical origin of the diffusive-like nonthermal motion, we consider three active force generation mechanisms--molecular motor transport, actomyosin contractility and microtubule polymerization forces. We test these mechanisms using pharmacological interventions. Experimental evidence and model calculations suggest that EGs are intimately linked to microtubules and that microtubule polymerization forces drive their dynamics. Thus, endogenous granules could serve as non-invasive probes for microtubule network dynamics in mammalian cells.

Cytoplasmic motion induced by cytoskeleton stretching and its effect on cell mechanics.

PubMed

Zhang, T

2011-09-01

Cytoplasmic motion assumed as a steady state laminar flow induced by cytoskeleton stretching in a cell is determined and its effect on the mechanical behavior of the cell under externally applied forces is demonstrated. Non-Newtonian fluid is assumed for the multiphase cytoplasmic fluid and the analytical velocity field around the macromolecular chain is obtained by solving the reduced nonlinear momentum equation using homotopy technique. The entropy generation by the fluid internal friction is calculated and incorporated into the entropic elasticity based 8-chain constitutive relations. Numerical examples showed strengthening behavior of cells in response to externally applied mechanical stimuli. The spatial distribution of the stresses within a cell under externally applied fluid flow forces were also studied.

The Role of MreB in Escherichia Coli's Cellular Rigidity

NASA Astrophysics Data System (ADS)

Shaevitz, Joshua W.

2009-03-01

Bacteria possess homologs of all three classes of eukaryotic cytoskeletal proteins. These filamentous proteins have been shown to localize proteins essential for a number of cell-biological processes in prokaryotes such as cell growth and division. However, to date, there has been no direct evidence that the cytoskeleton in bacteria bears mechanical loads or can generate physical forces than are used by the cell. I will present evidence from combined fluorescence and force microscopy measurements that MreB, an actin homolog, is responsible for half of Escherichia coli's cellular rigidity. These data support an interpretation in which the cytoskeleton, the peptidoglycan cell wall and a large turgor pressure work together to give gram-negative cells their mechanical properties.

Cell-cell and cell-extracellular matrix adhesions cooperate to organize actomyosin networks and maintain force transmission during dorsal closure.

PubMed

Goodwin, Katharine; Lostchuck, Emily E; Cramb, Kaitlyn M L; Zulueta-Coarasa, Teresa; Fernandez-Gonzalez, Rodrigo; Tanentzapf, Guy

2017-05-15

Tissue morphogenesis relies on the coordinated action of actin networks, cell-cell adhesions, and cell-extracellular matrix (ECM) adhesions. Such coordination can be achieved through cross-talk between cell-cell and cell-ECM adhesions. Drosophila dorsal closure (DC), a morphogenetic process in which an extraembryonic tissue called the amnioserosa contracts and ingresses to close a discontinuity in the dorsal epidermis of the embryo, requires both cell-cell and cell-ECM adhesions. However, whether the functions of these two types of adhesions are coordinated during DC is not known. Here we analyzed possible interdependence between cell-cell and cell-ECM adhesions during DC and its effect on the actomyosin network. We find that loss of cell-ECM adhesion results in aberrant distributions of cadherin-mediated adhesions and actin networks in the amnioserosa and subsequent disruption of myosin recruitment and dynamics. Moreover, loss of cell-cell adhesion caused up-regulation of cell-ECM adhesion, leading to reduced cell deformation and force transmission across amnioserosa cells. Our results show how interdependence between cell-cell and cell-ECM adhesions is important in regulating cell behaviors, force generation, and force transmission critical for tissue morphogenesis. © 2017 Goodwin, Lostchuck, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Measuring the Kinetic and Mechanical Properties of Non-Processive Myosins using Optical Tweezers

PubMed Central

Greenberg, Michael J.; Shuman, Henry; Ostap, E. Michael

2017-01-01

The myosin superfamily of molecular motors utilizes energy from ATP hydrolysis to generate force and motility along actin filaments in a diverse array of cellular processes. These motors are structurally, kinetically, and mechanically tuned to their specific molecular roles in the cell. Optical trapping techniques have played a central role in elucidating the mechanisms by which myosins generate force and in exposing the remarkable diversity of myosin functions. Here, we present thorough methods for measuring and analyzing interactions between actin and non-processive myosins using optical trapping techniques. PMID:27844441

Physics of Cellular Movements

NASA Astrophysics Data System (ADS)

Sackmann, Erich; Keber, Felix; Heinrich, Doris

2010-04-01

The survival of cells depends on perpetual active motions, including (a) bending excitations of the soft cell envelopes, (b) the bidirectional transport of materials and organelles between the cell center and the periphery, and (c) the ongoing restructuring of the intracellular macromolecular scaffolds mediating global cell changes associated with cell adhesion locomotion and phagocytosis. Central questions addressed are the following: How can this bustling motion of extremely complex soft structures be characterized and measured? What are the major driving forces? Further topics include (a) the active dynamic control of global shape changes by the interactive coupling of the aster-like soft scaffold of microtubules and the network of actin filaments associated with the cell envelope (the actin cortex) and (b) the generation of propulsion forces by solitary actin gelation waves propagating within the actin cortex.

Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes.

PubMed

Rai, Ashim; Pathak, Divya; Thakur, Shreyasi; Singh, Shampa; Dubey, Alok Kumar; Mallik, Roop

2016-02-11

Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Fluid mechanics as a driver of tissue-scale mechanical signaling in organogenesis.

PubMed

Gilbert, Rachel M; Morgan, Joshua T; Marcin, Elizabeth S; Gleghorn, Jason P

2016-12-01

Organogenesis is the process during development by which cells self-assemble into complex, multi-scale tissues. Whereas significant focus and research effort has demonstrated the importance of solid mechanics in organogenesis, less attention has been given to the fluid forces that provide mechanical cues over tissue length scales. Fluid motion and pressure is capable of creating spatial gradients of forces acting on cells, thus eliciting distinct and localized signaling patterns essential for proper organ formation. Understanding the multi-scale nature of the mechanics is critically important to decipher how mechanical signals sculpt developing organs. This review outlines various mechanisms by which tissues generate, regulate, and sense fluid forces and highlights the impact of these forces and mechanisms in case studies of normal and pathological development.

Reconstitution of Contractile FtsZ Rings in Liposomes

PubMed Central

Osawa, Masaki; Anderson, David E.; Erickson, Harold P.

2009-01-01

FtsZ is a tubulin homolog and the major cytoskeletal protein in bacterial cell division. It assembles into the Z ring, which contains FtsZ and a dozen other division proteins, and constricts to divide the cell. We have constructed a membrane-targeted FtsZ (FtsZ-mts) by splicing an amphipathic helix to its C terminus. When mixed with lipid vesicles, FtsZ-mts was incorporated into the interior of some tubular vesicles. There it formed multiple Z rings that could move laterally in both directions along the length of the liposome and coalesce into brighter Z rings. Brighter Z rings produced visible constrictions in the liposome, suggesting that FtsZ itself can assemble the Z ring and generate a force. No other proteins were needed for assembly and force generation. PMID:18420899

Forced expression of Hnf4a induces hepatic gene activation through directed differentiation.

PubMed

Yahoo, Neda; Pournasr, Behshad; Rostamzadeh, Jalal; Fathi, Fardin

2016-08-05

Embryonic stem (ES) cells are capable of unlimited self-renewal and have a diverse differentiation potential. These unique features make ES cells as an attractive source for developmental biology studies. Having the mature hepatocyte in the lab with functional activities is valuable in drug discovery studies. Overexpression of hepatocyte lineage-specific transcription factors (TFs) becomes a promising approach in pluripotent cell differentiation toward liver cells. Many studies generate transgenic ES cell lines to examine the effects of specific TFs overexpression in cell differentiation. In the present report, we have addressed whether a suspension or adherent model of differentiation is an appropriate way to study the role of Hnf4a overexpression. We generated ES cells that carried a doxycycline (Dox)-inducible Hnf4a using lentiviral vectors. The transduced cells were subjected to induced Hnf4a overexpression through both spontaneous and directed differentiation methods. Gene expression analysis showed substantially increased expression of hepatic gene markers, particularly Ttr and endogenous Hnf4a, in transduced cells differentiated by the directed approach. These results demonstrated that forced expression of TFs during directed differentiation would be an appropriate way to study relevant gene activation and the effects of overexpression in the context of hepatic differentiation. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanical Coupling of Smooth Muscle Cells Using Microengineered Substrates and Local Stimulation

NASA Astrophysics Data System (ADS)

Copeland, Craig; Hunter, David; Tung, Leslie; Chen, Christopher; Reich, Daniel

2013-03-01

Mechanical stresses directly affect many cellular processes, including signal transduction, growth, differentiation, and survival. Cells can themselves generate such stresses by activating myosin to contract the actin cytoskeleton, which in turn can regulate both cell-substrate and cell-cell interactions. We are studying mechanical forces at cell-cell and cell-substrate interactions using arrays of selectively patterned flexible PDMS microposts combined with the ability to apply local chemical stimulation. Micropipette ``spritzing'', a laminar flow technique, uses glass micropipettes mounted on a microscope stage to deliver drugs to controlled regions within a cellular construct while cell traction forces are recorded via the micropost array. The pipettes are controlled by micromanipulators allowing for rapid and precise movement across the array and the ability to treat multiple constructs within a sample. This technique allows for observing the propagation of a chemically induced mechanical stimulus through cell-cell and cell-substrate interactions. We have used this system to administer the acto-myosin inhibitors Blebbistatin and Y-27632 to single cells and observed the subsequent decrease in cell traction forces. Experiments using trypsin-EDTA have shown this system to be capable of single cell manipulation through removal of one cell within a pair configuration while leaving the other cell unaffected. This project is supported in part by NIH grant HL090747

Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen.

PubMed

Thievessen, Ingo; Fakhri, Nikta; Steinwachs, Julian; Kraus, Viola; McIsaac, R Scott; Gao, Liang; Chen, Bi-Chang; Baird, Michelle A; Davidson, Michael W; Betzig, Eric; Oldenbourg, Rudolf; Waterman, Clare M; Fabry, Ben

2015-11-01

Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation. © FASEB.

Targeted organ generation using Mixl1-inducible mouse pluripotent stem cells in blastocyst complementation.

PubMed

Kobayashi, Toshihiro; Kato-Itoh, Megumi; Nakauchi, Hiromitsu

2015-01-15

Generation of functional organs from patients' own cells is one of the ultimate goals of regenerative medicine. As a novel approach to creation of organs from pluripotent stem cells (PSCs), we employed blastocyst complementation in organogenesis-disabled animals and successfully generated PSC-derived pancreas and kidneys. Blastocyst complementation, which exploits the capacity of PSCs to participate in forming chimeras, does not, however, exclude contribution of PSCs to the development of tissues-including neural cells or germ cells-other than those specifically targeted by disabling of organogenesis. This fact provokes ethical controversy if human PSCs are to be used. In this study, we demonstrated that forced expression of Mix-like protein 1 (encoded by Mixl1) can be used to guide contribution of mouse embryonic stem cells to endodermal organs after blastocyst injection. We then succeeded in applying this method to generate functional pancreas in pancreatogenesis-disabled Pdx1 knockout mice using a newly developed tetraploid-based organ-complementation method. These findings hold promise for targeted organ generation from patients' own PSCs in livestock animals.

Force Exertion and Transmission in Cross-Linked Actin Networks

NASA Astrophysics Data System (ADS)

Stam, Samantha

Cells are responsive to external cues in their environment telling them to proliferate or migrate within their surrounding tissue. Sensing of cues that are mechanical in nature, such stiffness of a tissue or forces transmitted from other cells, is believed to involve the cytoskeleton of a cell. The cytoskeleton is a complex network of proteins consisting of polymers that provide structural support, motor proteins that remodel these structures, and many others. We do not yet have a complete understanding of how cytoskeletal components respond to either internal or external mechanical force and stiffness. Such an understanding should involve mechanisms by which constituent molecules, such as motor proteins, are responsive to mechanics. Additionally, physical models of how forces are transmitted through biopolymer networks are necessary. My research has focused on networks formed by the cytoskeletal filament actin and the molecular motor protein myosin II. Actin filaments form networks and bundles that form a structural framework of the cell, and myosin II slides actin filaments. In this thesis, we show that stiffness of an elastic load that opposes myosin-generated actin sliding has a very sharp effect on the myosin force output in simulations. Secondly, we show that the stiffness and connectivity of cytoskeletal filaments regulates the contractility and anisotropy of network deformations that transmit force on material length scales. Together, these results have implications for predicting and interpreting the deformations and forces in biopolymeric active materials.

InGaN working electrodes with assisted bias generated from GaAs solar cells for efficient water splitting.

PubMed

Liu, Shu-Yen; Sheu, J K; Lin, Yu-Chuan; Chen, Yu-Tong; Tu, S J; Lee, M L; Lai, W C

2013-11-04

Hydrogen generation through water splitting by n-InGaN working electrodes with bias generated from GaAs solar cell was studied. Instead of using an external bias provided by power supply, a GaAs-based solar cell was used as the driving force to increase the rate of hydrogen production. The water-splitting system was tuned using different approaches to set the operating points to the maximum power point of the GaAs solar cell. The approaches included changing the electrolytes, varying the light intensity, and introducing the immersed ITO ohmic contacts on the working electrodes. As a result, the hybrid system comprising both InGaN-based working electrodes and GaAs solar cells operating under concentrated illumination could possibly facilitate efficient water splitting.

The Toxoplasma Acto-MyoA Motor Complex Is Important but Not Essential for Gliding Motility and Host Cell Invasion

PubMed Central

Jackson, Allison J.; Whitelaw, Jamie A.; Pall, Gurman; Black, Jennifer Ann; Ferguson, David J. P.; Tardieux, Isabelle; Mogilner, Alex; Meissner, Markus

2014-01-01

Apicomplexan parasites are thought to actively invade the host cell by gliding motility. This movement is powered by the parasite's own actomyosin system, and depends on the regulated polymerisation and depolymerisation of actin to generate the force for gliding and host cell penetration. Recent studies demonstrated that Toxoplasma gondii can invade the host cell in the absence of several core components of the invasion machinery, such as the motor protein myosin A (MyoA), the microneme proteins MIC2 and AMA1 and actin, indicating the presence of alternative invasion mechanisms. Here the roles of MyoA, MLC1, GAP45 and Act1, core components of the gliding machinery, are re-dissected in detail. Although important roles of these components for gliding motility and host cell invasion are verified, mutant parasites remain invasive and do not show a block of gliding motility, suggesting that other mechanisms must be in place to enable the parasite to move and invade the host cell. A novel, hypothetical model for parasite gliding motility and invasion is presented based on osmotic forces generated in the cytosol of the parasite that are converted into motility. PMID:24632839

The Cytoskeleton and Force Response Mechanisms

NASA Technical Reports Server (NTRS)

Allen, Philip Goodwin

2003-01-01

The long term aim of this project was to define the mechanisms by which cells sense and respond to the physical forces experienced at 1g and missing in microgravity. Identification and characterization of the elements of the cells force response mechanism could provide pathways and molecules to serve as targets for pharmacological intervention to mitigate the pathologic effects of microgravity. Mechanical forces experienced by the organism can be transmitted to cells through molecules that allow cells to bind to the extracellular matrix and through other types of molecules which bind cells to each other. These molecules are coupled in large complexes of proteins to structural elements such as the actin cytoskeleton that give the cell the ability to sense, resist and respond to force. Application of small forces to tissue culture cells causes local elevation of intracellular calcium through stretch activated ion channels, increased tyrosine phosphorylation and a restructuring of the actin cytoskeleton. Using collagen coated iron oxide beads and strong magnets, we can apply different levels of force to cells in culture. We have found that force application causes the cells to polymerize actin at the site of mechanical deformation and unexpectedly, to depolymerize actin across the rest of the cell. Observations of GFP- actin expressing cells demonstrate that actin accumulates at the site of deformation within the first five minutes of force application and is maintained for many tens of minutes after force is removed. Consistent with the reinforcement of the cytoskeletal structures underlying the integrin-bead interaction, force also alters the motion of bound magnetic beads. This effect is seen following the removal of the magnetic field, and is only partially ablated by actin disruption with cytochalsin B. While actin is polymerizing locally at the site of force application, force also stimulates a global reduction in actin filament content within the cells. We have examined the roles of several actin filament disassembly factors in the global reduction of cellular actin filaments. The calcium regulated actin filament severing protein gelsolin is not necessary for the increased actin turnover, as cells derived from gelsolin null and wildtype mice still show a reduction in total actin filament content. Instead, our work suggests that the actin binding protein cofilin may be important for these changes in actin dynamics. Cofilin binds to and enhances the disassembly of actin filaments. Using immunological methods, we observe transient changes in the phosphorylation state of cofilin upon force application that suggests that cofilin may mediate actin filament turnover. Early after force application, cofilin is transiently dephosphorylated, activating its actin disassembly activity. Subsequently, we find a hyper-phosphorylation of cofilin, rendering it inactive. This reduction in cofilin activity may explain the stability of the force induced actin structuttes. In testing this hypothesis, we aimed to generate cells that express the constituitively active kinase (LIM-kinase) that phosphorylates cofilin. lnidial attempts in the cell lines used for the our previous studies proved unsuccessful. While we prepare this work for pubication, we are continuing to study other cell lines and tissue sources to determine whether they show a reduction in F-actin content after force application.

Force-activatable biosensor enables single platelet force mapping directly by fluorescence imaging.

PubMed

Wang, Yongliang; LeVine, Dana N; Gannon, Margaret; Zhao, Yuanchang; Sarkar, Anwesha; Hoch, Bailey; Wang, Xuefeng

2018-02-15

Integrin-transmitted cellular forces are critical for platelet adhesion, activation, aggregation and contraction during hemostasis and thrombosis. Measuring and mapping single platelet forces are desired in both research and clinical applications. Conventional force-to-strain based cell traction force microscopies have low resolution which is not ideal for cellular force mapping in small platelets. To enable platelet force mapping with submicron resolution, we developed a force-activatable biosensor named integrative tension sensor (ITS) which directly converts molecular tensions to fluorescent signals, therefore enabling cellular force mapping directly by fluorescence imaging. With ITS, we mapped cellular forces in single platelets at 0.4µm resolution. We found that platelet force distribution has strong polarization which is sensitive to treatment with the anti-platelet drug tirofiban, suggesting that the ITS force map can report anti-platelet drug efficacy. The ITS also calibrated integrin molecular tensions in platelets and revealed two distinct tension levels: 12-54 piconewton (nominal values) tensions generated during platelet adhesion and tensions above 54 piconewton generated during platelet contraction. Overall, the ITS is a powerful biosensor for the study of platelet mechanobiology, and holds great potential in antithrombotic drug development and assessing platelet activity in health and disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Energy level alignment in TiO2/metal sulfide/polymer interfaces for solar cell applications.

PubMed

Lindblad, Rebecka; Cappel, Ute B; O'Mahony, Flannan T F; Siegbahn, Hans; Johansson, Erik M J; Haque, Saif A; Rensmo, Håkan

2014-08-28

Semiconductor sensitized solar cell interfaces have been studied with photoelectron spectroscopy to understand the interfacial electronic structures. In particular, the experimental energy level alignment has been determined for complete TiO2/metal sulfide/polymer interfaces. For the metal sulfides CdS, Sb2S3 and Bi2S3 deposited from single source metal xanthate precursors, it was shown that both driving forces for electron injection into TiO2 and hole transfer to the polymer decrease for narrower bandgaps. The energy level alignment results were used in the discussion of the function of solar cells with the same metal sulfides as light absorbers. For example Sb2S3 showed the most favourable energy level alignment with 0.3 eV driving force for electron injection and 0.4 eV driving force for hole transfer and also the most efficient solar cells due to high photocurrent generation. The energy level alignment of the TiO2/Bi2S3 interface on the other hand showed no driving force for electron injection to TiO2, and the performance of the corresponding solar cell was very low.

Atomic force-multi-optical imaging integrated microscope for monitoring molecular dynamics in live cells.

PubMed

Trache, Andreea; Meininger, Gerald A

2005-01-01

A novel hybrid imaging system is constructed integrating atomic force microscopy (AFM) with a combination of optical imaging techniques that offer high spatial resolution. The main application of this instrument (the NanoFluor microscope) is the study of mechanotransduction with an emphasis on extracellular matrix-integrin-cytoskeletal interactions and their role in the cellular responses to changes in external chemical and mechanical factors. The AFM allows the quantitative assessment of cytoskeletal changes, binding probability, adhesion forces, and micromechanical properties of the cells, while the optical imaging applications allow thin sectioning of the cell body at the coverslip-cell interface, permitting the study of focal adhesions using total internal reflection fluorescence (TIRF) and internal reflection microscopy (IRM). Combined AFM-optical imaging experiments show that mechanical stimulation at the apical surface of cells induces a force-generating cytoskeletal response, resulting in focal contact reorganization on the basal surface that can be monitored in real time. The NanoFluor system is also equipped with a novel mechanically aligned dual camera acquisition system for synthesized Forster resonance energy transfer (FRET). The integrated NanoFluor microscope system is described, including its characteristics, applications, and limitations.

Entropic forces drive contraction of cytoskeletal networks.

PubMed

Braun, Marcus; Lansky, Zdenek; Hilitski, Feodor; Dogic, Zvonimir; Diez, Stefan

2016-05-01

The cytoskeleton is a network of interconnected protein filaments, which provide a three-dimensional scaffold for cells. Remodeling of the cytoskeleton is important for key cellular processes, such as cell motility, division, or morphogenesis. This remodeling is traditionally considered to be driven exclusively by processes consuming chemical energy, such as the dynamics of the filaments or the action of molecular motors. Here, we review two mechanisms of cytoskeletal network remodeling that are independent of the consumption of chemical energy. In both cases directed motion of overlapping filaments is driven by entropic forces, which arise from harnessing thermal energy present in solution. Entropic forces are induced either by macromolecular crowding agents or by diffusible crosslinkers confined to the regions where filaments overlap. Both mechanisms increase filament overlap length and lead to the contraction of filament networks. These force-generating mechanisms, together with the chemical energy-dependent mechanisms, need to be considered for the comprehensive quantitative picture of the remodeling of cytoskeletal networks in cells. © 2016 WILEY Periodicals, Inc.

Opto electronic tweezers based smart sweeper for cells/micro-particles sorting

NASA Astrophysics Data System (ADS)

Verma, R. S.; Kumar, N.

2018-04-01

We report on use of opto-electronic tweezers based sorting approach, termed as smart sweepers, for sorting the microscopic particles by using the Dielectrophoretic (DEP) force response of cells on applied a.c. bias frequency. The applied a.c. bias was kept in negative DEP region, close to the crossover frequency of one of the particles. A line shaped intensity pattern, generated by a cylindrical lens, was scanned across the mixture sample. The particles whose cross over frequency was close to the applied bias frequency, experienced negligible negative DEP(n-DEP) force. On the other hand, the other type of particle experienced large repelling force and were forced to move along the scanning direction of the line shaped intensity profile. We, as a proof of concept, demonstrated the working principle of opto electronic smart sweepers by sweeping out the polystyrene particles from a mixture consisting of polystyrene microspheres (PSM) and red blood cells (RBCs) and leaving RBCs in the region of interest.

Effects of G6PD activity inhibition on the viability, ROS generation and mechanical properties of cervical cancer cells.

PubMed

Fang, Zishui; Jiang, Chengrui; Feng, Yi; Chen, Rixin; Lin, Xiaoying; Zhang, Zhiqiang; Han, Luhao; Chen, Xiaodan; Li, Hongyi; Guo, Yibin; Jiang, Weiying

2016-09-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency has been revealed to be involved in the efficacy to anti-cancer therapy but the mechanism remains unclear. We aimed to investigate the anti-cancer mechanism of G6PD deficiency. In our study, dehydroepiandrosterone (DHEA) and shRNA technology were used for inhibiting the activity of G6PD of cervical cancer cells. Peak Force QNM Atomic Force Microscopy was used to assess the changes of topography and biomechanical properties of cells and detect the effects on living cells in a natural aqueous environment. Flow cytometry was used to detect the apoptosis and reactive oxygen species (ROS) generation. Scanning electron microscopy was used to observe cell morphology. Moreover, a laser scanning confocal microscope was used to observe the alterations in cytoskeleton to explore the involved mechanism. When G6PD was inhibited by DHEA or RNA interference, the abnormal Young's modulus and increased roughness of cell membrane were observed in HeLa cells, as well as the idioblasts. Simultaneously, G6PD deficiency resulted in decreased HeLa cells migration and proliferation ability but increased ROS generation inducing apoptosis. What's more, the inhibition of G6PD activity caused the disorganization of microfilaments and microtubules of cytoskeletons and cell shrinkage. Our results indicated the anti-cervix cancer mechanism of G6PD deficiency may be involved with the decreased cancer cells migration and proliferation ability as a result of abnormal reorganization of cell cytoskeleton and abnormal biomechanical properties caused by the increased ROS. Suppression of G6PD may be a promising strategy in developing novel therapeutic methods for cervical cancer. Copyright © 2016 Elsevier B.V. All rights reserved.

Apical constriction: themes and variations on a cellular mechanism driving morphogenesis

PubMed Central

Martin, Adam C.; Goldstein, Bob

2014-01-01

Apical constriction is a cell shape change that promotes tissue remodeling in a variety of homeostatic and developmental contexts, including gastrulation in many organisms and neural tube formation in vertebrates. In recent years, progress has been made towards understanding how the distinct cell biological processes that together drive apical constriction are coordinated. These processes include the contraction of actin-myosin networks, which generates force, and the attachment of actin networks to cell-cell junctions, which allows forces to be transmitted between cells. Different cell types regulate contractility and adhesion in unique ways, resulting in apical constriction with varying dynamics and subcellular organizations, as well as a variety of resulting tissue shape changes. Understanding both the common themes and the variations in apical constriction mechanisms promises to provide insight into the mechanics that underlie tissue morphogenesis. PMID:24803648

Fibroblasts Lead the Way: A Unified View of 3D Cell Motility.

PubMed

Petrie, Ryan J; Yamada, Kenneth M

2015-11-01

Primary human fibroblasts are remarkably adaptable, able to migrate in differing types of physiological 3D tissue and on rigid 2D tissue culture surfaces. The crawling behavior of these and other vertebrate cells has been studied intensively, which has helped generate the concept of the cell motility cycle as a comprehensive model of 2D cell migration. However, this model fails to explain how cells force their large nuclei through the confines of a 3D matrix environment and why primary fibroblasts can use more than one mechanism to move in 3D. Recent work shows that the intracellular localization of myosin II activity is governed by cell-matrix interactions to both force the nucleus through the extracellular matrix (ECM) and dictate the type of protrusions used to migrate in 3D. Published by Elsevier Ltd.

Fluid mechanics as a driver of tissue-scale mechanical signaling in organogenesis

PubMed Central

Gilbert, Rachel M.; Morgan, Joshua T.; Marcin, Elizabeth S.; Gleghorn, Jason P.

2016-01-01

Purpose of Review Organogenesis is the process during development by which cells self-assemble into complex, multi-scale tissues. Whereas significant focus and research effort has demonstrated the importance of solid mechanics in organogenesis, less attention has been given to the fluid forces that provide mechanical cues over tissue length scales. Recent Findings Fluid motion and pressure is capable of creating spatial gradients of forces acting on cells, thus eliciting distinct and localized signaling patterns essential for proper organ formation. Understanding the multi-scale nature of the mechanics is critically important to decipher how mechanical signals sculpt developing organs. Summary This review outlines various mechanisms by which tissues generate, regulate, and sense fluid forces and highlights the impact of these forces and mechanisms in case studies of normal and pathological development. PMID:28163984

Ground Vehicle Power and Mobility Overview - Germany Visit

DTIC Science & Technology

2011-11-10

the current and future force Survivability Robotics – Intelligent Systems Vehicle Electronics & Architecture Fuel, Water, Bridging ...Test Cell • Engine Generator Test Lab • Full Vehicle Environmental Test Cell • Hybrid Electric Reconfigurable Moveable Integration Testbed (HERMIT...Converter Conducted competitive runoff evaluations on Bridging Boat engine candidates Completed independent durability assessment of OEM

ERK reinforces actin polymerization to power persistent edge protrusion during motility

PubMed Central

Mendoza, Michelle C.; Vilela, Marco; Juarez, Jesus E.; Blenis, John; Danuser, Gaudenz

2016-01-01

Cells move through perpetual protrusion and retraction cycles at the leading edge. These cycles are coordinated with substrate adhesion and retraction of the cell rear. Here, we tracked spatial and temporal fluctuations in the molecular activities of individual moving cells to elucidate how extracellular regulated kinase (ERK) signaling controlled the dynamics of protrusion and retraction cycles. ERK is activated by many cell-surface receptors and we found that ERK signaling specifically reinforced cellular protrusions so that they translated into rapid, sustained forward motion of the leading edge. Using quantitative fluorescent speckle microscopy (qFSM) and cross-correlation analysis, we showed that ERK controlled the rate and timing of actin polymerization by promoting the recruitment of the actin nucleator Arp2/3 to the leading edge. Arp2/3 activity generates branched actin networks that can produce pushing force. These findings support a model in which surges in ERK activity induced by extracellular cues enhance Arp2/3-mediated actin polymerization to generate protrusion power phases with enough force to counteract increasing membrane tension and to promote sustained motility. PMID:25990957

F-actin cross-linking enhances the stability of force generation in disordered actomyosin networks

NASA Astrophysics Data System (ADS)

Jung, Wonyeong; Murrell, Michael P.; Kim, Taeyoon

2015-12-01

Myosin molecular motors and actin cross-linking proteins (ACPs) are known to mediate the generation and transmission of mechanical forces within the cortical F-actin cytoskeleton that drive major cellular processes such as cell division and migration. However, how motors and ACPs interact collectively over diverse timescales to modulate the time-dependent mechanical properties of the cytoskeleton remains unclear. In this study, we present a three-dimensional agent-based computational model of the cortical actomyosin network to quantitatively determine the effects of motor activity and the density and kinetics of ACPs on the accumulation and maintenance of mechanical tension within a disordered actomyosin network. We found that motors accumulate large stress quickly by behaving as temporary cross-linkers although this stress is relaxed over time unless there are sufficient passive ACPs to stabilize the network. Stabilization by ACPs helps motors to generate forces up to their maximum potential, leading to significant enhancement of the efficiency and stability of stress generation. Thus, we demonstrated that the force-dependent kinetics of ACP dissociation plays a critical role for the accumulation and sustainment of stress and the structural remodeling of networks.

Manipulation of cells' position across a microfluidic channel using a series of continuously varying herringbone structures

NASA Astrophysics Data System (ADS)

Jung, Yugyung; Hyun, Ji-chul; Choi, Jongchan; Atajanov, Arslan; Yang, Sung

2017-12-01

Controlling cells' movement is an important technique in biological analysis that is performed within a microfluidic system. Many external forces are utilized for manipulation of cells, including their position in the channel. These forces can effectively control cells in a desired manner. Most of techniques used to manipulate cells require sophisticated set-ups and equipment to generate desired effect. The exception to this is the use of hydrodynamic force. In this study, a series of continuously varying herringbone structures is proposed for positioning cells in a microfluidic channel using hydrodynamic force. This structure was experimentally developed by changing parameters, such as the length of the herringbone's apex, the length of the herringbone's base and the ratio of the height of the flat channel to the height of the herringbone structure. Results of this study, have demonstrated that the length of the herringbone's apex and the ratio of the heights of the flat channel and the herringbone structure were crucial parameters influencing positioning of cells at 100 μl/h flow rate. The final design was fixed at 170 and 80 μm for the length of herringbone's apex and the length of herringbone's base, respectively. The average position of cells in this device was 34 μm away from the side wall in a 200 μm wide channel. Finally, to substantiate a practical application of the herringbone structure for positioning, cells were randomly introduced into a microfluidic device, containing an array of trapping structures together with a series of herringbone structures along the channel. The cells were moved toward the trapping structure by the herringbone structure and the trapping efficiency was increased. Therefore, it is anticipated that this device will be utilized to continuously control cells' position without application of external forces.

Focal adhesion kinase is involved in mechanosensing during fibroblast migration

NASA Technical Reports Server (NTRS)

Wang, H. B.; Dembo, M.; Hanks, S. K.; Wang, Y.

2001-01-01

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase localized at focal adhesions and is believed to mediate adhesion-stimulated effects. Although ablation of FAK impairs cell movement, it is not clear whether FAK might be involved in the guidance of cell migration, a role consistent with its putative regulatory function. We have transfected FAK-null fibroblasts with FAK gene under the control of the tetracycline repression system. Cells were cultured on flexible polyacrylamide substrates for the detection of traction forces and the application of mechanical stimulation. Compared with control cells expressing wild-type FAK, FAK-null cells showed a decrease in migration speed and directional persistence. In addition, whereas FAK-expressing cells responded to exerted forces by reorienting their movements and forming prominent focal adhesions, FAK-null cells failed to show such responses. Furthermore, FAK-null cells showed impaired responses to decreases in substrate flexibility, which causes control cells to generate weaker traction forces and migrate away from soft substrates. Cells expressing Y397F FAK, which cannot be phosphorylated at a key tyrosine site, showed similar defects in migration pattern and force-induced reorientation as did FAK-null cells. However, other aspects of F397-FAK cells, including the responses to substrate flexibility and the amplification of focal adhesions upon mechanical stimulation, were similar to that of control cells. Our results suggest that FAK plays an important role in the response of migrating cells to mechanical input. In addition, phosphorylation at Tyr-397 is required for some, but not all, of the functions of FAK in cell migration.

Force-balance model of suppression of multipolar division in cancer cells with extra centrosomes

NASA Astrophysics Data System (ADS)

Zhu, Jie

2013-03-01

Cancer cells often possess extra centrosomes which have the potential to cause cell death due to catastrophic multipolar division. Many cancer cells, however, are able to escape multipolar mitosis by clustering the extra centrosomes to form bipolar spindles. The mechanism of centrosome clustering is therefore of great interest to the development of anti-cancer drugs because the de-clustering of extra centrosomes provides an appealing way to eliminate cancer cells while keeping healthy cells intact. We present a physical model assuming 1) dynamic centrosomal microtubules interact with chromosomes by both pushing on chromosome arms and pulling along kinetochores; 2) these microtubules interact with force generators associated with actin/adhesion structures at the cell boundary; and 3) motors act on anti-parallel microtubules from different centrosomes. We find via computer simulations that chromosomes tend to aggregate near the cell center while centrosomes can be either clustered to form bipolar spindles or scattered to form multipolar spindles, depending on the strengths of relative forces, cell shape and adhesion geometry. The model predictions agree with data from cells plated on adhesive micropatterns and from biochemically or genetically perturbed cells. Furthermore, our model is able to explain various microtubule distributions in interphase cells on patterned substrates. This work was supported by NSF

Magnetic Nanotweezers for Interrogating Biological Processes in Space and Time.

PubMed

Kim, Ji-Wook; Jeong, Hee-Kyung; Southard, Kaden M; Jun, Young-Wook; Cheon, Jinwoo

2018-04-17

The ability to sense and manipulate the state of biological systems has been extensively advanced during the past decade with the help of recent developments in physical tools. Unlike standard genetic and pharmacological perturbation techniques-knockdown, overexpression, small molecule inhibition-that provide a basic on/off switching capability, these physical tools provide the capacity to control the spatial, temporal, and mechanical properties of the biological targets. Among the various physical cues, magnetism offers distinct advantages over light or electricity. Magnetic fields freely penetrate biological tissues and are already used for clinical applications. As one of the unique features, magnetic fields can be transformed into mechanical stimuli which can serve as a cue in regulating biological processes. However, their biological applications have been limited due to a lack of high-performance magnetism-to-mechanical force transducers with advanced spatiotemporal capabilities. In this Account, we present recent developments in magnetic nanotweezers (MNTs) as a useful tool for interrogating the spatiotemporal control of cells in living tissue. MNTs are composed of force-generating magnetic nanoparticles and field generators. Through proper design and the integration of individual components, MNTs deliver controlled mechanical stimulation to targeted biomolecules at any desired space and time. We first discuss about MNT configuration with different force-stimulation modes. By modulating geometry of the magnetic field generator, MNTs exert pulling, dipole-dipole attraction, and rotational forces to the target specifically and quantitatively. We discuss the key physical parameters determining force magnitude, which include magnetic field strength, magnetic field gradient, magnetic moment of the magnetic particle, as well as distance between the field generator and the particle. MNTs also can be used over a wide range of biological time scales. By simply adjusting the amplitude and phase of the applied current, MNTs based on electromagnets allow for dynamic control of the magnetic field from microseconds to hours. Chemical design and the nanoscale effects of magnetic particles are also essential for optimizing MNT performance. We discuss key strategies to develop magnetic nanoparticles with improved force-generation capabilities with a particular focus on the effects of size, shape, and composition of the nanoparticles. We then introduce various strategies and design considerations for target-specific biomechanical stimulations with MNTs. One-to-one particle-receptor engagement for delivering a defined force to the targeted receptor and the small size of the nanoparticles are important. Finally, we demonstrate the utility of MNTs for manipulating biological functions and activities with various spatial (single molecule/cell to organisms) and temporal resolution (microseconds to days). MNTs have the potential to be utilized in many exciting applications across diverse biological systems spanning from fundamental biology investigations of spatial and mechanical signaling dynamics at the single-cell and systems levels to in vivo therapeutic applications.

Probing the compressibility of tumor cell nuclei by combined atomic force-confocal microscopy

NASA Astrophysics Data System (ADS)

Krause, Marina; te Riet, Joost; Wolf, Katarina

2013-12-01

The cell nucleus is the largest and stiffest organelle rendering it the limiting compartment during migration of invasive tumor cells through dense connective tissue. We here describe a combined atomic force microscopy (AFM)-confocal microscopy approach for measurement of bulk nuclear stiffness together with simultaneous visualization of the cantilever-nucleus contact and the fate of the cell. Using cantilevers functionalized with either tips or beads and spring constants ranging from 0.06-10 N m-1, force-deformation curves were generated from nuclear positions of adherent HT1080 fibrosarcoma cell populations at unchallenged integrity, and a nuclear stiffness range of 0.2 to 2.5 kPa was identified depending on cantilever type and the use of extended fitting models. Chromatin-decondensating agent trichostatin A (TSA) induced nuclear softening of up to 50%, demonstrating the feasibility of our approach. Finally, using a stiff bead-functionalized cantilever pushing at maximal system-intrinsic force, the nucleus was deformed to 20% of its original height which after TSA treatment reduced further to 5% remaining height confirming chromatin organization as an important determinant of nuclear stiffness. Thus, combined AFM-confocal microscopy is a feasible approach to study nuclear compressibility to complement concepts of limiting nuclear deformation in cancer cell invasion and other biological processes.

Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells.

PubMed

Zhang, Yuejin; Wei, Fuxiang; Poh, Yeh-Chuin; Jia, Qiong; Chen, Junjian; Chen, Junwei; Luo, Junyu; Yao, Wenting; Zhou, Wenwen; Huang, Wei; Yang, Fang; Zhang, Yao; Wang, Ning

2017-07-01

Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only a few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. 3D-magnetic twisting cytometry (3D-MTC) is a technique for applying local mechanical stresses to living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors, followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic-field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super-resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real-time acquisition of a living cell's mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC-microscopy platform takes ∼20 d to construct, and the experimental procedures require ∼4 d when carried out by a life sciences graduate student.

Engineering an artificial amoeba propelled by nanoparticle-triggered actin polymerization

NASA Astrophysics Data System (ADS)

Yi, Jinsoo; Schmidt, Jacob; Chien, Aichi; Montemagno, Carlo D.

2009-02-01

We have engineered an amoeba system combining nanofabricated inorganic materials with biological components, capable of propelling itself via actin polymerization. The nanofabricated materials have a mechanism similar to the locomotion of the Listeria monocytogenes, food poisoning bacteria. The propulsive force generation utilizes nanoparticles made from nickel and gold functionalized with the Listeria monocytogenes transmembrane protein, ActA. These Listeria-mimic nanoparticles were in concert with actin, actin binding proteins, ATP (adenosine triphosphate) and encapsulated within a lipid vesicle. This system is an artificial cell, such as a vesicle, where artificial nanobacteria and actin polymerization machinery are used in driving force generators inside the cell. The assembled structure was observed to crawl on a glass surface analogously to an amoeba, with the speed of the movement dependent on the amount of actin monomers and ATP present.

Engineering an artificial amoeba propelled by nanoparticle-triggered actin polymerization.

PubMed

Yi, Jinsoo; Schmidt, Jacob; Chien, Aichi; Montemagno, Carlo D

2009-02-25

We have engineered an amoeba system combining nanofabricated inorganic materials with biological components, capable of propelling itself via actin polymerization. The nanofabricated materials have a mechanism similar to the locomotion of the Listeria monocytogenes, food poisoning bacteria. The propulsive force generation utilizes nanoparticles made from nickel and gold functionalized with the Listeria monocytogenes transmembrane protein, ActA. These Listeria-mimic nanoparticles were in concert with actin, actin binding proteins, ATP (adenosine triphosphate) and encapsulated within a lipid vesicle. This system is an artificial cell, such as a vesicle, where artificial nanobacteria and actin polymerization machinery are used in driving force generators inside the cell. The assembled structure was observed to crawl on a glass surface analogously to an amoeba, with the speed of the movement dependent on the amount of actin monomers and ATP present.

Cellular Contraction and Polarization Drive Collective Cellular Motion.

PubMed

Notbohm, Jacob; Banerjee, Shiladitya; Utuje, Kazage J C; Gweon, Bomi; Jang, Hwanseok; Park, Yongdoo; Shin, Jennifer; Butler, James P; Fredberg, Jeffrey J; Marchetti, M Cristina

2016-06-21

Coordinated motions of close-packed multicellular systems typically generate cooperative packs, swirls, and clusters. These cooperative motions are driven by active cellular forces, but the physical nature of these forces and how they generate collective cellular motion remain poorly understood. Here, we study forces and motions in a confined epithelial monolayer and make two experimental observations: 1) the direction of local cellular motion deviates systematically from the direction of the local traction exerted by each cell upon its substrate; and 2) oscillating waves of cellular motion arise spontaneously. Based on these observations, we propose a theory that connects forces and motions using two internal state variables, one of which generates an effective cellular polarization, and the other, through contractile forces, an effective cellular inertia. In agreement with theoretical predictions, drugs that inhibit contractility reduce both the cellular effective elastic modulus and the frequency of oscillations. Together, theory and experiment provide evidence suggesting that collective cellular motion is driven by at least two internal variables that serve to sustain waves and to polarize local cellular traction in a direction that deviates systematically from local cellular velocity. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Effect of fuel concentration and force on collective transport by a team of dynein motors

PubMed Central

Takshak, Anjneya; Roy, Tanushree; Tandaiya, Parag

2016-01-01

Abstract Motor proteins are essential components of intracellular transport inside eukaryotic cells. These protein molecules use chemical energy obtained from hydrolysis of ATP to produce mechanical forces required for transporting cargos inside cells, from one location to another, in a directed manner. Of these motors, cytoplasmic dynein is structurally more complex than other motor proteins involved in intracellular transport, as it shows force and fuel (ATP) concentration dependent step‐size. Cytoplasmic dynein motors are known to work in a team during cargo transport and force generation. Here, we use a complete Monte‐Carlo model of single dynein constrained by in vitro experiments, which includes the effect of both force and ATP on stepping as well as detachment of motors under force. We then use our complete Monte‐Carlo model of single dynein motor to understand collective cargo transport by a team of dynein motors, such as dependence of cargo travel distance and velocity on applied force and fuel concentration. In our model, cargos pulled by a team of dynein motors do not detach rapidly under higher forces, confirming the experimental observation of longer persistence time of dynein team on microtubule under higher forces. PMID:27727483

Optogenetic control of RhoA reveals zyxin-mediated elasticity of stress fibres

NASA Astrophysics Data System (ADS)

Oakes, Patrick W.; Wagner, Elizabeth; Brand, Christoph A.; Probst, Dimitri; Linke, Marco; Schwarz, Ulrich S.; Glotzer, Michael; Gardel, Margaret L.

2017-06-01

Cytoskeletal mechanics regulates cell morphodynamics and many physiological processes. While contractility is known to be largely RhoA-dependent, the process by which localized biochemical signals are translated into cell-level responses is poorly understood. Here we combine optogenetic control of RhoA, live-cell imaging and traction force microscopy to investigate the dynamics of actomyosin-based force generation. Local activation of RhoA not only stimulates local recruitment of actin and myosin but also increased traction forces that rapidly propagate across the cell via stress fibres and drive increased actin flow. Surprisingly, this flow reverses direction when local RhoA activation stops. We identify zyxin as a regulator of stress fibre mechanics, as stress fibres are fluid-like without flow reversal in its absence. Using a physical model, we demonstrate that stress fibres behave elastic-like, even at timescales exceeding turnover of constituent proteins. Such molecular control of actin mechanics likely plays critical roles in regulating morphodynamic events.

Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions

PubMed Central

Swaminathan, Vinay; Kalappurakkal, Joseph Mathew; Moore, Travis I.; Koga, Nobuyasu; Baker, David A.; Oldenbourg, Rudolf; Tani, Tomomi; Springer, Timothy A.; Waterman, Clare M.

2017-01-01

Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven “retrograde flow” of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues. PMID:29073038

Characteristics of motive force derived from trajectory analysis of Amoeba proteus.

PubMed

Masaki, Noritaka; Miyoshi, Hiromi; Tsuchiya, Yoshimi

2007-01-01

We used a monochromatic charge-coupled-device camera to observe the migration behavior of Amoeba proteus every 5 s over a time course of 10000 s in order to investigate the characteristics of its centroid movement (cell velocity) over the long term. Fourier transformation of the time series of the cell velocity revealed that its power spectrum exhibits a Lorentz type profile with a relaxation time of a few hundred seconds. Moreover, some sharp peaks were found in the power spectrum, where the ratios of any two frequencies corresponding to the peaks were expressed as simple rational numbers. Analysis of the trajectory using a Langevin equation showed that the power spectrum reflects characteristics of the cell's motive force. These results suggest that some phenomena relating to the cell's motility, such as protoplasmic streaming and the sol-gel transformation of actin filaments, which seem to be independent phenomena and have different relaxation times, interact with each other and cooperatively participate in the generation process of the motive force.

Generation of a Listeria vaccine strain by enhanced Caspase-1 activation

PubMed Central

Warren, Sarah E.; Duong, Hien; Mao, Dat Phat; Armstrong, Abraham; Rajan, Jayant; Miao, Edward A.; Aderem, Alan

2012-01-01

The immunostimulatory properties conferred by vaccine adjuvants require Caspase-1 for processing of IL-1β and IL-18. Caspase-1 is activated in response to a breach of the cytosolic compartment by microbes and the process is initiated by intracellular pattern recognition receptors within inflammasomes. Listeria monocytogenes is detected in the cytosol by the NLRC4, NLRP3 and AIM2 inflammasomes. NLRC4 is activated by flagellin, and L. monocytogenes evades this detector by repressing flagellin expression. We generated an L. monocytogenes strain that was forced to express flagellin in the host cell cytosol. This strain hyperactivated Caspase-1 and was preferentially cleared via NLRC4 detection in an IL-1β/IL-18 independent manner. We also created a strain of L. monocytogenes with forced expression of another NLRC4 agonist, PrgJ from the Type III secretion system of S. typhimurium. Forced expression of flagellin or PrgJ resulted in attenuation, yet both strains conferred protective immunity in mice against lethal challenge with L. monocytogenes. This work is the first demonstration of specific targeting of the Caspase-1 activation pathway to generate a safe and potent L. monocytogenes based vaccine. Moreover, the attenuated strains with embedded flagellin or PrgJ adjuvants, represent attractive vectors for vaccines aimed at eliciting T cell responses. PMID:21538346

Inverse tissue mechanics of cell monolayer expansion.

PubMed

Kondo, Yohei; Aoki, Kazuhiro; Ishii, Shin

2018-03-01

Living tissues undergo deformation during morphogenesis. In this process, cells generate mechanical forces that drive the coordinated cell motion and shape changes. Recent advances in experimental and theoretical techniques have enabled in situ measurement of the mechanical forces, but the characterization of mechanical properties that determine how these forces quantitatively affect tissue deformation remains challenging, and this represents a major obstacle for the complete understanding of morphogenesis. Here, we proposed a non-invasive reverse-engineering approach for the estimation of the mechanical properties, by combining tissue mechanics modeling and statistical machine learning. Our strategy is to model the tissue as a continuum mechanical system and to use passive observations of spontaneous tissue deformation and force fields to statistically estimate the model parameters. This method was applied to the analysis of the collective migration of Madin-Darby canine kidney cells, and the tissue flow and force were simultaneously observed by the phase contrast imaging and traction force microscopy. We found that our monolayer elastic model, whose elastic moduli were reverse-engineered, enabled a long-term forecast of the traction force fields when given the tissue flow fields, indicating that the elasticity contributes to the evolution of the tissue stress. Furthermore, we investigated the tissues in which myosin was inhibited by blebbistatin treatment, and observed a several-fold reduction in the elastic moduli. The obtained results validate our framework, which paves the way to the estimation of mechanical properties of living tissues during morphogenesis.

Pitch, roll, and yaw moment generator for insect-like tailless flapping-wing MAV

NASA Astrophysics Data System (ADS)

Phan, Hoang Vu; Park, Hoon Cheol

2016-04-01

In this work, we proposed a control moment generator, which is called Trailing Edge Change (TEC) mechanism, for attitudes change in hovering insect-like tailless flapping-wing MAV. The control moment generator was installed to the flapping-wing mechanism to manipulate the wing kinematics by adjusting the wing roots location symmetrically or asymmetrically. As a result, the mean aerodynamic force center of each wing is relocated and control moments are generated. The three-dimensional wing kinematics captured by three synchronized high-speed cameras showed that the flapping-wing MAV can properly modify the wing kinematics. In addition, a series of experiments were performed using a multi-axis load cell to evaluate the forces and moments generation. The measurement demonstrated that the TEC mechanism produced reasonable amounts of pitch, roll and yaw moments by shifting position of the trailing edges at the wing roots of the flapping-wing MAV.

Validation of a Waste Heat Recovery Model for a 1kW PEM Fuel Cell using Thermoelectric Generator

NASA Astrophysics Data System (ADS)

Saufi Sulaiman, M.; Mohamed, W. A. N. W.; Singh, B.; Fitrie Ghazali, M.

2017-08-01

Fuel cell is a device that generates electricity through electrochemical reaction between hydrogen and oxygen. A major by-product of the exothermic reaction is waste heat. The recovery of this waste heat has been subject to research on order to improve the overall energy utilization. However, nearly all of the studies concentrate on high temperature fuel cells using advanced thermodynamic cycles due to the high quality of waste heat. The method, characteristics and challenges in harvesting waste heat from a low temperature fuel cell using a direct energy conversion device is explored in this publication. A heat recovery system for an open cathode 1kW Proton Exchange Membrane fuel cell (PEM FC) was developed using a single unit of thermoelectric generator (TEG) attached to a heat pipe. Power output of the fuel cell was varied to obtain the performance of TEG at different stack temperatures. Natural and forced convections modes of cooling were applied to the TEG cold side. This is to simulate the conditions of a mini fuel cell vehicle at rest and in motion. The experimental results were analysed and a mathematical model based on the thermal circuit analogy was developed and compared. Forced convection mode resulted in higher temperature difference, output voltage and maximum power which are 3.3°C, 33.5 mV, and 113.96mW respectively. The heat recovery system for 1 kW Proton Exchange Membrane fuel cell (PEM FC) using single TEG was successfully established and improved the electrical production of fuel cell. Moreover, the experimental results obtained was in a good agreement with theoretical results.

Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

PubMed Central

Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R.; Bryce, Nicole S.; Whan, Renee M.; Hardeman, Edna C.

2015-01-01

The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments. PMID:25978408

Extracellular matrix elasticity and topography: material-based cues that affect cell function via conserved mechanisms

PubMed Central

Janson, Isaac A.; Putnam, Andrew J.

2014-01-01

Chemical, mechanical, and topographic extracellular matrix (ECM) cues have been extensively studied for their influence on cell behavior. These ECM cues alter cell adhesion, cell shape, and cell migration, and activate signal transduction pathways to influence gene expression, proliferation, and differentiation. ECM elasticity and topography, in particular, have emerged as material properties of intense focus based on strong evidence these physical cue can partially dictate stem cell differentiation. Cells generate forces to pull on their adhesive contacts, and these tractional forces appear to be a common element of cells’ responses to both elasticity and topography. This review focuses on recently published work that links ECM topography and mechanics and their influence on differentiation and other cell behaviors, We also highlight signaling pathways typically implicated in mechanotransduction that are (or may be) shared by cells subjected to topographic cues. Finally, we conclude with a brief discussion of the potential implications of these commonalities for cell based therapies and biomaterial design. PMID:24910444

Design of flat pneumatic artificial muscles

NASA Astrophysics Data System (ADS)

Wirekoh, Jackson; Park, Yong-Lae

2017-03-01

Pneumatic artificial muscles (PAMs) have gained wide use in the field of robotics due to their ability to generate linear forces and motions with a simple mechanism, while remaining lightweight and compact. However, PAMs are limited by their traditional cylindrical form factors, which must increase radially to improve contraction force generation. Additionally, this form factor results in overly complicated fabrication processes when embedded fibers and sensor elements are required to provide efficient actuation and control of the PAMs while minimizing the bulkiness of the overall robotic system. In order to overcome these limitations, a flat two-dimensional PAM capable of being fabricated using a simple layered manufacturing process was created. Furthermore, a theoretical model was developed using Von Karman’s formulation for large deformations and the energy methods. Experimental characterizations of two different types of PAMs, a single-cell unit and a multi-cell unit, were performed to measure the maximum contraction lengths and forces at input pressures ranging from 0 to 150 kPa. Experimental data were then used to verify the fidelity of the theoretical model.

Large-scale generation of cell-derived nanovesicles

NASA Astrophysics Data System (ADS)

Jo, W.; Kim, J.; Yoon, J.; Jeong, D.; Cho, S.; Jeong, H.; Yoon, Y. J.; Kim, S. C.; Gho, Y. S.; Park, J.

2014-09-01

Exosomes are enclosed compartments that are released from cells and that can transport biological contents for the purpose of intercellular communications. Research into exosomes is hindered by their rarity. In this article, we introduce a device that uses centrifugal force and a filter with micro-sized pores to generate a large quantity of cell-derived nanovesicles. The device has a simple polycarbonate structure to hold the filter, and operates in a common centrifuge. Nanovesicles are similar in size and membrane structure to exosomes. Nanovesicles contain intracellular RNAs ranging from microRNA to mRNA, intracellular proteins, and plasma membrane proteins. The quantity of nanovesicles produced using the device is 250 times the quantity of naturally secreted exosomes. Also, the quantity of intracellular contents in nanovesicles is twice that in exosomes. Nanovesicles generated from murine embryonic stem cells can transfer RNAs to target cells. Therefore, this novel device and the nanovesicles that it generates are expected to be used in exosome-related research, and can be applied in various applications such as drug delivery and cell-based therapy.

The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles

PubMed Central

Levesque, Aime A.; Compton, Duane A.

2001-01-01

Chromokinesins have been postulated to provide the polar ejection force needed for chromosome congression during mitosis. We have evaluated that possibility by monitoring chromosome movement in vertebrate-cultured cells using time-lapse differential interference contrast microscopy after microinjection with antibodies specific for the chromokinesin Kid. 17.5% of cells injected with Kid-specific antibodies have one or more chromosomes that remain closely opposed to a spindle pole and fail to enter anaphase. In contrast, 82.5% of injected cells align chromosomes in metaphase, progress to anaphase, and display chromosome velocities not significantly different from control cells. However, injected cells lack chromosome oscillations, and chromosome orientation is atypical because chromosome arms extend toward spindle poles during both congression and metaphase. Furthermore, chromosomes cluster into a mass and fail to oscillate when Kid is perturbed in cells containing monopolar spindles. These data indicate that Kid generates the polar ejection force that pushes chromosome arms away from spindle poles in vertebrate-cultured cells. This force increases the efficiency with which chromosomes make bipolar spindle attachments and regulates kinetochore activities necessary for chromosome oscillation, but is not essential for chromosome congression. PMID:11564754

Shaping up synthetic cells

NASA Astrophysics Data System (ADS)

Mulla, Yuval; Aufderhorst-Roberts, Anders; Koenderink, Gijsje H.

2018-07-01

How do the cells in our body reconfigure their shape to achieve complex tasks like migration and mitosis, yet maintain their shape in response to forces exerted by, for instance, blood flow and muscle action? Cell shape control is defined by a delicate mechanical balance between active force generation and passive material properties of the plasma membrane and the cytoskeleton. The cytoskeleton forms a space-spanning fibrous network comprising three subsystems: actin, microtubules and intermediate filaments. Bottom-up reconstitution of minimal synthetic cells where these cytoskeletal subsystems are encapsulated inside a lipid vesicle provides a powerful avenue to dissect the force balance that governs cell shape control. Although encapsulation is technically demanding, a steady stream of advances in this technique has made the reconstitution of shape-changing minimal cells increasingly feasible. In this topical review we provide a route-map of the recent advances in cytoskeletal encapsulation techniques and outline recent reports that demonstrate shape change phenomena in simple biomimetic vesicle systems. We end with an outlook toward the next steps required to achieve more complex shape changes with the ultimate aim of building a fully functional synthetic cell with the capability to autonomously grow, divide and move.

Force Generation by Membrane-Associated Myosin-I

PubMed Central

Pyrpassopoulos, Serapion; Arpağ, Göker; Feeser, Elizabeth A.; Shuman, Henry; Tüzel, Erkan; Ostap, E. Michael

2016-01-01

Vertebrate myosin-IC (Myo1c) is a type-1 myosin that links cell membranes to the cytoskeleton via its actin-binding motor domain and its phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding tail domain. While it is known that Myo1c bound to PtdIns(4,5)P2 in fluid-lipid bilayers can propel actin filaments in an unloaded motility assay, its ability to develop forces against external load on actin while bound to fluid bilayers has not been explored. Using optical tweezers, we measured the diffusion coefficient of single membrane-bound Myo1c molecules by force-relaxation experiments, and the ability of ensembles of membrane-bound Myo1c molecules to develop and sustain forces. To interpret our results, we developed a computational model that recapitulates the basic features of our experimental ensemble data and suggests that Myo1c ensembles can generate forces parallel to lipid bilayers, with larger forces achieved when the myosin works away from the plane of the membrane or when anchored to slowly diffusing regions. PMID:27156719

Using the two-way shape memory effect of NiTi to control surface texture for cellular mechanotransduction

NASA Astrophysics Data System (ADS)

Liang, Yuan; Qin, Haifeng; Hou, Xiaoning; Doll, Gary L.; Ye, Chang; Dong, Yalin

2018-07-01

Mechanical force can crucially affect form and function of cells, and play critical roles in many diseases. While techniques to conveniently apply mechanical force to cells are limited, we fabricate a surface actuator prototype for cellular mechanotransduction by imparting severe plastic deformation into the surface of shape memory alloy (SMA). Using ultrasonic nanocrystal surface modification (UNSM), a deformation-based surface engineering technique with high controllability, micro surface patterns can be generated on the surface of SMA so that the micro-size cell can conform to the pattern; meanwhile, phase transformation can be induced in the subsurface by severe plastic deformation. By controlling plastic deformation and phase transformation, it is possible to establish a quantitative relation between deformation and temperature. When cells are cultured on the UNSM-treated surface, such surface can dynamically deform in response to external temperature change, and therefore apply controllable mechanical force to cells. Through this study, we demonstrate a novel way to fabricate a low-cost surface actuator that has the potential to be used for high-throughput cellular mechanotransduction.

Kinetochore-independent chromosome segregation driven by lateral microtubule bundles

PubMed Central

Muscat, Christina C; Torre-Santiago, Keila M; Tran, Michael V; Powers, James A; Wignall, Sarah M

2015-01-01

During cell division, chromosomes attach to spindle microtubules at sites called kinetochores, and force generated at the kinetochore-microtubule interface is the main driver of chromosome movement. Surprisingly, kinetochores are not required for chromosome segregation on acentrosomal spindles in Caenorhabditis elegans oocytes, but the mechanism driving chromosomes apart in their absence is not understood. In this study, we show that lateral microtubule–chromosome associations established during prometaphase remain intact during anaphase to facilitate separation, defining a novel form of kinetochore-independent segregation. Chromosome dynamics during congression and segregation are controlled by opposing forces; plus-end directed forces are mediated by a protein complex that forms a ring around the chromosome center and dynein on chromosome arms provides a minus-end force. At anaphase onset, ring removal shifts the balance between these forces, triggering poleward movement along lateral microtubule bundles. This represents an elegant strategy for controlling chromosomal movements during cell division distinct from the canonical kinetochore-driven mechanism. DOI: http://dx.doi.org/10.7554/eLife.06462.001 PMID:26026148

"Active" drops as phantom models for living cells: a mesoscopic particle-based approach.

PubMed

Dallavalle, Marco; Lugli, Francesca; Rapino, Stefania; Zerbetto, Francesco

2016-04-21

Drops and biological cells share some morphological features and visco-elastic properties. The modelling of drops by mesoscopic non-atomistic models has been carried out to a high degree of success in recent years. We extend such treatment and discuss a simple, drop-like model to describe the interactions of the outer layer of cells with the surfaces of materials. Cells are treated as active mechanical objects that are able to generate adhesion forces. They appear with their true size and are made of "parcels of fluids" or beads. The beads are described by (very) few quantities/parameters related to fundamental chemical forces such as hydrophilicity and lipophilicity that represent an average of the properties of a patch of material or an area of the cell(s) surface. The investigation of adhesion dynamics, motion of individual cells, and the collective behavior of clusters of cells on materials is possible. In the simulations, the drops become active soft matter objects and different from regular droplets they do not fuse when in contact, their trajectories are not Brownian, and they can be forced "to secrete" molecules, to name some of the properties targeted by the modeling. The behavior that emerges from the simulations allows ascribing some cell properties to their mechanics, which are related to their biological features.

FAK is required for tension-dependent organization of collective cell movements in Xenopus mesendoderm

PubMed Central

Bjerke, Maureen A.; Dzamba, Bette; Wang, Chong; DeSimone, Douglas W.

2014-01-01

Collective cell movements are integral to biological processes such as embryonic development and wound healing and also have a prominent role in some metastatic cancers. In migrating Xenopus mesendoderm, traction forces are generated by cells through integrin-based adhesions and tension transmitted across cadherin adhesions. This is accompanied by assembly of a mechanoresponsive cadherin adhesion complex containing keratin intermediate filaments and the catenin-family member plakoglobin. We demonstrate that focal adhesion kinase (FAK), a major component of integrin adhesion complexes, is required for normal morphogenesis at gastrulation, closure of the anterior neural tube, axial elongation and somitogenesis. Depletion of zygotically expressed FAK results in disruption of mesendoderm tissue polarity similar to that observed when expression of keratin or plakoglobin is inhibited. Both individual and collective migrations of mesendoderm cells from FAK depleted embryos are slowed, cell protrusions are disordered, and cell spreading and traction forces are decreased. Additionally, keratin filaments fail to organize at the rear of cells in the tissue and association of plakoglobin with cadherin is diminished. These findings suggest that FAK is required for the tension-dependent assembly of the cadherin adhesion complex that guides collective mesendoderm migration, perhaps by modulating the dynamic balance of substrate traction forces and cell cohesion needed to establish cell polarity. PMID:25127991

Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells

PubMed Central

Zhang, Yuejin; Wei, Fuxiang; Poh, Yeh-Chuin; Jia, Qiong; Chen, Junjian; Chen, Junwei; Luo, Junyu; Yao, Wenting; Zhou, Wenwen; Huang, Wei; Yang, Fang; Zhang, Yao; Wang, Ning

2017-01-01

Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. Three dimensional-Magnetic Twisting Cytometry (3D-MTC) is a technique for applying local mechanical stresses on living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real time acquisition of a living cell’s mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC – microscopy platform takes around 20 days to construct and the experimental procedures require ~4 days when carried out by a life sciences graduate student. PMID:28686583

Introduction to cell–hydrogel mechanosensing

PubMed Central

Ahearne, Mark

2014-01-01

The development of hydrogel-based biomaterials represents a promising approach to generating new strategies for tissue engineering and regenerative medicine. In order to develop more sophisticated cell-seeded hydrogel constructs, it is important to understand how cells mechanically interact with hydrogels. In this paper, we review the mechanisms by which cells remodel hydrogels, the influence that the hydrogel mechanical and structural properties have on cell behaviour and the role of mechanical stimulation in cell-seeded hydrogels. Cell-mediated remodelling of hydrogels is directed by several cellular processes, including adhesion, migration, contraction, degradation and extracellular matrix deposition. Variations in hydrogel stiffness, density, composition, orientation and viscoelastic characteristics all affect cell activity and phenotype. The application of mechanical force on cells encapsulated in hydrogels can also instigate changes in cell behaviour. By improving our understanding of cell–material mechano-interactions in hydrogels, this should enable a new generation of regenerative medical therapies to be developed. PMID:24748951

Receptor-mediated endocytosis generates nanomechanical force reflective of ligand identity and cellular property.

PubMed

Zhang, Xiao; Ren, Juan; Wang, Jingren; Li, Shixie; Zou, Qingze; Gao, Nan

2018-08-01

Whether environmental (thermal, chemical, and nutrient) signals generate quantifiable, nanoscale, mechanophysical changes in the cellular plasma membrane has not been well elucidated. Assessment of such mechanophysical properties of plasma membrane may shed lights on fundamental cellular process. Atomic force microscopic (AFM) measurement of the mechanical properties of live cells was hampered by the difficulty in accounting for the effects of the cantilever motion and the associated hydrodynamic force on the mechanical measurement. These challenges have been addressed in our recently developed control-based AFM nanomechanical measurement protocol, which enables a fast, noninvasive, broadband measurement of the real-time changes in plasma membrane elasticity in live cells. Here we show using this newly developed AFM platform that the plasma membrane of live mammalian cells exhibits a constant and quantifiable nanomechanical property, the membrane elasticity. This mechanical property sensitively changes in response to environmental factors, such as the thermal, chemical, and growth factor stimuli. We demonstrate that different chemical inhibitors of endocytosis elicit distinct changes in plasma membrane elastic modulus reflecting their specific molecular actions on the lipid configuration or the endocytic machinery. Interestingly, two different growth factors, EGF and Wnt3a, elicited distinct elastic force profiles revealed by AFM at the plasma membrane during receptor-mediated endocytosis. By applying this platform to genetically modified cells, we uncovered a previously unknown contribution of Cdc42, a key component of the cellular trafficking network, to EGF-stimulated endocytosis at plasma membrane. Together, this nanomechanical AFM study establishes an important foundation that is expandable and adaptable for investigation of cellular membrane evolution in response to various key extracellular signals. © 2017 Wiley Periodicals, Inc.

Feeling the force: how pollen tubes deal with obstacles.

PubMed

Burri, Jan T; Vogler, Hannes; Läubli, Nino F; Hu, Chengzhi; Grossniklaus, Ueli; Nelson, Bradley J

2018-06-15

Physical forces are involved in the regulation of plant development and morphogenesis by translating mechanical stress into the modification of physiological processes, which, in turn, can affect cellular growth. Pollen tubes respond rapidly to external stimuli and provide an ideal system to study the effect of mechanical cues at the single-cell level. Here, pollen tubes were exposed to mechanical stress while monitoring the reconfiguration of their growth and recording the generated forces in real-time. We combined a lab-on-a-chip device with a microelectromechanical systems (MEMS)-based capacitive force sensor to mimic and quantify the forces that are involved in pollen tube navigation upon confronting mechanical obstacles. Several stages of obstacle avoidance were identified, including force perception, growth adjustment and penetration. We have experimentally determined the perceptive force threshold, which is the force threshold at which the pollen tube reacts to an obstacle, for Lilium longiflorum and Arabidopsis thaliana. In addition, the method we developed provides a way to calculate turgor pressure based on force and optical data. Pollen tubes sense physical barriers and actively adjust their growth behavior to overcome them. Furthermore, our system offers an ideal platform to investigate intracellular activity during force perception and growth adaption in tip growing cells. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Generation and preservation of the slow underlying membrane potential oscillation in model bursting neurons.

PubMed

Franklin, Clarence C; Ball, John M; Schulz, David J; Nair, Satish S

2010-09-01

The underlying membrane potential oscillation of both forced and endogenous slow-wave bursting cells affects the number of spikes per burst, which in turn affects outputs downstream. We use a biophysical model of a class of slow-wave bursting cells with six active currents to investigate and generalize correlations among maximal current conductances that might generate and preserve its underlying oscillation. We propose three phases for the underlying oscillation for this class of cells: generation, maintenance, and termination and suggest that different current modules coregulate to preserve the characteristics of each phase. Coregulation of I(Burst) and I(A) currents within distinct boundaries maintains the dynamics during the generation phase. Similarly, coregulation of I(CaT) and I(Kd) maintains the peak and duration of the underlying oscillation, whereas the calcium-activated I(KCa) ensures appropriate termination of the oscillation and adjusts the duration independent of peak.

GDE2 regulates subtype-specific motor neuron generation through inhibition of Notch signaling.

PubMed

Sabharwal, Priyanka; Lee, Changhee; Park, Sungjin; Rao, Meenakshi; Sockanathan, Shanthini

2011-09-22

The specification of spinal interneuron and motor neuron identities initiates within progenitor cells, while motor neuron subtype diversification is regulated by hierarchical transcriptional programs implemented postmitotically. Here we find that mice lacking GDE2, a six-transmembrane protein that triggers motor neuron generation, exhibit selective losses of distinct motor neuron subtypes, specifically in defined subsets of limb-innervating motor pools that correlate with the loss of force-generating alpha motor neurons. Mechanistically, GDE2 is expressed by postmitotic motor neurons but utilizes extracellular glycerophosphodiester phosphodiesterase activity to induce motor neuron generation by inhibiting Notch signaling in neighboring motor neuron progenitors. Thus, neuronal GDE2 controls motor neuron subtype diversity through a non-cell-autonomous feedback mechanism that directly regulates progenitor cell differentiation, implying that subtype specification initiates within motor neuron progenitor populations prior to their differentiation into postmitotic motor neurons. Copyright © 2011 Elsevier Inc. All rights reserved.

GDE2 regulates subtype specific motor neuron generation through inhibition of Notch signaling

PubMed Central

Sabharwal, Priyanka; Lee, Changhee; Park, Sungjin; Rao, Meenakshi; Sockanathan, Shanthini

2011-01-01

The specification of spinal interneuron and motor neuron identities initiates within progenitor cells, while motor neuron subtype diversification is regulated by hierarchical transcriptional programs implemented postmitotically. Here, we find that mice lacking GDE2, a six-transmembrane protein that triggers motor neuron generation, exhibit selective losses of distinct motor neuron subtypes, specifically in defined subsets of limb-innervating motor pools that correlate with the loss of force-generating alpha motor neurons. Mechanistically, GDE2 is expressed by postmitotic motor neurons but utilizes extracellular glycerophosphodiester phosphodiesterase activity to induce motor neuron generation by inhibiting Notch signaling in neighboring motor neuron progenitors. Thus, neuronal GDE2 controls motor neuron subtype diversity through a non cell-autonomous feedback mechanism that directly regulates progenitor cell differentiation, implying that subtype specification initiates within motor neuron progenitor populations prior to their differentiation into postmitotic motor neurons. PMID:21943603

Active Materials Integrated with Actomyosin

NASA Astrophysics Data System (ADS)

Ito, Hiroaki; Makuta, Masahiro; Nishigami, Yukinori; Ichikawa, Masatoshi

2017-10-01

Muscles are the engine of our body, and actomyosin is the engine of a cell. Both muscle and the actomyosin use the same proteins, namely, actin, and myosin, which are the pair of cytoskeleton and motor proteins generating a force to realize deformation. The properties of force generation by actomyosin at a single-molecule level have been studied for many years. Moreover, the active properties of higher-order structures integrated by actomyosin are attracting the attention of researchers. Here, we review the recent progress in the study of reconstituted actomyosin systems in vitro toward real-space models of nonequilibrium systems, collective motion, biological phenomena, and active materials.

Biomechanical Characterization of Cardiomyocyte Using PDMS Pillar with Microgrooves

PubMed Central

Oyunbaatar, Nomin-Erdene; Lee, Deok-Hyu; Patil, Swati J.; Kim, Eung-Sam; Lee, Dong-Weon

2016-01-01

This paper describes the surface-patterned polydimethylsiloxane (PDMS) pillar arrays for enhancing cell alignment and contraction force in cardiomyocytes. The PDMS micropillar (μpillar) arrays with microgrooves (μgrooves) were fabricated using a unique micro-mold made using SU-8 double layer processes. The spring constant of the μpillar arrays was experimentally confirmed using atomic force microscopy (AFM). After culturing cardiac cells on the two different types of μpillar arrays, with and without grooves on the top of μpillar, the characteristics of the cardiomyocytes were analyzed using a custom-made image analysis system. The alignment of the cardiomyocytes on the μgrooves of the μpillars was clearly observed using a DAPI staining process. The mechanical force generated by the contraction force of the cardiomyocytes was derived from the displacement of the μpillar arrays. The contraction force of the cardiomyocytes aligned on the μgrooves was 20% higher than that of the μpillar arrays without μgrooves. The experimental results prove that applied geometrical stimulus is an effective method for aligning and improving the contraction force of cardiomyocytes. PMID:27517924

Sensor apparatus using an electrochemical cell

DOEpatents

Thakur, Mrinal

2002-01-01

A novel technology for sensing mechanical quantities such as force, stress, strain, pressure and acceleration has been invented. This technology is based on a change in the electrochemically generated voltage (electromotive force) with application of force, stress, strain, pressure or acceleration. The change in the voltage is due to a change in the internal resistance of the electrochemical cell with a change in the relative position or orientation of the electrodes (anode and cathode) in the cell. The signal to be detected (e.g. force, stress, strain, pressure or acceleration) is applied to one of the electrodes to cause a change in the relative position or orientation between the electrodes. Various materials, solid, semisolid, gel, paste or liquid can be utilized as the electrolyte. The electrolyte must be an ion conductor. The examples of solid electrolytes include specific polymer conductors, polymer composites, ion conducting glasses and ceramics. The electrodes are made of conductors such as metals with dissimilar electronegativities. Significantly enhanced sensitivities, up to three orders of magnitude higher than that of comparable commercial sensors, are obtained. The materials are substantially less expensive than commercially used materials for mechanical sensors.

A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells.

PubMed

Burnette, Dylan T; Shao, Lin; Ott, Carolyn; Pasapera, Ana M; Fischer, Robert S; Baird, Michelle A; Der Loughian, Christelle; Delanoe-Ayari, Helene; Paszek, Matthew J; Davidson, Michael W; Betzig, Eric; Lippincott-Schwartz, Jennifer

2014-04-14

How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers' attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions.

A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells

PubMed Central

Burnette, Dylan T.; Shao, Lin; Ott, Carolyn; Pasapera, Ana M.; Fischer, Robert S.; Baird, Michelle A.; Der Loughian, Christelle; Delanoe-Ayari, Helene; Paszek, Matthew J.; Davidson, Michael W.; Betzig, Eric

2014-01-01

How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers’ attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions. PMID:24711500

Mechanical Regulation in Cell Division and in Neurotransmitter Release

NASA Astrophysics Data System (ADS)

Thiyagarajan, Sathish

During their lifecycle, cells must produce forces which play important roles in several subcellular processes. Force-producing components are organized into macromolecular assemblies of proteins that are often dynamic, and are constructed or disassembled in response to various signals. The forces themselves may directly be involved in subcellular mechanics, or they may influence mechanosensing proteins either within or outside these structures. These proteins play different roles: they may ensure the stability of the force-producing structure, or they may send signals to a coupled process. The generation and sensing of subcellular forces is an active research topic, and this thesis focusses on the roles of these forces in two key areas: cell division and neurotransmitter release. The first part of the thesis deals with the effect of force on cell wall growth regulation during division in the fission yeast Schizosaccharomyces pombe, a cigar-shaped, unicellular organism. During cytokinesis, the last stage of cell division in which the cell physically divides into two, a tense cytokinetic ring anchored to the cellular membrane assembles and constricts, accompanied by the inward centripetal growth of new cell wall, called septum, in the wake of the inward-moving membrane. The contour of the septum hole maintains its circularity as it reduces in size--an indication of regulated growth. To characterize the cell wall growth process, we performed image analysis on contours of the leading edge of the septum obtained via fluorescence microscopy in the labs of our collaborators. We quantified the deviations from circularity using the edge roughness. The roughness was spatially correlated, suggestive of regulated growth. We hypothesized that the cell wall growers are mechanosensitive and respond to the force exerted by the ring. A mathematical model based on this hypothesis then showed that this leads to corrections of roughness in a curvature-dependent fashion. Thus, one of the roles of ring tension is to communicate with the mechanosensitive septum growth processes and coordinate growth to ensure the daughter cells have a functional cell wall. The second part of the thesis deals with how ring tension is produced and sustained, using experimentally measured ultrastructure of the cytokinetic ring itself. Recent super-resolution experiments have revealed that several key proteins of the fission yeast constricting ring are organized into membrane-anchored complexes called nodes. The force producing protein myosin-II in these nodes exerts pulling forces on polymeric actin filaments that are synthesized from polymerizers residing in the nodes. How these forces are marshalled to generate ring tension, and how such an organization maintains its stability is unclear. Using a mathematical model with coarse-grained representations of actin and myosin, we showed that such a node-based organization reproduces previously measured ring tension values. The model explains the origin of experimentally observed bidirectional motion of the nodes in the ring, and showed that turnover of the nodes rescues the ring from inherent contractile instabilities that would be expected when a force-producing structure is made up of small object that effectively attract one another. Finally, the third part of the thesis deals with the role of forces produced by SNARE proteins at synapses between two neurons during neurotransmission. A key step here is synaptic release, where inside a neuron, membrane-bound compartments called vesicles filled with neurotransmitter fuse with the membrane of the neuron forming a transient fusion pore, and release their contents to the outside of the cell. These neurotransmitter molecules are sensed by another neuron that is physically separate from the neuron in question and this neuron propagates the signal henceforth. Thus, regulation of neurotransmitter release is a key step in neurotransmission. A fusion machinery consisting of several proteins facilitates membrane fusion, and pore nucleation requires the formation of a SNARE protein complex in this machinery, whose role during pore dilation is unclear. Using electrophysiological measurements, our collaborators experimentally measured the statistics of the size of single fusion pores in vitro, and observed that average pore sizes increased with the number of SNARE proteins. Using mathematical modeling, we showed that this effect was due to an entropic crowding force that expands the pore and increases with the number of SNAREs, and counteracts the energy barrier to fusion pore expansion.

Induced pluripotent stem cells: advances to applications

PubMed Central

Nelson, Timothy J; Martinez-Fernandez, Almudena; Yamada, Satsuki; Ikeda, Yasuhiro; Perez-Terzic, Carmen; Terzic, Andre

2010-01-01

Induced pluripotent stem cell (iPS) technology has enriched the armamentarium of regenerative medicine by introducing autologous pluripotent progenitor pools bioengineered from ordinary somatic tissue. Through nuclear reprogramming, patient-specific iPS cells have been derived and validated. Optimizing iPS-based methodology will ensure robust applications across discovery science, offering opportunities for the development of personalized diagnostics and targeted therapeutics. Here, we highlight the process of nuclear reprogramming of somatic tissues that, when forced to ectopically express stemness factors, are converted into bona fide pluripotent stem cells. Bioengineered stem cells acquire the genuine ability to generate replacement tissues for a wide-spectrum of diseased conditions, and have so far demonstrated therapeutic benefit upon transplantation in model systems of sickle cell anemia, Parkinson’s disease, hemophilia A, and ischemic heart disease. The field of regenerative medicine is therefore primed to adopt and incorporate iPS cell-based advancements as a next generation stem cell platforms. PMID:21165156

Acetylcholine released by endothelial cells facilitates flow‐mediated dilatation

PubMed Central

Wilson, Calum; Lee, Matthew D.

2016-01-01

Key points The endothelium plays a pivotal role in the vascular response to chemical and mechanical stimuli.The endothelium is exquisitely sensitive to ACh, although the physiological significance of ACh‐induced activation of the endothelium is unknown.In the present study, we investigated the mechanisms of flow‐mediated endothelial calcium signalling.Our data establish that flow‐mediated endothelial calcium responses arise from the autocrine action of non‐neuronal ACh released by the endothelium. Abstract Circulating blood generates frictional forces (shear stress) on the walls of blood vessels. These frictional forces critically regulate vascular function. The endothelium senses these frictional forces and, in response, releases various vasodilators that relax smooth muscle cells in a process termed flow‐mediated dilatation. Although some elements of the signalling mechanisms have been identified, precisely how flow is sensed and transduced to cause the release of relaxing factors is poorly understood. By imaging signalling in large areas of the endothelium of intact arteries, we show that the endothelium responds to flow by releasing ACh. Once liberated, ACh acts to trigger calcium release from the internal store in endothelial cells, nitric oxide production and artery relaxation. Flow‐activated release of ACh from the endothelium is non‐vesicular and occurs via organic cation transporters. ACh is generated following mitochondrial production of acetylCoA. Thus, we show ACh is an autocrine signalling molecule released from endothelial cells, and identify a new role for the classical neurotransmitter in endothelial mechanotransduction. PMID:27730645

Conjugated Oligoelectrolytes Increase Current Response and Organic Containment Removal in Wastewater Microbial Fuel Cells

DTIC Science & Technology

2012-01-01

certain COEs have been demonstrated to sponta- neously insert into liposomes and within the membranes of yeast .14 The driving force for this...ofDSSN+ and a cartoon illustration of its incorporation into a cell membrane. Introduction of yeast modified withDSSN+ into microbial fuel cells (MFCs...leads to an increase of generated current, compared to unmodified yeast , which suggests improved interaction with the charge-collecting electrode.14

5. Exterior view, enclosure at walkin entry level between Test ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

5. Exterior view, enclosure at walk-in entry level between Test Cell 6 (right) and Test Cell 7 (left), Systems Integration Laboratory Building (T-28), looking southwest. High pressure gas tank and generator test firings are conducted in the enclosure. - Air Force Plant PJKS, Systems Integration Laboratory, Systems Integration Laboratory Building, Waterton Canyon Road & Colorado Highway 121, Lakewood, Jefferson County, CO

Formation and maintenance of tubular membrane projections require mechanical force, but their elongation and shortening do not require additional force.

PubMed

Inaba, Takehiko; Ishijima, Akihiko; Honda, Makoto; Nomura, Fumimasa; Takiguchi, Kingo; Hotani, Hirokazu

2005-04-29

Living cells develop their own characteristic shapes depending on their physiological functions, and their morphologies are based on the mechanical characteristics of the cytoskeleton and of membranes. To investigate the role of lipid membranes in morphogenesis, we constructed a simple system that can manipulate liposomes and measure the forces required to transform their shapes. Two polystyrene beads (1 microm in diameter) were encapsulated in giant liposomes and were manipulated using double-beam laser tweezers. Without any specific interaction between the lipid membrane and beads, mechanical forces could be applied to the liposome membrane from the inside. Spherical liposomes transformed into a lemon shape with increasing tension, and tubular membrane projections were subsequently generated in the tips at either end. This process is similar to the liposomal transformation caused by elongation of encapsulated cytoskeletons. In the elongation stage of lemon-shaped liposomes, the force required for the transformation became larger as the end-to-end length increased. Just before the tubular membrane was generated, the force reached the maximum strength (approximately 11 pN). However, immediately after the tubular membrane developed, the force suddenly decreased and was maintained at a constant strength (approximately 4 pN) that was independent of further tube elongation or shortening, even though there was no excess membrane reservoir as occurs in living cells. When the tube length was shortened to approximately 2 microm, the liposome reversed to a lemon shape and the force temporarily increased (to approximately 7 pN). These results indicate that the simple application of mechanical force is sufficient to form a protrusion in a membrane, that a critical force and length is needed to form and to maintain the protrusion, and suggest that the lipid bilayer itself has the ability to buffer the membrane tension.

Recent progress in stem cell differentiation directed by material and mechanical cues.

PubMed

Lin, Xunxun; Shi, Yuan; Cao, Yilin; Liu, Wei

2016-02-02

Stem cells play essential roles in tissue regeneration in vivo via specific lineage differentiation induced by environmental factors. In the past, biochemical signals were the focus of induced stem cell differentiation. As reported by Engler et al (2006 Cell 126 677-89), biophysical signal mediated stem cell differentiation could also serve as an important inducer. With the advancement of material science, it becomes a possible strategy to generate active biophysical signals for directing stem cell fate through specially designed material microstructures. In the past five years, significant progress has been made in this field, and these designed biophysical signals include material elasticity/rigidity, micropatterned structure, extracellular matrix (ECM) coated materials, material transmitted extracellular mechanical force etc. A large number of investigations involved material directed differentiation of mesenchymal stem cells, neural stem/progenitor cells, adipose derived stem cells, hematopoietic stem/progenitor cells, embryonic stem cells and other cells. Hydrogel based materials were commonly used to create varied mechanical properties via modifying the ratio of different components, crosslinking levels, matrix concentration and conjugation with other components. Among them, polyacrylamide (PAM) and polydimethylsiloxane (PDMS) hydrogels remained the major types of material. Specially designed micropatterning was not only able to create a unique topographical surface to control cell shape, alignment, cell-cell and cell-matrix contact for basic stem cell biology study, but also could be integrated with 3D bioprinting to generate micropattered 3D structure and thus to induce stem cell based tissue regeneration. ECM coating on a specific topographical structure was capable of inducing even more specific and potent stem cell differentiation along with soluble factors and mechanical force. The article overviews the progress of the past five years in this particular field.

Live-cell imaging of migrating cells expressing fluorescently-tagged proteins in a three-dimensional matrix.

PubMed

Shih, Wenting; Yamada, Soichiro

2011-12-22

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network. By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

The role of actin networks in cellular mechanosensing

NASA Astrophysics Data System (ADS)

Azatov, Mikheil

Physical processes play an important role in many biological phenomena, such as wound healing, organ development, and tumor metastasis. During these processes, cells constantly interact with and adapt to their environment by exerting forces to mechanically probe the features of their surroundings and generating appropriate biochemical responses. The mechanisms underlying how cells sense the physical properties of their environment are not well understood. In this thesis, I present my studies to investigate cellular responses to the stiffness and topography of the environment. In order to sense the physical properties of their environment, cells dynamically reorganize the structure of their actin cytoskeleton, a dynamic network of biopolymers, altering the shape and spatial distribution of protein assemblies. Several observations suggest that proteins that crosslink actin filaments may play an important role in cellular mechanosensitivity. Palladin is an actin-crosslinking protein that is found in the lamellar actin network, stress fibers and focal adhesions, cellular structures that are critical for mechanosensing of the physical environment. By virtue of its close interactions with these structures in the cell, palladin may play an important role in cell mechanics. However, the role of actin crosslinkers in general, and palladin in particular, in cellular force generation and mechanosensing is not well known. I have investigated the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. I have shown that the expression levels of palladin modulate the forces exerted by cells and their ability to sense substrate stiffness. Perturbation experiments also suggest that palladin levels in cells altered myosin motor activity. These results suggest that the actin crosslinkers, such as palladin, and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis. In addition to stiffness, the local geometry or topography of the surface has been shown to modulate the movement, morphology, and cytoskeletal organization of cells. However, the effect of topography on fluctuations of intracellular structures, which arise from motor driven activity on a viscoelastic actin network are not known. I have used nanofabricated substrates with parallel ridges to show that the cell shape, the actin cytoskeleton and focal adhesions all align along the direction of the ridges, exhibiting a biphasic dependence on the spacing between ridges. I further demonstrated that palladin bands along actin stress fibers undergo a complex diffusive motion with velocities aligned along the direction of ridges. These results provide insight into the mechanisms of cellular mechanosensing of the environment, suggesting a complex interplay between the actin cytoskeleton and cellular adhesions in coordinating cellular response to surface topography. Overall, this work has advanced our understanding of mechanisms that govern cellular responses to their physical environment.

Injection by hydrostatic pressure in conjunction with electrokinetic force on a microfluidic chip.

PubMed

Gai, Hongwei; Yu, Linfen; Dai, Zhongpeng; Ma, Yinfa; Lin, Bingcheng

2004-06-01

A simple method was developed for injecting a sample on a cross-form microfluidic chip by means of hydrostatic pressure combined with electrokinetic forces. The hydrostatic pressure was generated simply by adjusting the liquid level in different reservoirs without any additional driven equipment such as a pump. Two dispensing strategies using a floating injection and a gated injection, coupled with hydrostatic pressure loading, were tested. The fluorescence observation verified the feasibility of hydrostatic pressure loading in the separation of a mixture of fluorescein sodium salt and fluorescein isothiocyanate. This method was proved to be effective in leading cells to a separation channel for single cell analysis.

90-kilobar diamond-anvil high-pressure cell for use on an automatic diffractometer.

PubMed

Schiferl, D; Jamieson, J C; Lenko, J E

1978-03-01

A gasketed diamond-anvil high-pressure cell is described which can be used on a four-circle automatic diffractometer to collect x-ray intensity data from single-crystal samples subjected to truly hydrostatic pressures of over 90 kilobars. The force generating system exerts only forces normal to the diamond faces to obtain maximum reliability. A unique design allows exceptionally large open areas for maximum x-ray access and is particularly well suited for highly absorbing materials, as the x rays are not transmitted through the sample. Studies on ruby show that high-pressure crystal structure determinations may be done rapidly, reliably, and routinely with this system.

Simultaneous recordings of force and sliding movement between a myosin-coated glass microneedle and actin cables in vitro.

PubMed

Chaen, S; Oiwa, K; Shimmen, T; Iwamoto, H; Sugi, H

1989-03-01

To elucidate the molecular mechanism of muscle contraction resulting from the ATP-dependent actin-myosin interaction, we constructed an assay system with which both the force and the movement produced by the actin-myosin interaction in vitro can be simultaneously recorded and analyzed. The assay system consisted of the giant internodal cells of an alga, Nitellopsis obtusa, which contain well-organized arrays of actin filaments (actin cables) running along the cell long axis, and a glass microneedle (tip diameter, approximately 7 microns; elastic coefficient, approximately 40 pN/microns), which was coated with skeletal muscle myosin at the tip and extended from a micromanipulator at right angles with the actin cables. When the myosin-coated tip of the microneedle was brought into contact with the exposed surface of the actin cables, it exhibited ATP-dependent movement along the actin cables over a distance of 20-150 microns in 20-200 s (20-23 degrees C) and eventually stopped due to a balance between forces generated by the actin-myosin interaction (800-6000 pN) and by the bent microneedle. Since the load on the force-generating myosin molecules increased with the bending displacement of the microneedle (auxotonic condition), the relation between the load and the sliding velocity of the myosin heads past the actin cables was determined from the time course of the microneedle movement recorded with a video system. The shape of the force-velocity curve thus obtained was convex upwards, similar to that of the force-velocity curve of intact frog muscle fibers obtained under the auxotonic condition.

Cross-bridge elasticity in single smooth muscle cells

PubMed Central

1983-01-01

In smooth muscle, a cross-bridge mechanism is believed to be responsible for active force generation and fiber shortening. In the present studies, the viscoelastic and kinetic properties of the cross- bridge were probed by eliciting tension transients in response to small, rapid, step length changes (delta L = 0.3-1.0% Lcell in 2 ms). Tension transients were obtained in a single smooth muscle cell isolated from the toad (Bufo marinus) stomach muscularis, which was tied between a force transducer and a displacement device. To record the transients, which were of extremely small magnitude (0.1 microN), a high-frequency (400 Hz), ultrasensitive force transducer (18 mV/microN) was designed and built. The transients obtained during maximal force generation (Fmax = 2.26 microN) were characterized by a linear elastic response (Emax = 1.26 X 10(4) mN/mm2) coincident with the length step, which was followed by a biphasic tension recovery made up of two exponentials (tau fast = 5-20 ms, tau slow = 50-300 ms). During the development of force upon activation, transients were elicited. The relationship between stiffness and force was linear, which suggests that the transients originate within the cross-bridge and reflect the cross-bridge's viscoelastic and kinetic properties. The observed fiber elasticity suggests that the smooth muscle cross-bridge is considerably more compliant than in fast striated muscle. A thermodynamic model is presented that allows for an analysis of the factors contributing to the increased compliance of the smooth muscle cross-bridge. PMID:6413640

Grid generation in three dimensions by Poisson equations with control of cell size and skewness at boundary surfaces

NASA Technical Reports Server (NTRS)

Sorenson, R. L.; Steger, J. L.

1983-01-01

An algorithm for generating computational grids about arbitrary three-dimensional bodies is developed. The elliptic partial differential equation (PDE) approach developed by Steger and Sorenson and used in the NASA computer program GRAPE is extended from two to three dimensions. Forcing functions which are found automatically by the algorithm give the user the ability to control mesh cell size and skewness at boundary surfaces. This algorithm, as is typical of PDE grid generators, gives smooth grid lines and spacing in the interior of the grid. The method is applied to a rectilinear wind-tunnel case and to two body shapes in spherical coordinates.

Separation control by vortex generator devices in a transonic channel flow

NASA Astrophysics Data System (ADS)

Bur, Reynald; Coponet, Didier; Carpels, Yves

2009-12-01

An experimental study was conducted in a transonic channel to control by mechanical vortex generator devices the strong interaction between a shock wave and a separated turbulent boundary layer. Control devices—co-rotating and counter-rotating vane-type vortex generators—were implemented upstream of the shock foot region and tested both on a steady shock wave and on a forced shock oscillation configurations. The spanwise spacing of vortex generator devices along the channel appeared to be an important parameter to control the flow separation region. When the distance between each device is decreased, the vortices merging is more efficient to reduce the separation. Their placement upstream of the shock wave is determinant to ensure that vortices have mixed momentum all spanwise long before they reach the separation line, so as to avoid separation cells. Then, vortex generators slightly reduced the amplitude of the forced shock wave oscillation by delaying the upstream displacement of the leading shock.

An experimental investigation of flow around a vehicle passing through a tornado

NASA Astrophysics Data System (ADS)

Suzuki, Masahiro; Obara, Kouhei; Okura, Nobuyuki

2016-03-01

Flow around a vehicle running through a tornado was investigated experimentally. A tornado simulator was developed to generate a tornado-like swirl flow. PIV study confirmed that the simulator generates two-celled vortices which are observed in the natural tornadoes. A moving test rig was developed to run a 1/40 scaled train-shaped model vehicle under the tornado simulator. The car contained pressure sensors, a data logger with an AD converter to measure unsteady surface pressures during its run through the swirling flow. Aerodynamic forces acting on the vehicle were estimated from the pressure data. The results show that the aerodynamic forces change its magnitude and direction depending on the position of the car in the swirling flow. The asymmetry of the forces about the vortex centre suggests the vehicle itself may deform the flow field.

Mechanical forces as information: an integrated approach to plant and animal development

PubMed Central

Hernández-Hernández, Valeria; Rueda, Denisse; Caballero, Lorena; Alvarez-Buylla, Elena R.; Benítez, Mariana

2014-01-01

Mechanical forces such as tension and compression act throughout growth and development of multicellular organisms. These forces not only affect the size and shape of the cells and tissues but are capable of modifying the expression of genes and the localization of molecular components within the cell, in the plasma membrane, and in the plant cell wall. The magnitude and direction of these physical forces change with cellular and tissue properties such as elasticity. Thus, mechanical forces and the mesoscopic fields that emerge from their local action constitute important sources of positional information. Moreover, physical and biochemical processes interact in non-linear ways during tissue and organ growth in plants and animals. In this review we discuss how such mechanical forces are generated, transmitted, and sensed in these two lineages of multicellular organisms to yield long-range positional information. In order to do so we first outline a potentially common basis for studying patterning and mechanosensing that relies on the structural principle of tensegrity, and discuss how tensegral structures might arise in plants and animals. We then provide some examples of morphogenesis in which mechanical forces appear to act as positional information during development, offering a possible explanation for ubiquitous processes, such as the formation of periodic structures. Such examples, we argue, can be interpreted in terms of tensegral phenomena. Finally, we discuss the hypothesis of mechanically isotropic points as a potentially generic mechanism for the localization and maintenance of stem-cell niches in multicellular organisms. This comparative approach aims to help uncovering generic mechanisms of morphogenesis and thus reach a better understanding of the evolution and development of multicellular phenotypes, focusing on the role of physical forces in these processes. PMID:24959170

Mechanical forces as information: an integrated approach to plant and animal development.

PubMed

Hernández-Hernández, Valeria; Rueda, Denisse; Caballero, Lorena; Alvarez-Buylla, Elena R; Benítez, Mariana

2014-01-01

Mechanical forces such as tension and compression act throughout growth and development of multicellular organisms. These forces not only affect the size and shape of the cells and tissues but are capable of modifying the expression of genes and the localization of molecular components within the cell, in the plasma membrane, and in the plant cell wall. The magnitude and direction of these physical forces change with cellular and tissue properties such as elasticity. Thus, mechanical forces and the mesoscopic fields that emerge from their local action constitute important sources of positional information. Moreover, physical and biochemical processes interact in non-linear ways during tissue and organ growth in plants and animals. In this review we discuss how such mechanical forces are generated, transmitted, and sensed in these two lineages of multicellular organisms to yield long-range positional information. In order to do so we first outline a potentially common basis for studying patterning and mechanosensing that relies on the structural principle of tensegrity, and discuss how tensegral structures might arise in plants and animals. We then provide some examples of morphogenesis in which mechanical forces appear to act as positional information during development, offering a possible explanation for ubiquitous processes, such as the formation of periodic structures. Such examples, we argue, can be interpreted in terms of tensegral phenomena. Finally, we discuss the hypothesis of mechanically isotropic points as a potentially generic mechanism for the localization and maintenance of stem-cell niches in multicellular organisms. This comparative approach aims to help uncovering generic mechanisms of morphogenesis and thus reach a better understanding of the evolution and development of multicellular phenotypes, focusing on the role of physical forces in these processes.

A hybrid computational model to explore the topological characteristics of epithelial tissues.

PubMed

González-Valverde, Ismael; García-Aznar, José Manuel

2017-11-01

Epithelial tissues show a particular topology where cells resemble a polygon-like shape, but some biological processes can alter this tissue topology. During cell proliferation, mitotic cell dilation deforms the tissue and modifies the tissue topology. Additionally, cells are reorganized in the epithelial layer and these rearrangements also alter the polygon distribution. We present here a computer-based hybrid framework focused on the simulation of epithelial layer dynamics that combines discrete and continuum numerical models. In this framework, we consider topological and mechanical aspects of the epithelial tissue. Individual cells in the tissue are simulated by an off-lattice agent-based model, which keeps the information of each cell. In addition, we model the cell-cell interaction forces and the cell cycle. Otherwise, we simulate the passive mechanical behaviour of the cell monolayer using a material that approximates the mechanical properties of the cell. This continuum approach is solved by the finite element method, which uses a dynamic mesh generated by the triangulation of cell polygons. Forces generated by cell-cell interaction in the agent-based model are also applied on the finite element mesh. Cell movement in the agent-based model is driven by the displacements obtained from the deformed finite element mesh of the continuum mechanical approach. We successfully compare the results of our simulations with some experiments about the topology of proliferating epithelial tissues in Drosophila. Our framework is able to model the emergent behaviour of the cell monolayer that is due to local cell-cell interactions, which have a direct influence on the dynamics of the epithelial tissue. Copyright © 2017 John Wiley & Sons, Ltd.

Comparing contractile apparatus-driven cytokinesis mechanisms across kingdoms.

PubMed

Balasubramanian, Mohan K; Srinivasan, Ramanujam; Huang, Yinyi; Ng, Kian-Hong

2012-11-01

Cytokinesis is the final stage of the cell cycle during which a cell physically divides into two daughters through the assembly of new membranes (and cell wall in some cases) between the forming daughters. New membrane assembly can either proceed centripetally behind a contractile apparatus, as in the case of prokaryotes, archaea, fungi, and animals or expand centrifugally, as in the case of higher plants. In this article, we compare the mechanisms of cytokinesis in diverse organisms dividing through the use of a contractile apparatus. While an actomyosin ring participates in cytokinesis in almost all centripetally dividing eukaryotes, the majority of bacteria and archaea (except Crenarchaea) divide using a ring composed of the tubulin-related protein FtsZ. Curiously, despite molecular conservation of the division machinery components, division site placement and its cell cycle regulation occur by a variety of unrelated mechanisms even among organisms from the same kingdom. While molecular motors and cytoskeletal polymer dynamics contribute to force generation during eukaryotic cytokinesis, cytoskeletal polymer dynamics alone appears to be sufficient for force generation during prokaryotic cytokinesis. Intriguingly, there are life forms on this planet that appear to lack molecules currently known to participate in cytokinesis and how these cells perform cytokinesis remains a mystery waiting to be unravelled. Copyright © 2012 Wiley Periodicals, Inc.

Characterization of biomechanical properties of cells through dielectrophoresis-based cell stretching and actin cytoskeleton modeling.

PubMed

Bai, Guohua; Li, Ying; Chu, Henry K; Wang, Kaiqun; Tan, Qiulin; Xiong, Jijun; Sun, Dong

2017-04-04

Cytoskeleton is a highly dynamic network that helps to maintain the rigidity of a cell, and the mechanical properties of a cell are closely related to many cellular functions. This paper presents a new method to probe and characterize cell mechanical properties through dielectrophoresis (DEP)-based cell stretching manipulation and actin cytoskeleton modeling. Leukemia NB4 cells were used as cell line, and changes in their biological properties were examined after chemotherapy treatment with doxorubicin (DOX). DEP-integrated microfluidic chip was utilized as a low-cost and efficient tool to study the deformability of cells. DEP forces used in cell stretching were first evaluated through computer simulation, and the results were compared with modeling equations and with the results of optical stretching (OT) experiments. Structural parameters were then extracted by fitting the experimental data into the actin cytoskeleton model, and the underlying mechanical properties of the cells were subsequently characterized. The DEP forces generated under different voltage inputs were calculated and the results from different approaches demonstrate good approximations to the force estimation. Both DEP and OT stretching experiments confirmed that DOX-treated NB4 cells were stiffer than the untreated cells. The structural parameters extracted from the model and the confocal images indicated significant change in actin network after DOX treatment. The proposed DEP method combined with actin cytoskeleton modeling is a simple engineering tool to characterize the mechanical properties of cells.

Genetics Home Reference: early-onset myopathy with fatal cardiomyopathy

MedlinePlus

... they are made of proteins that generate the mechanical force needed for muscles to contract. Titin has several functions within sarcomeres. One of this protein's most important jobs is to provide structure, flexibility, and stability to these cell structures. Titin ...

Nonlinear coupled equations for electrochemical cells as developed by the general equation for nonequilibrium reversible-irreversible coupling.

PubMed

Bedeaux, Dick; Kjelstrup, Signe; Öttinger, Hans Christian

2014-09-28

We show how the Butler-Volmer and Nernst equations, as well as Peltier effects, are contained in the general equation for nonequilibrium reversible and irreversible coupling, GENERIC, with a unique definition of the overpotential. Linear flux-force relations are used to describe the transport in the homogeneous parts of the electrochemical system. For the electrode interface, we choose nonlinear flux-force relationships. We give the general thermodynamic basis for an example cell with oxygen electrodes and electrolyte from the solid oxide fuel cell. In the example cell, there are two activated chemical steps coupled also to thermal driving forces at the surface. The equilibrium exchange current density obtains contributions from both rate-limiting steps. The measured overpotential is identified at constant temperature and stationary states, in terms of the difference in electrochemical potential of products and reactants. Away from these conditions, new terms appear. The accompanying energy flux out of the surface, as well as the heat generation at the surface are formulated, adding to the general thermodynamic basis.

Nonlinear coupled equations for electrochemical cells as developed by the general equation for nonequilibrium reversible-irreversible coupling

NASA Astrophysics Data System (ADS)

Bedeaux, Dick; Kjelstrup, Signe; Öttinger, Hans Christian

2014-09-01

We show how the Butler-Volmer and Nernst equations, as well as Peltier effects, are contained in the general equation for nonequilibrium reversible and irreversible coupling, GENERIC, with a unique definition of the overpotential. Linear flux-force relations are used to describe the transport in the homogeneous parts of the electrochemical system. For the electrode interface, we choose nonlinear flux-force relationships. We give the general thermodynamic basis for an example cell with oxygen electrodes and electrolyte from the solid oxide fuel cell. In the example cell, there are two activated chemical steps coupled also to thermal driving forces at the surface. The equilibrium exchange current density obtains contributions from both rate-limiting steps. The measured overpotential is identified at constant temperature and stationary states, in terms of the difference in electrochemical potential of products and reactants. Away from these conditions, new terms appear. The accompanying energy flux out of the surface, as well as the heat generation at the surface are formulated, adding to the general thermodynamic basis.

Mechanical positioning of multiple nuclei in muscle cells.

PubMed

Manhart, Angelika; Windner, Stefanie; Baylies, Mary; Mogilner, Alex

2018-06-01

Many types of large cells have multiple nuclei. In skeletal muscle fibers, the nuclei are distributed along the cell to maximize their internuclear distances. This myonuclear positioning is crucial for cell function. Although microtubules, microtubule associated proteins, and motors have been implicated, mechanisms responsible for myonuclear positioning remain unclear. We used a combination of rough interacting particle and detailed agent-based modeling to examine computationally the hypothesis that a force balance generated by microtubules positions the muscle nuclei. Rather than assuming the nature and identity of the forces, we simulated various types of forces between the pairs of nuclei and between the nuclei and cell boundary to position the myonuclei according to the laws of mechanics. We started with a large number of potential interacting particle models and computationally screened these models for their ability to fit biological data on nuclear positions in hundreds of Drosophila larval muscle cells. This reverse engineering approach resulted in a small number of feasible models, the one with the best fit suggests that the nuclei repel each other and the cell boundary with forces that decrease with distance. The model makes nontrivial predictions about the increased nuclear density near the cell poles, the zigzag patterns of the nuclear positions in wider cells, and about correlations between the cell width and elongated nuclear shapes, all of which we confirm by image analysis of the biological data. We support the predictions of the interacting particle model with simulations of an agent-based mechanical model. Taken together, our data suggest that microtubules growing from nuclear envelopes push on the neighboring nuclei and the cell boundaries, which is sufficient to establish the nearly-uniform nuclear spreading observed in muscle fibers.

Microgravity-Enhanced Stem Cell Selection

NASA Technical Reports Server (NTRS)

Claudio, Pier Paolo; Valluri, Jagan

2011-01-01

Stem cells, both embryonic and adult, promise to revolutionize the practice of medicine in the future. In order to realize this potential, a number of hurdles must be overcome. Most importantly, the signaling mechanisms necessary to control the differentiation of stem cells into tissues of interest remain to be elucidated, and much of the present research on stem cells is focused on this goal. Nevertheless, it will also be essential to achieve large-scale expansion and, in many cases, assemble cells in 3D as transplantable tissues. To this end, microgravity analog bioreactors can play a significant role. Microgravity bioreactors were originally conceived as a tool to study the cellular responses to microgravity. However, the technology can address some of the shortcomings of conventional cell culture systems; namely, the deficiency of mass transport in static culture and high mechanical shear forces in stirred systems. Unexpectedly, the conditions created in the vessel were ideal for 3D cell culture. Recently, investigators have demonstrated the capability of the microgravity bioreactors to expand hematopoietic stem cells compared to static culture, and facilitate the differentiation of umbilical cord stem cells into 3D liver aggregates. Stem cells are capable of differentiating into functional cells. However, there are no reliable methods to induce the stem cells to form specific cells or to gain enough cells for transplantation, which limits their application in clinical therapy. The aim of this study is to select the best experimental setup to reach high proliferation levels by culturing these cells in a microgravity-based bioreactor. In typical cell culture, the cells sediment to the bottom surface of their container and propagate as a one-cell-layer sheet. Prevention of such sedimentation affords the freedom for self-assembly and the propagation of 3D tissue arrays. Suspension of cells is easily achievable using stirred technologies. Unfortunately, in conventional bioreactors, stirring invokes deleterious forces that disrupt cell aggregation and results in cell death. First-generation rotating bioreactors provided rotation on the horizontal axis, which resulted in the suspension of cells without stirring, thus providing a suitable environment to propagate cells without sedimentation to a surface. The rotating wall bioreactors did not provide a way to remove air bubbles that were causing shear and disrupting 3D cultures. Johnson Space Center successfully engineered the hydrofocusing bioreactor (HFB) that resolved the problem of removing the air bubbles from the fluid medium of NASA's rotating-wall space bioreactors. The HFB uses the principle of hydrodynamic focusing that simultaneously produces a low-shear fluid culture environment and a variable hydrofocusing force that can control the movement, location, and removal of suspended cells, tissues, and air bubbles from the bioreactor. The HFB is a rotating, domeshaped cell culture vessel with a centrally located sampling port and an internal viscous spinner. The vessel and spinner can rotate at different speeds either in the same or opposite directions. Rotation of the vessel and viscous interaction at the spinner generate a hydrofocusing force. Adjusting the differential rotation rate between vessel and spinner controls the magnitude of the force.

Mechanics of epithelial closure over non-adherent environments

NASA Astrophysics Data System (ADS)

Vedula, Sri Ram Krishna; Peyret, Grégoire; Cheddadi, Ibrahim; Chen, Tianchi; Brugués, Agustí; Hirata, Hiroaki; Lopez-Menendez, Horacio; Toyama, Yusuke; Neves de Almeida, Luís; Trepat, Xavier; Lim, Chwee Teck; Ladoux, Benoit

2015-01-01

The closure of gaps within epithelia is crucial to maintain its integrity during biological processes such as wound healing and gastrulation. Depending on the distribution of extracellular matrix, gap closure occurs through assembly of multicellular actin-based contractile cables or protrusive activity of border cells into the gap. Here we show that the supracellular actomyosin contractility of cells near the gap edge exerts sufficient tension on the surrounding tissue to promote closure of non-adherent gaps. Using traction force microscopy, we observe that cell-generated forces on the substrate at the gap edge first point away from the centre of the gap and then increase in the radial direction pointing into the gap as closure proceeds. Combining with numerical simulations, we show that the increase in force relies less on localized purse-string contractility and more on large-scale remodelling of the suspended tissue around the gap. Our results provide a framework for understanding the assembly and the mechanics of cellular contractility at the tissue level.

Mechanics of epithelial closure over non-adherent environments

PubMed Central

Vedula, Sri Ram Krishna; Peyret, Grégoire; Cheddadi, Ibrahim; Chen, Tianchi; Brugués, Agustí; Hirata, Hiroaki; Lopez-Menendez, Horacio; Toyama, Yusuke; Neves de Almeida, Luís; Trepat, Xavier; Lim, Chwee Teck; Ladoux, Benoit

2015-01-01

The closure of gaps within epithelia is crucial to maintain its integrity during biological processes such as wound healing and gastrulation. Depending on the distribution of extracellular matrix, gap closure occurs through assembly of multicellular actin-based contractile cables or protrusive activity of border cells into the gap. Here we show that the supracellular actomyosin contractility of cells near the gap edge exerts sufficient tension on the surrounding tissue to promote closure of non-adherent gaps. Using traction force microscopy, we observe that cell-generated forces on the substrate at the gap edge first point away from the centre of the gap and then increase in the radial direction pointing into the gap as closure proceeds. Combining with numerical simulations, we show that the increase in force relies less on localized purse-string contractility and more on large-scale remodelling of the suspended tissue around the gap. Our results provide a framework for understanding the assembly and the mechanics of cellular contractility at the tissue level. PMID:25608921

Digital holographic microscopy long-term and real-time monitoring of cell division and changes under simulated zero gravity.

PubMed

Pan, Feng; Liu, Shuo; Wang, Zhe; Shang, Peng; Xiao, Wen

2012-05-07

The long-term and real-time monitoring the cell division and changes of osteoblasts under simulated zero gravity condition were succeed by combing a digital holographic microscopy (DHM) with a superconducting magnet (SM). The SM could generate different magnetic force fields in a cylindrical cavity, where the gravitational force of biological samples could be canceled at a special gravity position by a high magnetic force. Therefore the specimens were levitated and in a simulated zero gravity environment. The DHM was modified to fit with SM by using single mode optical fibers and a vertically-configured jig designed to hold specimens and integrate optical device in the magnet's bore. The results presented the first-phase images of living cells undergoing dynamic divisions and changes under simulated zero gravity environment for a period of 10 hours. The experiments demonstrated that the SM-compatible DHM setup could provide a highly efficient and versatile method for research on the effects of microgravity on biological samples.

Viscous damping and spring force in periodic perforated planar microstructures when the Reynolds’ equation cannot be applied

PubMed Central

Homentcovschi, Dorel; Miles, Ronald N.

2010-01-01

A model of squeeze-film behavior is developed based on Stokes’ equations for viscous, compressible isothermal flows. The flow domain is an axisymmetrical, unit cell approximation of a planar, periodic, perforated microstructure. The model is developed for cases when the lubrication approximation cannot be applied. The complex force generated by vibrations of the diaphragm driving the flow has two components: the damping force and the spring force. While for large frequencies the spring force dominates, at low (acoustical) frequencies the damping force is the most important part. The analytical approach developed here yields an explicit formula for both forces. In addition, using a finite element software package, the damping force is also obtained numerically. A comparison is made between the analytic result, numerical solution, and some experimental data found in the literature, which validates the analytic formula and provides compelling arguments about its value in designing microelectomechanical devices. PMID:20329828

Molecular pathophysiology of cerebral edema

PubMed Central

Gerzanich, Volodymyr; Simard, J Marc

2015-01-01

Advancements in molecular biology have led to a greater understanding of the individual proteins responsible for generating cerebral edema. In large part, the study of cerebral edema is the study of maladaptive ion transport. Following acute CNS injury, cells of the neurovascular unit, particularly brain endothelial cells and astrocytes, undergo a program of pre- and post-transcriptional changes in the activity of ion channels and transporters. These changes can result in maladaptive ion transport and the generation of abnormal osmotic forces that, ultimately, manifest as cerebral edema. This review discusses past models and current knowledge regarding the molecular and cellular pathophysiology of cerebral edema. PMID:26661240

Regulation of muscle contraction by Drebrin-like protein 1 probed by atomic force microscopy

NASA Astrophysics Data System (ADS)

Garces, Renata; Butkevich, Eugenia; Platen, Mitja; Schmidt, Christoph F.; Biophysics Team

Sarcomeres are the fundamental contractile units of striated muscle cells. They are composed of a variety of structural and regulatory proteins functioning in a precisely orchestrated fashion to enable coordinated force generation in striated muscles. Recently, we have identified a C. elegans drebrin-like protein 1 (DBN-1) as a novel sarcomere component, which stabilizes actin filaments during muscle contraction. To further characterize the function of DBN-1 in muscle cells, we generated a new dbn-1 loss-of-function allele. Absence of DBN-1 resulted in a unique worm movement phenotype, characterized by hyper-bending. It is not clear yet if DBN-1 acts to enhance or reduce the capacity for contraction. We present here an experimental mechanical study on C. elegans muscle mechanics. We measured the stiffness of the worm by indenting living C. eleganswith a micron-sized sphere adhered to the cantilever of an atomic force microscope (AFM). Modeling the worm as a pressurized elastic shell allows us to monitor the axial tension in the muscle through the measured stiffness. We compared responses of wild-type and mutant C. elegans in which DBN-1 is not expressed..

Ionic mechanisms subserving mechanosensory transduction and neural integration in statocyst hair cells of Hermissenda

NASA Technical Reports Server (NTRS)

Farley, Joseph

1988-01-01

The neural processing of gravitational-produced sensory stimulation of statocyst hair cells in the nudibranch mollusk Hermissenda was studied. The goal in these studies was to understand how: gravireceptor neurons sense or transduce gravitational forces, gravitational stimulation is integrated so as to produce a graded receptor potential, and ultimately the generation of an action potential, and various neural adaptation phenomena which hair cells exhibit arise. The approach to these problems was primarily electrophysical.

Electromagnetic tweezers with independent force and torque control

NASA Astrophysics Data System (ADS)

Jiang, Chang; Lionberger, Troy A.; Wiener, Diane M.; Meyhofer, Edgar

2016-08-01

Magnetic tweezers are powerful tools to manipulate and study the mechanical properties of biological molecules and living cells. In this paper we present a novel, bona fide electromagnetic tweezer (EMT) setup that allows independent control of the force and torque applied via micrometer-sized magnetic beads to a molecule under study. We implemented this EMT by combining a single solenoid that generates force (f-EMT) with a set of four solenoids arranged into a symmetric quadrupole to generate torque (τ-EMT). To demonstrate the capability of the tweezers, we attached optically asymmetric Janus beads to single, tethered DNA molecules. We show that tension in the piconewton force range can be applied to single DNA molecules and the molecule can simultaneously be twisted with torques in the piconewton-nanometer range. Furthermore, the EMT allows the two components to be independently controlled. At various force levels applied to the Janus bead, the trap torsional stiffness can be continuously changed simply by varying the current magnitude applied to the τ-EMT. The flexible and independent control of force and torque by the EMT makes it an ideal tool for a range of measurements where tensional and torsional properties need to be studied simultaneously on a molecular or cellular level.

Self-Organization and Forces in the Mitotic Spindle.

PubMed

Pavin, Nenad; Tolić, Iva M

2016-07-05

At the onset of division, the cell forms a spindle, a precise self-constructed micromachine composed of microtubules and the associated proteins, which divides the chromosomes between the two nascent daughter cells. The spindle arises from self-organization of microtubules and chromosomes, whose different types of motion help them explore the space and eventually approach and interact with each other. Once the interactions between the chromosomes and the microtubules have been established, the chromosomes are moved to the equatorial plane of the spindle and ultimately toward the opposite spindle poles. These transport processes rely on directed forces that are precisely regulated in space and time. In this review, we discuss how microtubule dynamics and their rotational movement drive spindle self-organization, as well as how the forces acting in the spindle are generated, balanced, and regulated.

Toward the Structure of Dynamic Membrane-Anchored Actin Networks

PubMed Central

Weber, Igor

2007-01-01

In the cortex of a motile cell, membrane-anchored actin filaments assemble into structures of varying shape and function. Filopodia are distinguished by a core of bundled actin filaments within finger-like extensions of the membrane. In a recent paper by Medalia et al1 cryo-electron tomography has been used to reconstruct, from filopodia of Dictyostelium cells, the 3-dimensional organization of actin filaments in connection with the plasma membrane. A special arrangement of short filaments converging toward the filopod's tip has been called a “terminal cone”. In this region force is applied for protrusion of the membrane. Here we discuss actin organization in the filopodia of Dictyostelium in the light of current views on forces that are generated by polymerizing actin filaments, and on the resistance of membranes against deformation that counteracts these forces. PMID:19262130

Actomyosin Pulls to Advance the Nucleus in a Migrating Tissue Cell

PubMed Central

Wu, Jun; Kent, Ian A.; Shekhar, Nandini; Chancellor, T.J.; Mendonca, Agnes; Dickinson, Richard B.; Lele, Tanmay P.

2014-01-01

The cytoskeletal forces involved in translocating the nucleus in a migrating tissue cell remain unresolved. Previous studies have variously implicated actomyosin-generated pushing or pulling forces on the nucleus, as well as pulling by nucleus-bound microtubule motors. We found that the nucleus in an isolated migrating cell can move forward without any trailing-edge detachment. When a new lamellipodium was triggered with photoactivation of Rac1, the nucleus moved toward the new lamellipodium. This forward motion required both nuclear-cytoskeletal linkages and myosin activity. Apical or basal actomyosin bundles were found not to translate with the nucleus. Although microtubules dampen fluctuations in nuclear position, they are not required for forward translocation of the nucleus during cell migration. Trailing-edge detachment and pulling with a microneedle produced motion and deformation of the nucleus suggestive of a mechanical coupling between the nucleus and the trailing edge. Significantly, decoupling the nucleus from the cytoskeleton with KASH overexpression greatly decreased the frequency of trailing-edge detachment. Collectively, these results explain how the nucleus is moved in a crawling fibroblast and raise the possibility that forces could be transmitted from the front to the back of the cell through the nucleus. PMID:24411232

Growing cells push back under pressure.

PubMed

Gibson, W T; Gibson, M C

2012-04-13

In both plants and animals, the interplay between mechanical force generation and mechanical sensing plays a stabilizing role in many developmental processes. Uyttewaal et al. now demonstrate that cells in the Arabidopsis shoot apical meristem respond to local mechanical stresses by reorienting their growth, thereby guiding morphogenesis. Notably, the mechanism underlying such guidance is amplification--not suppression--of growth-rate heterogeneity. Copyright © 2012 Elsevier Inc. All rights reserved.

Probing mechanobiology with laser-induced shockwaves

NASA Astrophysics Data System (ADS)

Carmona, Christopher; Preece, Daryl C.; Gomez-Godinez, Veronica; Shi, Linda Z.; Berns, Michael W.

2017-08-01

Traumatic Brain Injury (TBI) occurs when an external force injures the brain. While clinical outcomes of TBI can vary widely in severity, few mechanisms of neurodegeneration following TBI have been identified for treatment. We propose a model for studying TBI using laser-induced shockwaves (LISs). An optical system was developed that allows single cells to be studied in response to LISs. Our system utilizes an optically-coupled force measurement component that allows for the visualization of shockwave dynamics. Here, the force measurement system is characterized by imaging stages over the period of violent expansion and collapse of microbubbles responsible for shockwave generation.

Hybrid mechanosensing system to generate the polarity needed for migration in fish keratocytes

PubMed Central

Okimura, Chika; Iwadate, Yoshiaki

2016-01-01

ABSTRACT Crawling cells can generate polarity for migration in response to forces applied from the substratum. Such reaction varies according to cell type: there are both fast- and slow-crawling cells. In response to periodic stretching of the elastic substratum, the intracellular stress fibers in slow-crawling cells, such as fibroblasts, rearrange themselves perpendicular to the direction of stretching, with the result that the shape of the cells extends in that direction; whereas fast-crawling cells, such as neutrophil-like differentiated HL-60 cells and Dictyostelium cells, which have no stress fibers, migrate perpendicular to the stretching direction. Fish epidermal keratocytes are another type of fast-crawling cell. However, they have stress fibers in the cell body, which gives them a typical slow-crawling cell structure. In response to periodic stretching of the elastic substratum, intact keratocytes rearrange their stress fibers perpendicular to the direction of stretching in the same way as fibroblasts and migrate parallel to the stretching direction, while blebbistatin-treated stress fiber-less keratocytes migrate perpendicular to the stretching direction, in the same way as seen in HL-60 cells and Dictyostelium cells. Our results indicate that keratocytes have a hybrid mechanosensing system that comprises elements of both fast- and slow-crawling cells, to generate the polarity needed for migration. PMID:27124267

Mesenchymal stem cell mechanobiology and emerging experimental platforms

PubMed Central

MacQueen, Luke; Sun, Yu; Simmons, Craig A.

2013-01-01

Experimental control over progenitor cell lineage specification can be achieved by modulating properties of the cell's microenvironment. These include physical properties of the cell adhesion substrate, such as rigidity, topography and deformation owing to dynamic mechanical forces. Multipotent mesenchymal stem cells (MSCs) generate contractile forces to sense and remodel their extracellular microenvironments and thereby obtain information that directs broad aspects of MSC function, including lineage specification. Various physical factors are important regulators of MSC function, but improved understanding of MSC mechanobiology requires novel experimental platforms. Engineers are bridging this gap by developing tools to control mechanical factors with improved precision and throughput, thereby enabling biological investigation of mechanics-driven MSC function. In this review, we introduce MSC mechanobiology and review emerging cell culture platforms that enable new insights into mechanobiological control of MSCs. Our main goals are to provide engineers and microtechnology developers with an up-to-date description of MSC mechanobiology that is relevant to the design of experimental platforms and to introduce biologists to these emerging platforms. PMID:23635493

Measuring the Adhesion Forces for the Multivalent Binding of Vancomycin-Conjugated Dendrimer to Bacterial Cell-Wall Peptide.

PubMed

Peterson, Elizabeth; Joseph, Christine; Peterson, Hannah; Bouwman, Rachael; Tang, Shengzhuang; Cannon, Jayme; Sinniah, Kumar; Choi, Seok Ki

2018-06-19

Multivalent ligand-receptor interaction provides the fundamental basis for the hypothetical notion that high binding avidity relates to the strong force of adhesion. Despite its increasing importance in the design of targeted nanoconjugates, an understanding of the physical forces underlying the multivalent interaction remains a subject of urgent investigation. In this study, we designed three vancomycin (Van)-conjugated dendrimers G5(Van) n ( n = mean valency = 0, 1, 4) for bacterial targeting with generation 5 (G5) poly(amidoamine) dendrimer as a multivalent scaffold and evaluated both their binding avidity and physical force of adhesion to a bacterial model surface by employing surface plasmon resonance (SPR) spectroscopy and atomic force microscopy. The SPR experiment for these conjugates was performed in a biosensor chip surface immobilized with a bacterial cell-wall peptide Lys-d-Ala-d-Ala. Of these, G5(Van) 4 bound most tightly with a K D of 0.34 nM, which represents an increase in avidity by 2 or 3 orders of magnitude relative to a monovalent conjugate G5(Van) 1 or free vancomycin, respectively. By single-molecule force spectroscopy, we measured the adhesion force between G5(Van) n and the same cell-wall peptide immobilized on the surface. The distribution of adhesion forces increased in proportion to vancomycin valency with the mean force of 134 pN at n = 4 greater than 96 pN at n = 1 at a loading rate of 5200 pN/s. In summary, our results are strongly supportive of the positive correlation between the avidity and adhesion force in the multivalent interaction of vancomycin nanoconjugates.

Cavity-induced microstreaming for simultaneous on-chip pumping and size-based separation of cells and particles.

PubMed

Patel, Maulik V; Nanayakkara, Imaly A; Simon, Melinda G; Lee, Abraham P

2014-10-07

We present a microfluidic platform for simultaneous on-chip pumping and size-based separation of cells and particles without external fluidic control systems required for most existing platforms. The device utilizes an array of acoustically actuated air/liquid interfaces generated using dead-end side channels termed Lateral Cavity Acoustic Transducers (LCATs). The oscillating interfaces generate local streaming flow while the angle of the LCATs relative to the main channel generates a global bulk flow from the inlet to the outlet. The interaction of these two competing velocity fields (i.e. global bulk velocity vs. local streaming velocity) is responsible for the observed separation. It is shown that the separation of 5 μm and 10 μm polystyrene beads is dependent on the ratio of these two competing velocity fields. The experimental and simulation results suggest that particle trajectories based only on Stokes drag force cannot fully explain the separation behavior and that the impact of additional forces due to the oscillating flow field must be considered to determine the trajectory of the beads and ultimately the separation behavior of the device. To demonstrate an application of this separation platform with cellular components, smaller red blood cells (7.5 ± 0.8 μm) are separated from larger K562 cells (16.3 ± 2.0 μm) with viabilities comparable to those of controls based on a trypan blue exclusion assay.

Actin Is Crucial for All Kinetically Distinguishable Forms of Endocytosis at Synapses.

PubMed

Wu, Xin-Sheng; Lee, Sung Hoon; Sheng, Jiansong; Zhang, Zhen; Zhao, Wei-Dong; Wang, Dongsheng; Jin, Yinghui; Charnay, Patrick; Ervasti, James M; Wu, Ling-Gang

2016-12-07

Mechanical force is needed to mediate endocytosis. Whether actin, the most abundant force-generating molecule, is essential for endocytosis is highly controversial in mammalian cells, particularly synapses, likely due to the use of actin blockers, the efficiency and specificity of which are often unclear in the studied cell. Here we addressed this issue using a knockout approach combined with measurements of membrane capacitance and fission pore conductance, imaging of vesicular protein endocytosis, and electron microscopy. We found that two actin isoforms, β- and γ-actin, are crucial for slow, rapid, bulk, and overshoot endocytosis at large calyx-type synapses, and for slow endocytosis and bulk endocytosis at small hippocampal synapses. Polymerized actin provides mechanical force to form endocytic pits. Actin also facilitates replenishment of the readily releasable vesicle pool, likely via endocytic clearance of active zones. We conclude that polymerized actin provides mechanical force essential for all kinetically distinguishable forms of endocytosis at synapses. Published by Elsevier Inc.

Actin is crucial for all kinetically distinguishable forms of endocytosis at synapses

PubMed Central

Wu, Xin-Sheng; Lee, Sunghoon; Sheng, Jiansong; Zhang, Zhen; Zhao, Weidong; Wang, Dongsheng; Jin, Yinghui; Charnay, Patrick; Ervasti, James M.; Wu, Ling-Gang

2016-01-01

Summary Mechanical force is needed to mediate endocytosis. Whether actin, the most abundant force-generating molecule, is essential for endocytosis is highly controversial in mammalian cells, particularly synapses, likely due to the use of actin blockers, the efficiency and specificity of which are often unclear in the studied cell. Here we addressed this issue using knockout approach combined with measurements of membrane capacitance and fission pore conductance, imaging of vesicular protein endocytosis, and electron microscopy. We found that two actin isoforms, β- and γ-actin, are crucial for slow, rapid, bulk, and overshoot endocytosis at large calyx-type synapses, and for slow endocytosis and bulk endocytosis at small hippocampal synapses. Polymerized actin provides mechanical force to form endocytic pits. Actin also facilitates replenishment of the readily releasable vesicle pool, likely via endocytic clearance of active zones. We conclude that polymerized actin provides mechanical force essential for all kinetically distinguishable forms of endocytosis at synapses. PMID:27840001

The bifoil photodyne: a photonic crystal oscillator.

PubMed

Lugo, J E; Doti, R; Sanchez, N; de la Mora, M B; del Rio, J A; Faubert, J

2014-01-15

Optical tweezers is an example how to use light to generate a physical force. They have been used to levitate viruses, bacteria, cells, and sub cellular organisms. Nonetheless it would be beneficial to use such force to develop a new kind of applications. However the radiation pressure usually is small to think in moving larger objects. Currently, there is some research investigating novel photonic working principles to generate a higher force. Here, we studied theoretically and experimentally the induction of electromagnetic forces in one-dimensional photonic crystals when light impinges on the off-axis direction. The photonic structure consists of a micro-cavity like structure formed of two one-dimensional photonic crystals made of free-standing porous silicon, separated by a variable air gap and the working wavelength is 633 nm. We show experimental evidence of this force when the photonic structure is capable of making auto-oscillations and forced-oscillations. We measured peak displacements and velocities ranging from 2 up to 35 microns and 0.4 up to 2.1 mm/s with a power of 13 mW. Recent evidence showed that giant resonant light forces could induce average velocity values of 0.45 mm/s in microspheres embedded in water with 43 mW light power.

The bifoil photodyne: a photonic crystal oscillator

PubMed Central

Lugo, J. E.; Doti, R.; Sanchez, N.; de la Mora, M. B.; del Rio, J. A.; Faubert, J.

2014-01-01

Optical tweezers is an example how to use light to generate a physical force. They have been used to levitate viruses, bacteria, cells, and sub cellular organisms. Nonetheless it would be beneficial to use such force to develop a new kind of applications. However the radiation pressure usually is small to think in moving larger objects. Currently, there is some research investigating novel photonic working principles to generate a higher force. Here, we studied theoretically and experimentally the induction of electromagnetic forces in one-dimensional photonic crystals when light impinges on the off-axis direction. The photonic structure consists of a micro-cavity like structure formed of two one-dimensional photonic crystals made of free-standing porous silicon, separated by a variable air gap and the working wavelength is 633 nm. We show experimental evidence of this force when the photonic structure is capable of making auto-oscillations and forced-oscillations. We measured peak displacements and velocities ranging from 2 up to 35 microns and 0.4 up to 2.1 mm/s with a power of 13 mW. Recent evidence showed that giant resonant light forces could induce average velocity values of 0.45 mm/s in microspheres embedded in water with 43 mW light power. PMID:24423985

Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen

PubMed Central

Thievessen, Ingo; Fakhri, Nikta; Steinwachs, Julian; Kraus, Viola; McIsaac, R. Scott; Gao, Liang; Chen, Bi-Chang; Baird, Michelle A.; Davidson, Michael W.; Betzig, Eric; Oldenbourg, Rudolf; Waterman, Clare M.; Fabry, Ben

2015-01-01

Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton–ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell–ECM adhesion and traction force generation.—Thievessen, I., Fakhri, N., Steinwachs, J., Kraus, V., McIsaac, R. S., Gao, L., Chen, B.-C., Baird, M. A., Davidson, M. W., Betzig, E., Oldenbourg, R., Waterman, C., M., Fabry, B. Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen. PMID:26195589

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids.

PubMed

Meraviglia, Viviana; Zanon, Alessandra; Lavdas, Alexandros A; Schwienbacher, Christine; Silipigni, Rosamaria; Di Segni, Marina; Chen, Huei-Sheng Vincent; Pramstaller, Peter P; Hicks, Andrew A; Rossini, Alessandra

2015-06-05

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation. In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes.

PubMed

Shafa, Mehdi; Krawetz, Roman; Zhang, Yuan; Rattner, Jerome B; Godollei, Anna; Duff, Henry J; Rancourt, Derrick E

2011-12-14

Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Murine D3-MHC-neo(r) ESCs formed embyroid bodies (EBs) and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin) was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC differentiation protocols within suspension bioreactors demands a more complete understanding of the impacts of shear forces on the regulation of pluripotency and differentiation in pluripotent stem cells.

Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes

PubMed Central

2011-01-01

Background Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Results Murine D3-MHC-neor ESCs formed embyroid bodies (EBs) and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin) was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. Conclusions This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC differentiation protocols within suspension bioreactors demands a more complete understanding of the impacts of shear forces on the regulation of pluripotency and differentiation in pluripotent stem cells. PMID:22168552

Synaptopodin couples epithelial contractility to α-actinin-4–dependent junction maturation

PubMed Central

Kannan, Nivetha

2015-01-01

The epithelial junction experiences mechanical force exerted by endogenous actomyosin activities and from interactions with neighboring cells. We hypothesize that tension generated at cell–cell adhesive contacts contributes to the maturation and assembly of the junctional complex. To test our hypothesis, we used a hydraulic apparatus that can apply mechanical force to intercellular junction in a confluent monolayer of cells. We found that mechanical force induces α-actinin-4 and actin accumulation at the cell junction in a time- and tension-dependent manner during junction development. Intercellular tension also induces α-actinin-4–dependent recruitment of vinculin to the cell junction. In addition, we have identified a tension-sensitive upstream regulator of α-actinin-4 as synaptopodin. Synaptopodin forms a complex containing α-actinin-4 and β-catenin and interacts with myosin II, indicating that it can physically link adhesion molecules to the cellular contractile apparatus. Synaptopodin depletion prevents junctional accumulation of α-actinin-4, vinculin, and actin. Knockdown of synaptopodin and α-actinin-4 decreases the strength of cell–cell adhesion, reduces the monolayer permeability barrier, and compromises cellular contractility. Our findings underscore the complexity of junction development and implicate a control process via tension-induced sequential incorporation of junctional components. PMID:26504173

Magnetic force micropiston: an integrated force/microfluidic device for the application of compressive forces in a confined environment.

PubMed

Fisher, J K; Kleckner, N

2014-02-01

Cellular biology takes place inside confining spaces. For example, bacteria grow in crevices, red blood cells squeeze through capillaries, and chromosomes replicate inside the nucleus. Frequently, the extent of this confinement varies. Bacteria grow longer and divide, red blood cells move through smaller and smaller passages as they travel to capillary beds, and replication doubles the amount of DNA inside the nucleus. This increase in confinement, either due to a decrease in the available space or an increase in the amount of material contained in a constant volume, has the potential to squeeze and stress objects in ways that may lead to changes in morphology, dynamics, and ultimately biological function. Here, we describe a device developed to probe the interplay between confinement and the mechanical properties of cells and cellular structures, and forces that arise due to changes in a structure's state. In this system, the manipulation of a magnetic bead exerts a compressive force upon a target contained in the confining space of a microfluidic channel. This magnetic force microfluidic piston is constructed in such a way that we can measure (a) target compliance and changes in compliance as induced by changes in buffer, extract, or biochemical composition, (b) target expansion force generated by changes in the same parameters, and (c) the effects of compression stress on a target's structure and function. Beyond these issues, our system has general applicability to a variety of questions requiring the combination of mechanical forces, confinement, and optical imaging.

Magnetic force micropiston: An integrated force/microfluidic device for the application of compressive forces in a confined environment

NASA Astrophysics Data System (ADS)

Fisher, J. K.; Kleckner, N.

2014-02-01

Cellular biology takes place inside confining spaces. For example, bacteria grow in crevices, red blood cells squeeze through capillaries, and chromosomes replicate inside the nucleus. Frequently, the extent of this confinement varies. Bacteria grow longer and divide, red blood cells move through smaller and smaller passages as they travel to capillary beds, and replication doubles the amount of DNA inside the nucleus. This increase in confinement, either due to a decrease in the available space or an increase in the amount of material contained in a constant volume, has the potential to squeeze and stress objects in ways that may lead to changes in morphology, dynamics, and ultimately biological function. Here, we describe a device developed to probe the interplay between confinement and the mechanical properties of cells and cellular structures, and forces that arise due to changes in a structure's state. In this system, the manipulation of a magnetic bead exerts a compressive force upon a target contained in the confining space of a microfluidic channel. This magnetic force microfluidic piston is constructed in such a way that we can measure (a) target compliance and changes in compliance as induced by changes in buffer, extract, or biochemical composition, (b) target expansion force generated by changes in the same parameters, and (c) the effects of compression stress on a target's structure and function. Beyond these issues, our system has general applicability to a variety of questions requiring the combination of mechanical forces, confinement, and optical imaging.

Rapid fabricating technique for multi-layered human hepatic cell sheets by forceful contraction of the fibroblast monolayer.

PubMed

Sakai, Yusuke; Koike, Makiko; Hasegawa, Hideko; Yamanouchi, Kosho; Soyama, Akihiko; Takatsuki, Mitsuhisa; Kuroki, Tamotsu; Ohashi, Kazuo; Okano, Teruo; Eguchi, Susumu

2013-01-01

Cell sheet engineering is attracting attention from investigators in various fields, from basic research scientists to clinicians focused on regenerative medicine. However, hepatocytes have a limited proliferation potential in vitro, and it generally takes a several days to form a sheet morphology and multi-layered sheets. We herein report our rapid and efficient technique for generating multi-layered human hepatic cell (HepaRG® cell) sheets using pre-cultured fibroblast monolayers derived from human skin (TIG-118 cells) as a feeder layer on a temperature-responsive culture dish. Multi-layered TIG-118/HepaRG cell sheets with a thick morphology were harvested on day 4 of culturing HepaRG cells by forceful contraction of the TIG-118 cells, and the resulting sheet could be easily handled. In addition, the human albumin and alpha 1-antitrypsin synthesis activities of TIG-118/HepaRG cells were approximately 1.2 and 1.3 times higher than those of HepaRG cells, respectively. Therefore, this technique is considered to be a promising modality for rapidly fabricating multi-layered human hepatocyte sheets from cells with limited proliferation potential, and the engineered cell sheet could be used for cell transplantation with highly specific functions.

A beginner's guide to atomic force microscopy probing for cell mechanics

PubMed Central

2016-01-01

Abstract Atomic Force microscopy (AFM) is becoming a prevalent tool in cell biology and biomedical studies, especially those focusing on the mechanical properties of cells and tissues. The newest generation of bio‐AFMs combine ease of use and seamless integration with live‐cell epifluorescence or more advanced optical microscopies. As a unique feature with respect to other bionanotools, AFM provides nanometer‐resolution maps for cell topography, stiffness, viscoelasticity, and adhesion, often overlaid with matching optical images of the probed cells. This review is intended for those about to embark in the use of bio‐AFMs, and aims to assist them in designing an experiment to measure the mechanical properties of adherent cells. In addition to describing the main steps in a typical cell mechanics protocol and explaining how data is analysed, this review will also discuss some of the relevant contact mechanics models available and how they have been used to characterize specific features of cellular and biological samples. Microsc. Res. Tech. 80:75–84, 2017. © 2016 Wiley Periodicals, Inc. PMID:27676584

Probing the non-equilibrium force fluctuation spectrum of actomyosin cortices in vivo

NASA Astrophysics Data System (ADS)

Tan, Tzer Han; Swartz, Zachary; Keren, Kinneret; Fakhri, Nikta

Mechanics of the cortex govern the shape of animal cells, and its dynamics underlie cell migration, cytokinesis and embryogenesis. The molecular players involved are largely known, yet it is unclear how their collective dynamics give rise to large scale behavior. This is mostly due to the lack of experimental tools to probe the spatially varying active mechanical properties of the cortex. Here, we introduce a novel technique based on fluorescent single walled carbon nanotubes to generate non-equilibrium force fluctuation spectrum of actomysion cortices in starfish oocytes. The quantitative measurements combined with a theoretical model reveal the role of stress organization in active mechanics and dynamics of the cortex.

Charge transport in CdTe solar cells revealed by conductive tomographic atomic force microscopy

DOE PAGES

Luria, Justin; Kutes, Yasemin; Moore, Andrew; ...

2016-09-26

Polycrystalline photovoltaics comprising cadmium telluride (CdTe) represent a growing portion of the solar cell market, yet the physical picture of charge transport through the meso-scale grain morphology remains a topic of debate. It is unknown how thin film morphology affects the transport of electron-hole pairs. Accordingly this study is the first to generate three dimensional images of photocurrent throughout a thin-film solar cell, revealing the profound influence of grain boundaries and stacking faults on device efficiency.

DIRECTIONAL FLUID TRANSPORT ACROSS ORGAN-BLOOD BARRIERS: PHYSIOLOGY AND CELL BIOLOGY

PubMed Central

Caceres, Paulo S.; Benedicto, Ignacio; Lehmann, Guillermo L.; Rodriguez-Boulan, Enrique J.

2018-01-01

Directional fluid flow is an essential process for embryo development as well as for organ and organism homeostasis. Here, we review the diverse structure of various organ-blood barriers, the driving forces, transporters and polarity mechanisms that regulate fluid transport across them, focusing on kidney-, eye- and brain-blood barriers. We end by discussing how cross-talk between barrier epithelial and endothelial cells, perivascular cells and basement membrane signaling contribute to generate and maintain organ-blood barriers. PMID:28003183

Concise review: growing hearts in the right place: on the design of biomimetic materials for cardiac stem cell differentiation.

PubMed

Farouz, Yohan; Chen, Yong; Terzic, André; Menasché, Philippe

2015-04-01

Tissue engineering aims at recapitulating permissive conditions that enable cells to collaborate and form functional tissues. Applications range from human tissue modeling for diagnostic purposes to therapeutic solutions in regenerative medicine and surgery. Across this spectrum, human stem cells are the active ingredient, expandable virtually indefinitely and with the propensity to generate new tissue. Engaging lineage-specific differentiation requires a precise concerto of key spatial and temporal factors, such as soluble molecules and growth factors, but also physical and mechanical stimuli. These stimuli compete to modulate distinct developmental signaling pathways and ultimately affect the differentiation efficiency. The heart is a chemo-mechano-electrical biological system that behaves as both a sensor and an actuator. It can transduce electrical inputs to generate mechanical contraction and electrical wave propagation. Such a complex organ arises from multipart developmental events that interact with one another to self-regulate. Here, we overview the main events of heart development and the role of mechanical forces in modifying the microenvironment of the progenitor cells. We analyze the cascades regulating cardiac gene activation to illustrate how mechanotransduction is already involved in the most popular protocols for stem cell differentiation (SCD) into cardiomyocytes. We then review how forces are transmitted to embryonic stem cells by cell-substrate or cell-cell communications, and how biomaterials can be designed to mimic these interactions and help reproduce key features of the developmental milieu. Putting this back in a clinical perspective, many challenges need to be overcome before biomaterials-based SCD protocols can be scaled up and marketed. © AlphaMed Press.

Simultaneous recordings of force and sliding movement between a myosin-coated glass microneedle and actin cables in vitro.

PubMed Central

Chaen, S; Oiwa, K; Shimmen, T; Iwamoto, H; Sugi, H

1989-01-01

To elucidate the molecular mechanism of muscle contraction resulting from the ATP-dependent actin-myosin interaction, we constructed an assay system with which both the force and the movement produced by the actin-myosin interaction in vitro can be simultaneously recorded and analyzed. The assay system consisted of the giant internodal cells of an alga, Nitellopsis obtusa, which contain well-organized arrays of actin filaments (actin cables) running along the cell long axis, and a glass microneedle (tip diameter, approximately 7 microns; elastic coefficient, approximately 40 pN/microns), which was coated with skeletal muscle myosin at the tip and extended from a micromanipulator at right angles with the actin cables. When the myosin-coated tip of the microneedle was brought into contact with the exposed surface of the actin cables, it exhibited ATP-dependent movement along the actin cables over a distance of 20-150 microns in 20-200 s (20-23 degrees C) and eventually stopped due to a balance between forces generated by the actin-myosin interaction (800-6000 pN) and by the bent microneedle. Since the load on the force-generating myosin molecules increased with the bending displacement of the microneedle (auxotonic condition), the relation between the load and the sliding velocity of the myosin heads past the actin cables was determined from the time course of the microneedle movement recorded with a video system. The shape of the force-velocity curve thus obtained was convex upwards, similar to that of the force-velocity curve of intact frog muscle fibers obtained under the auxotonic condition. Images PMID:2922395

Quantification of surface tension and internal pressure generated by single mitotic cells

NASA Astrophysics Data System (ADS)

Fischer-Friedrich, Elisabeth; Hyman, Anthony A.; Jülicher, Frank; Müller, Daniel J.; Helenius, Jonne

2014-08-01

During mitosis, adherent cells round up, by increasing the tension of the contractile actomyosin cortex while increasing the internal hydrostatic pressure. In the simple scenario of a liquid cell interior, the surface tension is related to the local curvature and the hydrostatic pressure difference by Laplace's law. However, verification of this scenario for cells requires accurate measurements of cell shape. Here, we use wedged micro-cantilevers to uniaxially confine single cells and determine confinement forces while concurrently determining cell shape using confocal microscopy. We fit experimentally measured confined cell shapes to shapes obeying Laplace's law with uniform surface tension and find quantitative agreement. Geometrical parameters derived from fitting the cell shape, and the measured force were used to calculate hydrostatic pressure excess and surface tension of cells. We find that HeLa cells increase their internal hydrostatic pressure excess and surface tension from ~ 40 Pa and 0.2 mNm-1 during interphase to ~ 400 Pa and 1.6 mNm-1 during metaphase. The method introduced provides a means to determine internal pressure excess and surface tension of rounded cells accurately and with minimal cellular perturbation, and should be applicable to characterize the mechanical properties of various cellular systems.

Floating electrode dielectrophoresis.

PubMed

Golan, Saar; Elata, David; Orenstein, Meir; Dinnar, Uri

2006-12-01

In practice, dielectrophoresis (DEP) devices are based on micropatterned electrodes. When subjected to applied voltages, the electrodes generate nonuniform electric fields that are necessary for the DEP manipulation of particles. In this study, electrically floating electrodes are used in DEP devices. It is demonstrated that effective DEP forces can be achieved by using floating electrodes. Additionally, DEP forces generated by floating electrodes are different from DEP forces generated by excited electrodes. The floating electrodes' capabilities are explained theoretically by calculating the electric field gradients and demonstrated experimentally by using test-devices. The test-devices show that floating electrodes can be used to collect erythrocytes (red blood cells). DEP devices which contain many floating electrodes ought to have fewer connections to external signal sources. Therefore, the use of floating electrodes may considerably facilitate the fabrication and operation of DEP devices. It can also reduce device dimensions. However, the key point is that DEP devices can integrate excited electrodes fabricated by microtechnology processes and floating electrodes fabricated by nanotechnology processes. Such integration is expected to promote the use of DEP devices in the manipulation of nanoparticles.

Simple display system of mechanical properties of cells and their dispersion.

PubMed

Shimizu, Yuji; Kihara, Takanori; Haghparast, Seyed Mohammad Ali; Yuba, Shunsuke; Miyake, Jun

2012-01-01

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.

Simple Display System of Mechanical Properties of Cells and Their Dispersion

PubMed Central

Shimizu, Yuji; Kihara, Takanori; Haghparast, Seyed Mohammad Ali; Yuba, Shunsuke; Miyake, Jun

2012-01-01

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. PMID:22479595

The response of the East Asia summer precipitation to greenhouse gases and anthropogenic aerosols

NASA Astrophysics Data System (ADS)

Tian, Fangxing; Dong, Buwen; Robson, Jon; Sutton, Rowan

2017-04-01

The changes of precipitation over China since the mid-20th century display a dipole trend pattern over eastern China, which is known as Southern-Flood-Northern-Drought (SFND) pattern. The trends have been attributed to different factors, such as the changes of aerosol and greenhouse gas emissions. However much less is known about the different effects of these factors on generating the SFND pattern. This work investigated the drivers and dynamical mechanisms by using a atmosphere-ocean-mixed-layer model forced by anthropogenic greenhouse gase (GHG), anthropogenic aerosol (AA) and the combined effects. The model experiments with different forcings indicates that the GHG forcing dominates the precipitation increase, which is stronger over south China than over north China. On the other hand, the drought over north China is dominated by the AA forcing. Analysis of physical processes indicates that the GHG forcing increases the moisture and leads to strong convergence over east China, and then more precipitation. The AA forcing leads to north wind anomalies and generates divergent anomalies over north China, which reduces the precipitation. Further analysis indicates that the changes of the circulation which related to the SFND pattern are forced by the enhancement of the Western North Pacific Subtropical High (WNPSH). Both GHG and AA forcing can enhance the WNPSH by changing the local Hadley cell.

Air Force Research Laboratory Success Stories: A Review of 1997/1998

DTIC Science & Technology

1999-03-01

one year). Assuming that 60 percent of the electrical power required for these sites could be generated with solar cells , the higher efficiency AMTEC ...engineers ability to meet the cost goal of the Mars Pathfinder program. Accomplishment High efficiency solar cells and non-rechargeable batteries developed...integrated manufacturing system to efficiently mix low volume and high volume processing. MPCL metrics have been defined in categories which reflect

Personalized nanomedicine advancements for stem cell tracking☆

PubMed Central

Janowski, Mirek; Bulte, Jeff W.M.; Walczak, Piotr

2012-01-01

Recent technological developments in biomedicine have facilitated the generation of data on the anatomical, physiological and molecular level for individual patients and thus introduces opportunity for therapy to be personalized in an unprecedented fashion. Generation of patient-specific stem cells exemplifies the efforts toward this new approach. Cell-based therapy is a highly promising treatment paradigm; however, due to the lack of consistent and unbiased data about the fate of stem cells in vivo, interpretation of therapeutic remains challenging hampering the progress in this field. The advent of nanotechnology with a wide palette of inorganic and organic nanostructures has expanded the arsenal of methods for tracking transplanted stem cells. The diversity of nanomaterials has revolutionized personalized nanomedicine and enables individualized tailoring of stem cell labeling materials for the specific needs of each patient. The successful implementation of stem cell tracking will likely be a significant driving force that will contribute to the further development of nanotheranostics. The purpose of this review is to emphasize the role of cell tracking using currently available nanoparticles. PMID:22820528

Laboratory and theoretical models of planetary-scale instabilities and waves

NASA Technical Reports Server (NTRS)

Hart, John E.; Toomre, Juri

1990-01-01

Meteorologists and planetary astronomers interested in large-scale planetary and solar circulations recognize the importance of rotation and stratification in determining the character of these flows. In the past it has been impossible to accurately model the effects of sphericity on these motions in the laboratory because of the invariant relationship between the uni-directional terrestrial gravity and the rotation axis of an experiment. Researchers studied motions of rotating convecting liquids in spherical shells using electrohydrodynamic polarization forces to generate radial gravity, and hence centrally directed buoyancy forces, in the laboratory. The Geophysical Fluid Flow Cell (GFFC) experiments performed on Spacelab 3 in 1985 were analyzed. Recent efforts at interpretation led to numerical models of rotating convection with an aim to understand the possible generation of zonal banding on Jupiter and the fate of banana cells in rapidly rotating convection as the heating is made strongly supercritical. In addition, efforts to pose baroclinic wave experiments for future space missions using a modified version of the 1985 instrument led to theoretical and numerical models of baroclinic instability. Rather surprising properties were discovered, which may be useful in generating rational (rather than artificially truncated) models for nonlinear baroclinic instability and baroclinic chaos.

[Role of proton-motive force in the conjugative DNA transport in Staphylococci].

PubMed

Gavriliuk, V G; Vinnikov, A I

1997-01-01

Sensitivity of the conjugative process in staphylococci to the action of uncouplers of oxidative phosphorylation and inhibitors of electron transport systems have been proved, that testifies to the energy-dependent character of conjugative transport of DNA. Proceeding of the conjugation process depends upon the generation of delta microH+ on the membrane of both the donor and recipient cells. contribution of protonmotive forces to providing for the transfer of plasmids during conjugation to staphylococci has been defined.

Shear Forces during Blast, Not Abrupt Changes in Pressure Alone, Generate Calcium Activity in Human Brain Cells

DTIC Science & Technology

2012-06-29

the tissue-force interaction(s) and the cellular damage properties remain unresolved. Studies on a mechanical head model demonstrated high transient...that pressure transient. In vitro models of primary blast injury [5,18,19] are likewise limited by an absence of real-time, high spatial and temporal... models , as well as with human injuries in which expression of bTBI symptoms among different individuals that are exposed to the same blast is

Myosin XI-dependent formation of tubular structures from endoplasmic reticulum isolated from tobacco cultured BY-2 cells.

PubMed

Yokota, Etsuo; Ueda, Haruko; Hashimoto, Kohsuke; Orii, Hidefumi; Shimada, Tomoo; Hara-Nishimura, Ikuko; Shimmen, Teruo

2011-05-01

The reticular network of the endoplasmic reticulum (ER) consists of tubular and lamellar elements and is arranged in the cortical region of plant cells. This network constantly shows shape change and remodeling motion. Tubular ER structures were formed when GTP was added to the ER vesicles isolated from tobacco (Nicotiana tabacum) cultured BY-2 cells expressing ER-localized green fluorescent protein. The hydrolysis of GTP during ER tubule formation was higher than that under conditions in which ER tubule formation was not induced. Furthermore, a shearing force, such as the flow of liquid, was needed for the elongation/extension of the ER tubule. The shearing force was assumed to correspond to the force generated by the actomyosin system in vivo. To confirm this hypothesis, the S12 fraction was prepared, which contained both cytosol and microsome fractions, including two classes of myosins, XI (175-kD myosin) and VIII (BY-2 myosin VIII-1), and ER-localized green fluorescent protein vesicles. The ER tubules and their mesh-like structures were arranged in the S12 fraction efficiently by the addition of ATP, GTP, and exogenous filamentous actin. The tubule formation was significantly inhibited by the depletion of 175-kD myosin from the S12 fraction but not BY-2 myosin VIII-1. Furthermore, a recombinant carboxyl-terminal tail region of 175-kD myosin also suppressed ER tubule formation. The tips of tubules moved along filamentous actin during tubule elongation. These results indicated that the motive force generated by the actomyosin system contributes to the formation of ER tubules, suggesting that myosin XI is responsible not only for the transport of ER in cytoplasm but also for the reticular organization of cortical ER.

Myosin XI-Dependent Formation of Tubular Structures from Endoplasmic Reticulum Isolated from Tobacco Cultured BY-2 Cells1[W][OA

PubMed Central

Yokota, Etsuo; Ueda, Haruko; Hashimoto, Kohsuke; Orii, Hidefumi; Shimada, Tomoo; Hara-Nishimura, Ikuko; Shimmen, Teruo

2011-01-01

The reticular network of the endoplasmic reticulum (ER) consists of tubular and lamellar elements and is arranged in the cortical region of plant cells. This network constantly shows shape change and remodeling motion. Tubular ER structures were formed when GTP was added to the ER vesicles isolated from tobacco (Nicotiana tabacum) cultured BY-2 cells expressing ER-localized green fluorescent protein. The hydrolysis of GTP during ER tubule formation was higher than that under conditions in which ER tubule formation was not induced. Furthermore, a shearing force, such as the flow of liquid, was needed for the elongation/extension of the ER tubule. The shearing force was assumed to correspond to the force generated by the actomyosin system in vivo. To confirm this hypothesis, the S12 fraction was prepared, which contained both cytosol and microsome fractions, including two classes of myosins, XI (175-kD myosin) and VIII (BY-2 myosin VIII-1), and ER-localized green fluorescent protein vesicles. The ER tubules and their mesh-like structures were arranged in the S12 fraction efficiently by the addition of ATP, GTP, and exogenous filamentous actin. The tubule formation was significantly inhibited by the depletion of 175-kD myosin from the S12 fraction but not BY-2 myosin VIII-1. Furthermore, a recombinant carboxyl-terminal tail region of 175-kD myosin also suppressed ER tubule formation. The tips of tubules moved along filamentous actin during tubule elongation. These results indicated that the motive force generated by the actomyosin system contributes to the formation of ER tubules, suggesting that myosin XI is responsible not only for the transport of ER in cytoplasm but also for the reticular organization of cortical ER. PMID:21427277

YAP is essential for tissue tension to ensure vertebrate 3D body shape.

PubMed

Porazinski, Sean; Wang, Huijia; Asaoka, Yoichi; Behrndt, Martin; Miyamoto, Tatsuo; Morita, Hitoshi; Hata, Shoji; Sasaki, Takashi; Krens, S F Gabriel; Osada, Yumi; Asaka, Satoshi; Momoi, Akihiro; Linton, Sarah; Miesfeld, Joel B; Link, Brian A; Senga, Takeshi; Shimizu, Nobuyoshi; Nagase, Hideaki; Matsuura, Shinya; Bagby, Stefan; Kondoh, Hisato; Nishina, Hiroshi; Heisenberg, Carl-Philipp; Furutani-Seiki, Makoto

2015-05-14

Vertebrates have a unique 3D body shape in which correct tissue and organ shape and alignment are essential for function. For example, vision requires the lens to be centred in the eye cup which must in turn be correctly positioned in the head. Tissue morphogenesis depends on force generation, force transmission through the tissue, and response of tissues and extracellular matrix to force. Although a century ago D'Arcy Thompson postulated that terrestrial animal body shapes are conditioned by gravity, there has been no animal model directly demonstrating how the aforementioned mechano-morphogenetic processes are coordinated to generate a body shape that withstands gravity. Here we report a unique medaka fish (Oryzias latipes) mutant, hirame (hir), which is sensitive to deformation by gravity. hir embryos display a markedly flattened body caused by mutation of YAP, a nuclear executor of Hippo signalling that regulates organ size. We show that actomyosin-mediated tissue tension is reduced in hir embryos, leading to tissue flattening and tissue misalignment, both of which contribute to body flattening. By analysing YAP function in 3D spheroids of human cells, we identify the Rho GTPase activating protein ARHGAP18 as an effector of YAP in controlling tissue tension. Together, these findings reveal a previously unrecognised function of YAP in regulating tissue shape and alignment required for proper 3D body shape. Understanding this morphogenetic function of YAP could facilitate the use of embryonic stem cells to generate complex organs requiring correct alignment of multiple tissues.

The Drosophila actin regulator ENABLED regulates cell shape and orientation during gonad morphogenesis.

PubMed

Sano, Hiroko; Kunwar, Prabhat S; Renault, Andrew D; Barbosa, Vitor; Clark, Ivan B N; Ishihara, Shuji; Sugimura, Kaoru; Lehmann, Ruth

2012-01-01

Organs develop distinctive morphologies to fulfill their unique functions. We used Drosophila embryonic gonads as a model to study how two different cell lineages, primordial germ cells (PGCs) and somatic gonadal precursors (SGPs), combine to form one organ. We developed a membrane GFP marker to image SGP behaviors live. These studies show that a combination of SGP cell shape changes and inward movement of anterior and posterior SGPs leads to the compaction of the spherical gonad. This process is disrupted in mutants of the actin regulator, enabled (ena). We show that Ena coordinates these cell shape changes and the inward movement of the SGPs, and Ena affects the intracellular localization of DE-cadherin (DE-cad). Mathematical simulation based on these observations suggests that changes in DE-cad localization can generate the forces needed to compact an elongated structure into a sphere. We propose that Ena regulates force balance in the SGPs by sequestering DE-cad, leading to the morphogenetic movement required for gonad compaction.

Biased migration of confined neutrophil-like cells in asymmetric hydraulic environments.

PubMed

Prentice-Mott, Harrison V; Chang, Chi-Han; Mahadevan, L; Mitchison, Timothy J; Irimia, Daniel; Shah, Jagesh V

2013-12-24

Cells integrate multiple measurement modalities to navigate their environment. Soluble and substrate-bound chemical gradients and physical cues have all been shown to influence cell orientation and migration. Here we investigate the role of asymmetric hydraulic pressure in directional sensing. Cells confined in microchannels identified and chose a path of lower hydraulic resistance in the absence of chemical cues. In a bifurcating channel with asymmetric hydraulic resistances, this choice was preceded by the elaboration of two leading edges with a faster extension rate along the lower resistance channel. Retraction of the "losing" edge appeared to precipitate a final choice of direction. The pressure differences altering leading edge protrusion rates were small, suggesting weak force generation by leading edges. The response to the physical asymmetry was able to override a dynamically generated chemical cue. Motile cells may use this bias as a result of hydraulic resistance, or "barotaxis," in concert with chemotaxis to navigate complex environments.

Improved design of a cone-shaped rotating disk for shear force loading in a cell culture plate

NASA Astrophysics Data System (ADS)

Keawprachum, Boonrit; Limjeerajarus, Nuttapol; Nakalekha Limjeerajarus, Chalida; Srisungsitthisunti, Pornsak

2018-01-01

In our previous study, a cone-shaped rotating disk had been designed and proposed for generating shear force on the cell in a cell culture plate. This study aims to improve the design of the rotating disk that could provide a better uniformity of shear stress distribution. The top of the cone was designed to be trimmed off to obtain a flat head area. The effect of tilt angle (θ) was numerically studied using computational fluid dynamics (CFD) technique in ANSYS-Fluent software. The results revealed that for 500 rpm, the new designed rotating disk with a height of cone-shaped top to the plate bottom h = 1 mm and θ = 25° provided the best uniformity of 0.820 which was better than that of the previously designed.

Physical limits of cell migration: control by ECM space and nuclear deformation and tuning by proteolysis and traction force.

PubMed

Wolf, Katarina; Te Lindert, Mariska; Krause, Marina; Alexander, Stephanie; Te Riet, Joost; Willis, Amanda L; Hoffman, Robert M; Figdor, Carl G; Weiss, Stephen J; Friedl, Peter

2013-06-24

Cell migration through 3D tissue depends on a physicochemical balance between cell deformability and physical tissue constraints. Migration rates are further governed by the capacity to degrade ECM by proteolytic enzymes, particularly matrix metalloproteinases (MMPs), and integrin- and actomyosin-mediated mechanocoupling. Yet, how these parameters cooperate when space is confined remains unclear. Using MMP-degradable collagen lattices or nondegradable substrates of varying porosity, we quantitatively identify the limits of cell migration by physical arrest. MMP-independent migration declined as linear function of pore size and with deformation of the nucleus, with arrest reached at 10% of the nuclear cross section (tumor cells, 7 µm²; T cells, 4 µm²; neutrophils, 2 µm²). Residual migration under space restriction strongly depended upon MMP-dependent ECM cleavage by enlarging matrix pore diameters, and integrin- and actomyosin-dependent force generation, which jointly propelled the nucleus. The limits of interstitial cell migration thus depend upon scaffold porosity and deformation of the nucleus, with pericellular collagenolysis and mechanocoupling as modulators.

Magnetically actuated tissue engineered scaffold: insights into mechanism of physical stimulation

NASA Astrophysics Data System (ADS)

Sapir-Lekhovitser, Yulia; Rotenberg, Menahem Y.; Jopp, Juergen; Friedman, Gary; Polyak, Boris; Cohen, Smadar

2016-02-01

Providing the right stimulatory conditions resulting in efficient tissue promoting microenvironment in vitro and in vivo is one of the ultimate goals in tissue development for regenerative medicine. It has been shown that in addition to molecular signals (e.g. growth factors) physical cues are also required for generation of functional cell constructs. These cues are particularly relevant to engineering of biological tissues, within which mechanical stress activates mechano-sensitive receptors, initiating biochemical pathways which lead to the production of functionally mature tissue. Uniform magnetic fields coupled with magnetizable nanoparticles embedded within three dimensional (3D) scaffold structures remotely create transient physical forces that can be transferrable to cells present in close proximity to the nanoparticles. This study investigated the hypothesis that magnetically responsive alginate scaffold can undergo reversible shape deformation due to alignment of scaffold's walls in a uniform magnetic field. Using custom made Helmholtz coil setup adapted to an Atomic Force Microscope we monitored changes in matrix dimensions in situ as a function of applied magnetic field, concentration of magnetic particles within the scaffold wall structure and rigidity of the matrix. Our results show that magnetically responsive scaffolds exposed to an externally applied time-varying uniform magnetic field undergo a reversible shape deformation. This indicates on possibility of generating bending/stretching forces that may exert a mechanical effect on cells due to alternating pattern of scaffold wall alignment and relaxation. We suggest that the matrix structure deformation is produced by immobilized magnetic nanoparticles within the matrix walls resulting in a collective alignment of scaffold walls upon magnetization. The estimated mechanical force that can be imparted on cells grown on the scaffold wall at experimental conditions is in the order of 1 pN, which correlates well with reported threshold to induce mechanotransduction effects on cellular level. This work is our next step in understanding of how to accurately create proper stimulatory microenvironment for promotion of cellular organization to form mature tissue engineered constructs.

Magnetically actuated tissue engineered scaffold: insights into mechanism of physical stimulation

PubMed Central

Sapir-Lekhovitser, Yulia; Rotenberg, Menahem Y.; Jopp, Juergen; Friedman, Gary; Polyak, Boris; Cohen, Smadar

2016-01-01

Providing the right stimulatory conditions resulting in efficient tissue promoting microenvironment in vitro and in vivo is one of the ultimate goals in tissue development for regenerative medicine. It has been shown that in addition to molecular signals (e.g. growth factors) physical cues are also required for generation of functional cell constructs. These cues are particularly relevant to engineering of biological tissues, within which mechanical stress activates mechano-sensitive receptors, initiating biochemical pathways which lead to the production of functionally mature tissue. Uniform magnetic fields coupled with magnetizable nanoparticles embedded within three dimensional (3D) scaffold structures remotely create transient physical forces that can be transferrable to cells present in close proximity to the nanoparticles. This study investigated the hypothesis that magnetically responsive alginate scaffold can undergo reversible shape deformation due to alignment of scaffold’s walls in a uniform magnetic field. Using custom made Helmholtz coil setup adapted to an Atomic Force Microscope we monitored changes in matrix dimensions in situ as a function of applied magnetic field, concentration of magnetic particles within the scaffold wall structure and rigidity of the matrix. Our results show that magnetically responsive scaffolds exposed to an externally applied time-varying uniform magnetic field undergo a reversible shape deformation. This indicates on possibility of generating bending/stretching forces that may exert a mechanical effect on cells due to alternating pattern of scaffold wall alignment and relaxation. We suggest that the matrix structure deformation is produced by immobilized magnetic nanoparticles within the matrix walls resulting in a collective alignment of scaffold walls upon magnetization. The estimated mechanical force that can be imparted on cells grown on the scaffold wall at experimental conditions is in the order of 1 pN, which correlates well with reported threshold to induce mechanotransduction effects on cellular level. This work is our next step in understanding of how to accurately create proper stimulatory microenvironment for promotion of cellular organization to form mature tissue engineered constructs. PMID:26790538

Optical tweezers force measurements to study parasites chemotaxis

NASA Astrophysics Data System (ADS)

de Thomaz, A. A.; Pozzo, L. Y.; Fontes, A.; Almeida, D. B.; Stahl, C. V.; Santos-Mallet, J. R.; Gomes, S. A. O.; Feder, D.; Ayres, D. C.; Giorgio, S.; Cesar, C. L.

2009-07-01

In this work, we propose a methodology to study microorganisms chemotaxis in real time using an Optical Tweezers system. Optical Tweezers allowed real time measurements of the force vectors, strength and direction, of living parasites under chemical or other kinds of gradients. This seems to be the ideal tool to perform observations of taxis response of cells and microorganisms with high sensitivity to capture instantaneous responses to a given stimulus. Forces involved in the movement of unicellular parasites are very small, in the femto-pico-Newton range, about the same order of magnitude of the forces generated in an Optical Tweezers. We applied this methodology to investigate the Leishmania amazonensis (L. amazonensis) and Trypanossoma cruzi (T. cruzi) under distinct situations.

Interaction of Dendritic Polymers with Synthetic Lipid and Cell Membranes

NASA Astrophysics Data System (ADS)

Mecke, Almut; Hong, Seungpyo; Bielinska, Anna U.; Banaszak Holl, Mark M.; Orr, Bradford G.; Baker, James R., Jr.

2004-03-01

Polyamidoamine (PAMAM) dendrimers are promising candidates for the development of nanoscale therapeutic transport agents. Here we present studies on dendrimer-membrane interactions leading to a better understanding of possible uptake mechanisms into cells. Using synthetic lipid and natural cell membranes as model systems it is shown that the effect of PAMAM dendrimers on a membrane strongly depends on the dendrimer generation, architecture and chemical properties of the branch end groups. Atomic force microscopy data indicates that generation 7 dendrimers have the ability to form small ( 10-100 nm) holes in a lipid bilayer. When dendrimers with otherwise identical chemical properties are arranged in a covalently linked cluster, no hole formation occurs. Dendrimer-lipid micelle formation is proposed and investigated as a possible mechanism for this behavior. Smaller dendrimers (generation 5) have a greatly reduced ability to remove lipid molecules from a bilayer. In addition to the size of the dendrimer, the charge of the branch end groups plays a significant role for dendrimer-membrane interactions. These results agree well with biological studies using cultured cells and point to a new mechanism of specific targeting and uptake into cells.

Trespassing cancer cells: ‘fingerprinting’ invasive protrusions reveals metastatic culprits

PubMed Central

Klemke, Richard L.

2012-01-01

Metastatic cancer cells produce invasive membrane protrusions called invadopodia and pseudopodia, which play a central role in driving cancer cell dissemination in the body. Malignant cells use these structures to attach to and degrade extracellular matrix proteins, generate force for cell locomotion, and to penetrate the vasculature. Recent work using unique subcellular fractionation methodologies combined with spatial genomic, proteomic, and phosphoproteomic profiling has provided insight into the invadopodiome and pseudopodiome signaling networks that control the protrusion of invasive membranes. Here I highlight how these powerful spatial “omics” approaches reveal important signatures of metastatic cancer cells and possible new therapeutic targets aimed at treating metastatic disease. PMID:22980730

Bacterial Colony from Two-Dimensional Division to Three-Dimensional Development

PubMed Central

Su, Pin-Tzu; Liao, Chih-Tang; Roan, Jiunn-Ren; Wang, Shao-Hung; Chiou, Arthur; Syu, Wan-Jr

2012-01-01

On agar surface, bacterial daughter cells form a 4-cell array after the first two rounds of division, and this phenomenon has been previously attributed to a balancing of interactions among the daughter bacteria and the underneath agar. We studied further the organization and development of colony after additional generations. By confocal laser scanning microscopy and real-time imaging, we observed that bacterial cells were able to self-organize and resulted in a near circular micro-colony consisting of monolayer cells. After continuous dividing, bacteria transited from two-dimensional expansion into three-dimensional growth and formed two to multi-layers in the center but retained a monolayer in the outer ring of the circular colony. The transverse width of this outer ring appeared to be approximately constant once the micro-colony reached a certain age. This observation supports the notion that balanced interplays of the forces involved lead to a gross morphology as the bacteria divide into offspring on agar surface. In this case, the result is due to a balance between the expansion force of the dividing bacteria, the non-covalent force among bacterial offspring and that between bacteria and substratum. PMID:23155376

Entropy-driven motility of Sinorhizobium meliloti on a semi-solid surface

PubMed Central

Dilanji, Gabriel E.; Teplitski, Max; Hagen, Stephen J.

2014-01-01

Sinorhizobium meliloti growing on soft agar can exhibit an unusual surface spreading behaviour that differs from other bacterial surface motilities. Bacteria in the colony secrete an exopolysaccharide-rich mucoid fluid that expands outward on the surface, carrying within it a suspension of actively dividing cells. The moving slime disperses the cells in complex and dynamic patterns indicative of simultaneous bacterial growth, swimming and aggregation. We find that while flagellar swimming is required to maintain the cells in suspension, the spreading and the associated pattern formation are primarily driven by the secreted exopolysaccharide EPS II, which creates two entropy-increasing effects: an osmotic flow of water from the agar to the mucoid fluid and a crowding or depletion attraction between the cells. Activation of these physical/chemical phenomena may be a useful function for the high molecular weight EPS II, a galactoglucan whose biosynthesis is tightly regulated by the ExpR/SinI/SinR quorum-sensing system: unlike bacterial colonies that spread via bacterium-generated, physical propulsive forces, S. meliloti under quorum conditions may use EPS II to activate purely entropic forces within its environment, so that it can disperse by passively ‘surfing’ on those forces. PMID:24741008

Carcinogenesis explained within the context of a theory of organisms.

PubMed

Sonnenschein, Carlos; Soto, Ana M

2016-10-01

For a century, the somatic mutation theory (SMT) has been the prevalent theory to explain carcinogenesis. According to the SMT, cancer is a cellular problem, and thus, the level of organization where it should be studied is the cellular level. Additionally, the SMT proposes that cancer is a problem of the control of cell proliferation and assumes that proliferative quiescence is the default state of cells in metazoa. In 1999, a competing theory, the tissue organization field theory (TOFT), was proposed. In contraposition to the SMT, the TOFT posits that cancer is a tissue-based disease whereby carcinogens (directly) and mutations in the germ-line (indirectly) alter the normal interactions between the diverse components of an organ, such as the stroma and its adjacent epithelium. The TOFT explicitly acknowledges that the default state of all cells is proliferation with variation and motility. When taking into consideration the principle of organization, we posit that carcinogenesis can be explained as a relational problem whereby release of the constraints created by cell interactions and the physical forces generated by cellular agency lead cells within a tissue to regain their default state of proliferation with variation and motility. Within this perspective, what matters both in morphogenesis and carcinogenesis is not only molecules, but also biophysical forces generated by cells and tissues. Herein, we describe how the principles for a theory of organisms apply to the TOFT and thus to the study of carcinogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Computational Framework for 3D Mechanical Modeling of Plant Morphogenesis with Cellular Resolution

PubMed Central

Gilles, Benjamin; Hamant, Olivier; Boudaoud, Arezki; Traas, Jan; Godin, Christophe

2015-01-01

The link between genetic regulation and the definition of form and size during morphogenesis remains largely an open question in both plant and animal biology. This is partially due to the complexity of the process, involving extensive molecular networks, multiple feedbacks between different scales of organization and physical forces operating at multiple levels. Here we present a conceptual and modeling framework aimed at generating an integrated understanding of morphogenesis in plants. This framework is based on the biophysical properties of plant cells, which are under high internal turgor pressure, and are prevented from bursting because of the presence of a rigid cell wall. To control cell growth, the underlying molecular networks must interfere locally with the elastic and/or plastic extensibility of this cell wall. We present a model in the form of a three dimensional (3D) virtual tissue, where growth depends on the local modulation of wall mechanical properties and turgor pressure. The model shows how forces generated by turgor-pressure can act both cell autonomously and non-cell autonomously to drive growth in different directions. We use simulations to explore lateral organ formation at the shoot apical meristem. Although different scenarios lead to similar shape changes, they are not equivalent and lead to different, testable predictions regarding the mechanical and geometrical properties of the growing lateral organs. Using flower development as an example, we further show how a limited number of gene activities can explain the complex shape changes that accompany organ outgrowth. PMID:25569615

Physics of phagocytosis of foreign versus self-tolerance

NASA Astrophysics Data System (ADS)

Tsai, Richard; Rodriguez, Pia; Discher, Dennis

2009-03-01

The first cells to `attack' an implanted or injected foreign material or microbe are phagocytic cells of the innate immune system. These cells actively and rapidly phagocytose foreign cells, surfaces, or particles, but the process that is inefficient when faced with ``self'' cells. We have examined the biochemistry and some of the physics of this decision to eat or not eat. One particular protein on all animal cell membranes, called CD47, seems to engage phagocytic cell couter-receptors, and deactivate the force-generating myosin machinery that otherwise makes phagocytosis efficient. We will map the phagocytic synapse between phagocytes and particles or cells and describe the physicochemical dynamics that mediate this key decision in compatability.

Mechanical Detection of a Long-Range Actin Network Emanating from a Biomimetic Cortex

PubMed Central

Bussonnier, Matthias; Carvalho, Kevin; Lemière, Joël; Joanny, Jean-François; Sykes, Cécile; Betz, Timo

2014-01-01

Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that participate in the force generation of eukaryotic cells. Such forces are used for cell motility, cytokinesis, and tissue remodeling. Among those actin networks, we focus on the actin cortex, a dense branched network beneath the plasma membrane that is of particular importance for the mechanical properties of the cell. Here we reproduce the cellular cortex by activating actin filament growth on a solid surface. We unveil the existence of a sparse actin network that emanates from the surface and extends over a distance that is at least 10 times larger than the cortex itself. We call this sparse actin network the “actin cloud” and characterize its mechanical properties with optical tweezers. We show, both experimentally and theoretically, that the actin cloud is mechanically relevant and that it should be taken into account because it can sustain forces as high as several picoNewtons (pN). In particular, it is known that in plant cells, actin networks similar to the actin cloud have a role in positioning the nucleus; in large oocytes, they play a role in driving chromosome movement. Recent evidence shows that such networks even prevent granule condensation in large cells. PMID:25140420

High-throughput particle manipulation by hydrodynamic, electrokinetic, and dielectrophoretic effects in an integrated microfluidic chip

PubMed Central

Li, Shunbo; Li, Ming; Bougot-Robin, Kristelle; Cao, Wenbin; Yeung Yeung Chau, Irene; Li, Weihua; Wen, Weijia

2013-01-01

Integrating different steps on a chip for cell manipulations and sample preparation is of foremost importance to fully take advantage of microfluidic possibilities, and therefore make tests faster, cheaper and more accurate. We demonstrated particle manipulation in an integrated microfluidic device by applying hydrodynamic, electroosmotic (EO), electrophoretic (EP), and dielectrophoretic (DEP) forces. The process involves generation of fluid flow by pressure difference, particle trapping by DEP force, and particle redirect by EO and EP forces. Both DC and AC signals were applied, taking advantages of DC EP, EO and AC DEP for on-chip particle manipulation. Since different types of particles respond differently to these signals, variations of DC and AC signals are capable to handle complex and highly variable colloidal and biological samples. The proposed technique can operate in a high-throughput manner with thirteen independent channels in radial directions for enrichment and separation in microfluidic chip. We evaluated our approach by collecting Polystyrene particles, yeast cells, and E. coli bacteria, which respond differently to electric field gradient. Live and dead yeast cells were separated successfully, validating the capability of our device to separate highly similar cells. Our results showed that this technique could achieve fast pre-concentration of colloidal particles and cells and separation of cells depending on their vitality. Hydrodynamic, DC electrophoretic and DC electroosmotic forces were used together instead of syringe pump to achieve sufficient fluid flow and particle mobility for particle trapping and sorting. By eliminating bulky mechanical pumps, this new technique has wide applications for in situ detection and analysis. PMID:24404011

High-throughput particle manipulation by hydrodynamic, electrokinetic, and dielectrophoretic effects in an integrated microfluidic chip.

PubMed

Li, Shunbo; Li, Ming; Bougot-Robin, Kristelle; Cao, Wenbin; Yeung Yeung Chau, Irene; Li, Weihua; Wen, Weijia

2013-01-01

Integrating different steps on a chip for cell manipulations and sample preparation is of foremost importance to fully take advantage of microfluidic possibilities, and therefore make tests faster, cheaper and more accurate. We demonstrated particle manipulation in an integrated microfluidic device by applying hydrodynamic, electroosmotic (EO), electrophoretic (EP), and dielectrophoretic (DEP) forces. The process involves generation of fluid flow by pressure difference, particle trapping by DEP force, and particle redirect by EO and EP forces. Both DC and AC signals were applied, taking advantages of DC EP, EO and AC DEP for on-chip particle manipulation. Since different types of particles respond differently to these signals, variations of DC and AC signals are capable to handle complex and highly variable colloidal and biological samples. The proposed technique can operate in a high-throughput manner with thirteen independent channels in radial directions for enrichment and separation in microfluidic chip. We evaluated our approach by collecting Polystyrene particles, yeast cells, and E. coli bacteria, which respond differently to electric field gradient. Live and dead yeast cells were separated successfully, validating the capability of our device to separate highly similar cells. Our results showed that this technique could achieve fast pre-concentration of colloidal particles and cells and separation of cells depending on their vitality. Hydrodynamic, DC electrophoretic and DC electroosmotic forces were used together instead of syringe pump to achieve sufficient fluid flow and particle mobility for particle trapping and sorting. By eliminating bulky mechanical pumps, this new technique has wide applications for in situ detection and analysis.

Large gradient high magnetic field affects the association of MACF1 with actin and microtubule cytoskeleton.

PubMed

Qian, Ai-Rong; Hu, Li-Fang; Gao, Xiang; Zhang, Wei; Di, Sheng-Meng; Tian, Zong-Cheng; Yang, Peng-Fei; Yin, Da-Chuan; Weng, Yuan-Yuan; Shang, Peng

2009-10-01

The intense inhomogeneous magnetic fields acting on the diamagnetic materials naturally present in cells can generate strong magnetic forces. We have developed a superconducting magnet platform with large gradient high magnetic field (LG-HMF), which can produce three magnetic force fields of -1360, 0, and 1312 T(2)/m, and three corresponding apparent gravity levels, namely 0, 1, and 2-g for diamagnetic materials. In this study, the effects of different magnetic force fields on osteoblast-like cells (MG-63 and MC3T3-E1) viability, microtubule actin crosslinking factor 1 (MACF1) expression and its association with cytoskeleton were investigated. Results showed that cell viability increased to different degrees after exposure to 0 or 1-g conditions for 24 h, but it decreased by about 30% under 2-g conditions compared with control conditions. An increase in MACF1 expression at the RNA or protein level was observed in osteoblast-like cells under the magnetic force field of -1360 T(2)/m (0-g) relative to 1312 T(2)/m (2-g). Under control conditions, anti-MACF1 staining was scattered in the cytoplasm and partially colocalized with actin filaments (AFs) or microtubules (MTs) in the majority of osteoblast-like cells. Under 0-g conditions, MACF1 labeling was concentrated at perinuclear region and colocalization was not apparent. The patterns of anti-MACF1 labeling on MTs varied with MTs' changing under LG-HMF environment. In conclusion, LG-HMF affects osteoblast-like cell viability, MACF1 distribution, expression, and its association with cytoskeleton to some extent.

Mechanical output of myosin II motors is regulated by myosin filament size and actin network mechanics

NASA Astrophysics Data System (ADS)

Stam, Samantha; Alberts, Jonathan; Gardel, Margaret; Munro, Edwin

2013-03-01

The interactions of bipolar myosin II filaments with actin arrays are a predominate means of generating forces in numerous physiological processes including muscle contraction and cell migration. However, how the spatiotemporal regulation of these forces depends on motor mechanochemistry, bipolar filament size, and local actin mechanics is unknown. Here, we simulate myosin II motors with an agent-based model in which the motors have been benchmarked against experimental measurements. Force generation occurs in two distinct regimes characterized either by stable tension maintenance or by stochastic buildup and release; transitions between these regimes occur by changes to duty ratio and myosin filament size. The time required for building force to stall scales inversely with the stiffness of a network and the actin gliding speed of a motor. Finally, myosin motors are predicted to contract a network toward stiffer regions, which is consistent with experimental observations. Our representation of myosin motors can be used to understand how their mechanical and biochemical properties influence their observed behavior in a variety of in vitro and in vivo contexts.

Mapping of Mechanical Strains and Stresses around Quiescent Engineered Three-Dimensional Epithelial Tissues

PubMed Central

Gjorevski, Nikolce; Nelson, Celeste M.

2012-01-01

Understanding how physical signals guide biological processes requires qualitative and quantitative knowledge of the mechanical forces generated and sensed by cells in a physiologically realistic three-dimensional (3D) context. Here, we used computational modeling and engineered epithelial tissues of precise geometry to define the experimental parameters that are required to measure directly the mechanical stress profile of 3D tissues embedded within native type I collagen. We found that to calculate the stresses accurately in these settings, we had to account for mechanical heterogeneities within the matrix, which we visualized and quantified using confocal reflectance and atomic force microscopy. Using this technique, we were able to obtain traction forces at the epithelium-matrix interface, and to resolve and quantify patterns of mechanical stress throughout the surrounding matrix. We discovered that whereas single cells generate tension by contracting and pulling on the matrix, the contraction of multicellular tissues can also push against the matrix, causing emergent compression. Furthermore, tissue geometry defines the spatial distribution of mechanical stress across the epithelium, which communicates mechanically over distances spanning hundreds of micrometers. Spatially resolved mechanical maps can provide insight into the types and magnitudes of physical parameters that are sensed and interpreted by multicellular tissues during normal and pathological processes. PMID:22828342

Photo- and electropatterning of hydrogel-encapsulated living cell arrays.

PubMed

Albrecht, Dirk R; Tsang, Valerie Liu; Sah, Robert L; Bhatia, Sangeeta N

2005-01-01

Living cells have the potential to serve as sensors, naturally integrating the response to stimuli to generate predictions about cell fate (e.g., differentiation, migration, proliferation, apoptosis). Miniaturized arrays of living cells further offer the capability to interrogate many cells in parallel and thereby enable high-throughput and/or combinatorial assays. However, the interface between living cells and synthetic chip platforms is a critical one wherein the cellular phenotype must be preserved to generate useful signals. While some cell types retain tissue-specific features on a flat (2-D) surface, it has become increasingly apparent that a 3-D physical environment will be required for others. In this paper, we present two independent methods for creating living cell arrays that are encapsulated within a poly(ethylene glycol)-based hydrogel to create a local 3-D microenvironment. First, 'photopatterning' selectively crosslinks hydrogel microstructures containing living cells with approximately 100 microm feature size. Second, 'electropatterning' utilizes dielectrophoretic forces to position cells within a prepolymer solution prior to crosslinking, forming cell patterns with micron resolution. We further combine these methods to obtain hierarchical control of cell positioning over length scales ranging from microns to centimeters. This level of microenvironmental control should enable the fabrication of next-generation cellular microarrays in which robust 3-D cultures of cells are presented with appropriate physical and chemical cues and, consequently, report on cellular responses that resemble in vivo behavior.

Applications of optical manipulation in plant biology

NASA Astrophysics Data System (ADS)

Buer, Charles S.

Measuring small forces in biology is important for determining basic physiological parameters of a cell. The plant cell wall provides a primary defense and presents a barrier to research. Magnitudes of small forces are impossible to measure with mechanical transducers, glass needles, atomic force microscopy, or micropipet-based force transduction due to the cell wall. Therefore, a noninvasive method of breaching the plant cell wall to access the symplastic region of the cell is required. Laser light provides sub-micrometer positioning, particle manipulation without mechanical contact, and piconewton force determination. Consequently, the extension of laser microsurgery to expand an experimental tool for plant biology encompassed the overall objective. A protocol was developed for precisely inserting microscopic objects into the periplasmic region of plant callus cells using laser microsurgery. Ginkgo biloba and Agrobacterium rhizogenes were used as the model system for developing the optical tweezers and scalpel techniques. Better than 95% survival was achieved after plasmolyzing G. biloba cells, ablating a 2-4 μm hole through the cell wall using a pulsed UV laser beam, trapping and manipulating bacteria into the periplasmic region, and deplasmolyzing the cells. Optical trapping experiments implied a difference existed between the bacteria models. Determining the optical trapping efficiency of Agrobacterium rhizogenes and A. tumefaciens strains indicated the A. rhizogenes strain, ATCC 11325, was significantly less efficiently trapped than strains A4 and ATCC 15834 and the A. tumefaciens strain LBA4404. Differences were also found in capsule generation, growth media viscosity, and transmission electron microscopy negative staining implying that a difference in surface structure exists. Calcofluor fluorescence suggests the difference involves an exopolysaccharide. Callus cell plasmolysis revealed Hechtian strands interconnecting the plasma membrane and the cell wall. The spring tension of these strands was measured in normal and cold-hardened G. biloba and N. tabacum callus cells. There was little change in flexibility between the groups of cultured cells in either species studied. Microspheres were attached to Hechtian strands in normal cultured Nicotiana tabacum and the cells were deplasmolyzed and replasmolyzed to determine the fate of Hechtian strands. The microspheres either moved to the plasma membrane and adhered or moved to the cell wall and adhered. The attached microspheres occasionally moved independently on the same strand. Inserted microspheres provided a visual probe to follow physiological events within a plant cell.

Children with Heavy Prenatal Alcohol Exposure Experience Reduced Control of Isotonic Force

PubMed Central

Nguyen, Tanya T.; Levy, Susan S.; Riley, Edward P.; Thomas, Jennifer D.; Simmons, Roger W.

2013-01-01

Background Heavy prenatal alcohol exposure can result in diverse and extensive damage to the central nervous system, including the cerebellum, basal ganglia, and cerebral cortex. Given that these brain regions are involved in the generation and maintenance of motor force, we predicted that prenatal alcohol exposure would adversely affect this parameter of motor control. We previously reported that children with gestational alcohol exposure experience significant deficits in regulating isometric (i.e., constant) force. The purpose of the present study was to determine if these children exhibit similar deficits when producing isotonic (i.e., graded) force. Methods Children with heavy prenatal alcohol exposure and typically developing children completed a series of isotonic force contractions by exerting force on a load cell to match a criterion target force displayed on a computer monitor. Two levels of target force (5% or 20% of maximum voluntary force) were investigated in combination with varying levels of visual feedback. Results Compared to controls, children with heavy prenatal alcohol exposure generated isotonic force signals that were less accurate, more variable, and less complex in the time domain compared to control children. Specifically, interactions were found between group and visual feedback for response accuracy and signal complexity, suggesting that these children have greater difficulty altering their motor output when visual feedback is low. Conclusions These data suggest that prenatal alcohol exposure produces deficits in regulating isotonic force, which presumably result from alcohol-related damage to developing brain regions involved in motor control. These children will most likely experience difficulty performing basic motor skills and daily functional skills that require coordination of finely graded force. Therapeutic strategies designed to increase feedback and, consequently, facilitate visual-motor integration could improve isotonic force production in these children. PMID:22834891

Alkaline phosphatase and OCT-3/4 as useful markers for predicting susceptibility of human deciduous teeth-derived dental pulp cells to reprogramming factor-induced iPS cells.

PubMed

Inada, Emi; Saitoh, Issei; Kubota, Naoko; Soda, Miki; Matsueda, Kazunari; Murakami, Tomoya; Sawami, Tadashi; Kagoshima, Akiko; Yamasaki, Youichi; Sato, Masahiro

2017-11-01

The aim of the present study was to prove that primary cells enriched with stem cells are more easily reprogrammed to generate induced pluripotent stem (iPS) cells than those with scarce numbers of stem cells. We surveyed the alkaline phosphatase (ALP) activity in five primarily-isolated human deciduous teeth-derived dental pulp cells (HDDPC) with cytochemical staining to examine the possible presence of stem cells. Next, the expression of stemness-specific factors, such as OCT(Octumer-binding transcription factor)3/4, NANOG, SOX2(SRY (sex determining region Y)-box 2), CD90, muscle segment homeodomain homeobox (MSX) 1, and MSX2, was assessed with a reverse transcription polymerase chain reaction method. Finally, these isolated HDDPC were transfected with plasmids carrying genes coding Yamanaka factors to determine whether these cells could be reprogrammed to generate iPS cells. Of the five primarily-isolated HDDPC, two (HDDPC-1 and -5) exhibited higher degrees of ALP activity. OCT-3/4 expression was also prominent in those two lines. Furthermore, these two lines proliferated faster than the other three lines. The transfection of HDDPC with Yamanaka factors resulted in the generation of iPS cells from HDDPC-1 and -5. The number of cells with the stemness property of HDDPC differs among individuals, which suggests that HDDPC showing an increased expression of both ALP and OCT-3/4 can be more easily reprogrammed to generate iPS cells after the forced expression of reprogramming factors. © 2016 John Wiley & Sons Australia, Ltd.

A 'Good' muscle in a 'Bad' environment: the importance of airway smooth muscle force adaptation to airway hyperresponsiveness.

PubMed

Bossé, Ynuk; Chapman, David G; Paré, Peter D; King, Gregory G; Salome, Cheryl M

2011-12-15

Asthma is characterized by airway inflammation, with a consequent increase in spasmogens, and exaggerated airway narrowing in response to stimuli, termed airway hyperresponsiveness (AHR). The nature of any relationship between inflammation and AHR is less clear. Recent ex vivo data has suggested a novel mechanism by which inflammation may lead to AHR, in which increased basal ASM-tone, due to the presence of spasmogens in the airways, may "strengthen" the ASM and ultimately lead to exaggerated airway narrowing. This phenomenon was termed "force adaptation" [Bossé, Y., Chin, L.Y., Paré, P.D., Seow, C.Y., 2009. Adaptation of airway smooth muscle to basal tone: relevance to airway hyperresponsiveness. Am. J. Respir. Cell Mol. Biol. 40, 13-18]. However, it is unknown whether the magnitude of the effect of force adaptation ex vivo could contribute to exaggerated airway narrowing in vivo. Our aim was to utilize a computational model of ASM shortening in order to quantify the potential effect of force adaptation on airway narrowing when all other mechanical factors were kept constant. The shortening in the model is dictated by a balance between physiological loads and ASM force-generating capacity at different lengths. The results suggest that the magnitude of the effect of force adaptation on ASM shortening would lead to substantially more airway narrowing during bronchial challenge at any given airway generation. We speculate that the increased basal ASM-tone in asthma, due to the presence of inflammation-derived spasmogens, produces an increase in the force-generating capacity of ASM, predisposing to AHR during subsequent challenge. Copyright © 2011 Elsevier B.V. All rights reserved.

Improved throughput traction microscopy reveals pivotal role for matrix stiffness in fibroblast contractility and TGF-β responsiveness

PubMed Central

Marinković, Aleksandar; Mih, Justin D.; Park, Jin-Ah; Liu, Fei

2012-01-01

Lung fibroblast functions such as matrix remodeling and activation of latent transforming growth factor-β1 (TGF-β1) are associated with expression of the myofibroblast phenotype and are directly linked to fibroblast capacity to generate force and deform the extracellular matrix. However, the study of fibroblast force-generating capacities through methods such as traction force microscopy is hindered by low throughput and time-consuming procedures. In this study, we improved at the detail level methods for higher-throughput traction measurements on polyacrylamide hydrogels using gel-surface-bound fluorescent beads to permit autofocusing and automated displacement mapping, and transduction of fibroblasts with a fluorescent label to streamline cell boundary identification. Together these advances substantially improve the throughput of traction microscopy and allow us to efficiently compute the forces exerted by lung fibroblasts on substrates spanning the stiffness range present in normal and fibrotic lung tissue. Our results reveal that lung fibroblasts dramatically alter the forces they transmit to the extracellular matrix as its stiffness changes, with very low forces generated on matrices as compliant as normal lung tissue. Moreover, exogenous TGF-β1 selectively accentuates tractions on stiff matrices, mimicking fibrotic lung, but not on physiological stiffness matrices, despite equivalent changes in Smad2/3 activation. Taken together, these results demonstrate a pivotal role for matrix mechanical properties in regulating baseline and TGF-β1-stimulated contraction of lung fibroblasts and suggest that stiff fibrotic lung tissue may promote myofibroblast activation through contractility-driven events, whereas normal lung tissue compliance may protect against such feedback amplification of fibroblast activation. PMID:22659883

Power transduction of actin filaments ratcheting in vitro against a load.

PubMed

Démoulin, Damien; Carlier, Marie-France; Bibette, Jérôme; Baudry, Jean

2014-12-16

The actin cytoskeleton has the unique capability of producing pushing forces at the leading edge of motile cells without the implication of molecular motors. This phenomenon has been extensively studied theoretically, and molecular models, including the widely known Brownian ratchet, have been proposed. However, supporting experimental work is lacking, due in part to hardly accessible molecular length scales. We designed an experiment to directly probe the mechanism of force generation in a setup where a population of actin filaments grows against a load applied by magnetic microparticles. The filaments, arranged in stiff bundles by fascin, are constrained to point toward the applied load. In this protrusion-like geometry, we are able to directly measure the velocity of filament elongation and its dependence on force. Using numerical simulations, we provide evidence that our experimental data are consistent with a Brownian ratchet-based model. We further demonstrate the existence of a force regime far below stalling where the mechanical power transduced by the ratcheting filaments to the load is maximal. The actin machinery in migrating cells may tune the number of filaments at the leading edge to work in this force regime.

Analysis of microtubule growth dynamics arising from altered actin network structure and contractility in breast tumor cells

NASA Astrophysics Data System (ADS)

Ory, Eleanor C.; Bhandary, Lekhana; E Boggs, Amanda; Chakrabarti, Kristi R.; Parker, Joshua; Losert, Wolfgang; Martin, Stuart S.

2017-04-01

The periphery of epithelial cells is shaped by opposing cytoskeletal physical forces generated predominately by two dynamic force generating systems—growing microtubule ends push against the boundary from the cell center, and the actin cortex contracts the attached plasma membrane. Here we investigate how changes to the structure and dynamics of the actin cortex alter the dynamics of microtubules. Current drugs target actin polymerization and contraction to reduce cell division and invasiveness; however, the impacts on microtubule dynamics remain incompletely understood. Using human MCF-7 breast tumor cells expressing GFP-tagged microtubule end-binding-protein-1 (EB1) and coexpression of cytoplasmic fluorescent protein mCherry, we map the trajectories of growing microtubule ends and cytoplasmic boundary respectively. Based on EB1 tracks and cytoplasmic boundary outlines, we calculate the speed, distance from cytoplasmic boundary, and straightness of microtubule growth. Actin depolymerization with Latrunculin-A reduces EB1 growth speed as well as allows the trajectories to extend beyond the cytoplasmic boundary. Blebbistatin, a direct myosin-II inhibitor, reduced EB1 speed and yielded less straight EB1 trajectories. Inhibiting signaling upstream of myosin-II contractility via the Rho-kinase inhibitor, Y-27632, altered EB1 dynamics differently from Blebbistatin. These results indicate that reduced actin cortex integrity can induce distinct alterations in microtubule dynamics. Given recent findings that tumor stem cell characteristics are increased by drugs which reduce actin contractility or stabilize microtubules, it remains important to clearly define how cytoskeletal drugs alter the interactions between these two filament systems in tumor cells.

Rapid Fabricating Technique for Multi-Layered Human Hepatic Cell Sheets by Forceful Contraction of the Fibroblast Monolayer

PubMed Central

Sakai, Yusuke; Koike, Makiko; Hasegawa, Hideko; Yamanouchi, Kosho; Soyama, Akihiko; Takatsuki, Mitsuhisa; Kuroki, Tamotsu; Ohashi, Kazuo; Okano, Teruo; Eguchi, Susumu

2013-01-01

Cell sheet engineering is attracting attention from investigators in various fields, from basic research scientists to clinicians focused on regenerative medicine. However, hepatocytes have a limited proliferation potential in vitro, and it generally takes a several days to form a sheet morphology and multi-layered sheets. We herein report our rapid and efficient technique for generating multi-layered human hepatic cell (HepaRG® cell) sheets using pre-cultured fibroblast monolayers derived from human skin (TIG-118 cells) as a feeder layer on a temperature-responsive culture dish. Multi-layered TIG-118/HepaRG cell sheets with a thick morphology were harvested on day 4 of culturing HepaRG cells by forceful contraction of the TIG-118 cells, and the resulting sheet could be easily handled. In addition, the human albumin and alpha 1-antitrypsin synthesis activities of TIG-118/HepaRG cells were approximately 1.2 and 1.3 times higher than those of HepaRG cells, respectively. Therefore, this technique is considered to be a promising modality for rapidly fabricating multi-layered human hepatocyte sheets from cells with limited proliferation potential, and the engineered cell sheet could be used for cell transplantation with highly specific functions. PMID:23923035

Isolation of Precursor Cells from Waste Solid Fat Tissue

NASA Technical Reports Server (NTRS)

Byerly, Diane; Sognier, Marguerite A.

2009-01-01

A process for isolating tissue-specific progenitor cells exploits solid fat tissue obtained as waste from such elective surgical procedures as abdominoplasties (tummy tucks) and breast reductions. Until now, a painful and risky process of aspiration of bone marrow has been used to obtain a limited number of tissue- specific progenitor cells. The present process yields more tissue-specific progenitor cells and involves much less pain and risk for the patient. This process includes separation of fat from skin, mincing of the fat into small pieces, and forcing a fat saline mixture through a sieve. The mixture is then digested with collagenase type I in an incubator. After centrifugation tissue-specific progenitor cells are recovered and placed in a tissue-culture medium in flasks or Petri dishes. The tissue-specific progenitor cells can be used for such purposes as (1) generating three-dimensional tissue equivalent models for studying bone loss and muscle atrophy (among other deficiencies) and, ultimately, (2) generating replacements for tissues lost by the fat donor because of injury or disease.

A Miniature Couette to Generate Shear for Flow Cytometry: Studying Real-Time Modulation of Intracellular Calcium in Monocytic Cells

PubMed Central

Zwartz, Gordon J.; Chigaev, Alexandre; Foutz, Terry D.; Edwards, Bruce; Sklar, Larry A.

2013-01-01

Extracellular hydrodynamic forces may be transmitted to the interior of cells through the alteration of integrin conformation and affinity. Integrin activation regulates leukocyte recruitment, cell activation, and transmigration. The cellular and molecular mechanisms for integrin activation are not precisely known, although intracellular calcium signaling is involved. Flow cytometry offers a versatile way to study intracellular calcium signaling in real-time. We report a novel method to generate defined shear by using a miniature Couette. Testing involved measuring shear induced intracellular calcium signals of human monoblastoid U937 cells in suspension. The Couette was connected externally to a flow cytometer and pressurized at 6 PSI (4.1 N/m2). Cells were subjected to well-defined shear between 0 and 1000 s−1 and delivered continuously within 10 s to a FACScan at 1 μl/s. Intracellular calcium levels and the percentage of cells activated increased as shear increased in duration and intensity. PMID:22045643

Cell Protrusion and Retraction Driven by Fluctuations in Actin Polymerization: A Two-Dimensional Model

PubMed Central

Ryan, Gillian L.; Holz, Danielle; Yamashiro, Sawako; Taniguchi, Daisuke; Watanabe, Naoki; Vavylonis, Dimitrios

2017-01-01

Animal cells that spread onto a surface often rely on actin-rich lamellipodial extensions to execute protrusion. Many cell types recently adhered on a two-dimensional substrate exhibit protrusion and retraction of their lamellipodia, even though the cell is not translating. Traveling waves of protrusion have also been observed, similar to those observed in crawling cells. These regular patterns of protrusion and retraction allow quantitative analysis for comparison to mathematical models. The periodic fluctuations in leading edge position of XTC cells have been linked to excitable actin dynamics using a one-dimensional model of actin dynamics, as a function of arc-length along the cell. In this work we extend this earlier model of actin dynamics into two dimensions (along the arc-length and radial directions of the cell) and include a model membrane that protrudes and retracts in response to the changing number of free barbed ends of actin filaments near the membrane. We show that if the polymerization rate at the barbed ends changes in response to changes in their local concentration at the leading edge and/or the opposing force from the cell membrane, the model can reproduce the patterns of membrane protrusion and retraction seen in experiment. We investigate both Brownian ratchet and switch-like force-velocity relationships between the membrane load forces and actin polymerization rate. The switch-like polymerization dynamics recover the observed patterns of protrusion and retraction as well as the fluctuations in F-actin concentration profiles. The model generates predictions for the behavior of cells after local membrane tension perturbations. PMID:28752950

Three-dimensional rapid visualization of matrix deformations around angiogenic sprouts (Conference Presentation)

NASA Astrophysics Data System (ADS)

Steuwe, Christian; Vayens, Marie-Mo; Jorge Peñas, Alvaro; Krajnik, Bartosz; Van Oosterwyck, Hans; Roeffaers, Maarten B. J.

2017-02-01

At the cell - extracellular matrix interface, physiologically important traction forces exerted by angiogenic sprouts can be investigated indirectly by mapping the consecutive matrix deformations. In this paper we present an approach to study these forces in three dimensions and with high time resolution. The technique employs lightsheet microscopy, in which a sheet of light is used to illuminate the sample - resulting in z-sectioning capability, superior image recording speed and reduced phototoxicity. For this study, human umbilical vein endothelial cells (HUVEC) are transduced with a LifeAct adenoviral vector to visualize the actin cytoskeleton during live sprouting into a collagen type I hydrogel. The calculation of the matrix deformations is formulated as a B-spline-based 3D non-rigid image registration process that warps the image of beads inside the stressed gel to match the image after stress relaxation. Using this approach we study the role of fast moving actin filaments for filopodia- and tip-cell dynamics in 3D under chemically defined culture conditions such as inhibited acto-myosin force generation. With a time resolution in the range of ten seconds, we find that our technique is at least 20 times faster than conventional traction force microscopy based on confocal imaging. Ultimately, this approach will shed light on rapid mechano-chemical feedback mechanisms important for sprouting angiogenesis.

Fission yeast Ags1 confers the essential septum strength needed for safe gradual cell abscission

PubMed Central

Sato, Mamiko; Muñoz, Javier; Moreno, M. Belén; Clemente-Ramos, Jose Angel; Ramos, Mariona; Okada, Hitoshi; Osumi, Masako; Durán, Angel; Ribas, Juan Carlos

2012-01-01

Fungal cytokinesis requires the assembly of a dividing septum wall. In yeast, the septum has to be selectively digested during the critical cell separation process. Fission yeast cell wall α(1-3)glucan is essential, but nothing is known about its localization and function in the cell wall or about cooperation between the α- and β(1-3)glucan synthases Ags1 and Bgs for cell wall and septum assembly. Here, we generate a physiological Ags1-GFP variant and demonstrate a tight colocalization with Bgs1, suggesting a cooperation in the important early steps of septum construction. Moreover, we define the essential functions of α(1-3)glucan in septation and cell separation. We show that α(1-3)glucan is essential for both secondary septum formation and the primary septum structural strength needed to support the physical forces of the cell turgor pressure during cell separation. Consequently, the absence of Ags1 and therefore α(1-3)glucan generates a special and unique side-explosive cell separation due to an instantaneous primary septum tearing caused by the turgor pressure. PMID:22891259

Generation of organized germ layers from a single mouse embryonic stem cell.

PubMed

Poh, Yeh-Chuin; Chen, Junwei; Hong, Ying; Yi, Haiying; Zhang, Shuang; Chen, Junjian; Wu, Douglas C; Wang, Lili; Jia, Qiong; Singh, Rishi; Yao, Wenting; Tan, Youhua; Tajik, Arash; Tanaka, Tetsuya S; Wang, Ning

2014-05-30

Mammalian inner cell mass cells undergo lineage-specific differentiation into germ layers of endoderm, mesoderm and ectoderm during gastrulation. It has been a long-standing challenge in developmental biology to replicate these organized germ layer patterns in culture. Here we present a method of generating organized germ layers from a single mouse embryonic stem cell cultured in a soft fibrin matrix. Spatial organization of germ layers is regulated by cortical tension of the colony, matrix dimensionality and softness, and cell-cell adhesion. Remarkably, anchorage of the embryoid colony from the 3D matrix to collagen-1-coated 2D substrates of ~1 kPa results in self-organization of all three germ layers: ectoderm on the outside layer, mesoderm in the middle and endoderm at the centre of the colony, reminiscent of generalized gastrulating chordate embryos. These results suggest that mechanical forces via cell-matrix and cell-cell interactions are crucial in spatial organization of germ layers during mammalian gastrulation. This new in vitro method could be used to gain insights on the mechanisms responsible for the regulation of germ layer formation.

Force Control Characteristics for Generation and Relaxation in the Lower Limb.

PubMed

Ohtaka, Chiaki; Fujiwara, Motoko

2018-05-29

We investigated the characteristics for force generation and relaxation using graded isometric contractions of the knee extensors. Participants performed the following tasks as quickly and accurately as possible. For the force generation task, force was increased from 0% to 20%, 40% and 60% of the maximal voluntary force (MVF). For the force relaxation task, force was decreased from 60% to 40%, 20% and 0%. The following parameters of the recorded force were calculated: error, time, and rate of force development. The error was consistently greater for force relaxation than generation. Reaction and adjustment times were independent of the tasks. The control strategy was markedly different for force relaxation and generation, this tendency was particularly evident for the lower limb compared to the upper limb.

Simple Cell Balance Circuit

NASA Technical Reports Server (NTRS)

Johnson, Steven D.; Byers, Jerry W.; Martin, James A.

2012-01-01

A method has been developed for continuous cell voltage balancing for rechargeable batteries (e.g. lithium ion batteries). A resistor divider chain is provided that generates a set of voltages representing the ideal cell voltage (the voltage of each cell should be as if the cells were perfectly balanced). An operational amplifier circuit with an added current buffer stage generates the ideal voltage with a very high degree of accuracy, using the concept of negative feedback. The ideal voltages are each connected to the corresponding cell through a current- limiting resistance. Over time, having the cell connected to the ideal voltage provides a balancing current that moves the cell voltage very close to that ideal level. In effect, it adjusts the current of each cell during charging, discharging, and standby periods to force the cell voltages to be equal to the ideal voltages generated by the resistor divider. The device also includes solid-state switches that disconnect the circuit from the battery so that it will not discharge the battery during storage. This solution requires relatively few parts and is, therefore, of lower cost and of increased reliability due to the fewer failure modes. Additionally, this design uses very little power. A preliminary model predicts a power usage of 0.18 W for an 8-cell battery. This approach is applicable to a wide range of battery capacities and voltages.

Force loading explains spatial sensing of ligands by cells

NASA Astrophysics Data System (ADS)

Oria, Roger; Wiegand, Tina; Escribano, Jorge; Elosegui-Artola, Alberto; Uriarte, Juan Jose; Moreno-Pulido, Cristian; Platzman, Ilia; Delcanale, Pietro; Albertazzi, Lorenzo; Navajas, Daniel; Trepat, Xavier; García-Aznar, José Manuel; Cavalcanti-Adam, Elisabetta Ada; Roca-Cusachs, Pere

2017-12-01

Cells can sense the density and distribution of extracellular matrix (ECM) molecules by means of individual integrin proteins and larger, integrin-containing adhesion complexes within the cell membrane. This spatial sensing drives cellular activity in a variety of normal and pathological contexts. Previous studies of cells on rigid glass surfaces have shown that spatial sensing of ECM ligands takes place at the nanometre scale, with integrin clustering and subsequent formation of focal adhesions impaired when single integrin-ligand bonds are separated by more than a few tens of nanometres. It has thus been suggested that a crosslinking ‘adaptor’ protein of this size might connect integrins to the actin cytoskeleton, acting as a molecular ruler that senses ligand spacing directly. Here, we develop gels whose rigidity and nanometre-scale distribution of ECM ligands can be controlled and altered. We find that increasing the spacing between ligands promotes the growth of focal adhesions on low-rigidity substrates, but leads to adhesion collapse on more-rigid substrates. Furthermore, disordering the ligand distribution drastically increases adhesion growth, but reduces the rigidity threshold for adhesion collapse. The growth and collapse of focal adhesions are mirrored by, respectively, the nuclear or cytosolic localization of the transcriptional regulator protein YAP. We explain these findings not through direct sensing of ligand spacing, but by using an expanded computational molecular-clutch model, in which individual integrin-ECM bonds—the molecular clutches—respond to force loading by recruiting extra integrins, up to a maximum value. This generates more clutches, redistributing the overall force among them, and reducing the force loading per clutch. At high rigidity and high ligand spacing, maximum recruitment is reached, preventing further force redistribution and leading to adhesion collapse. Measurements of cellular traction forces and actin flow speeds support our model. Our results provide a general framework for how cells sense spatial and physical information at the nanoscale, precisely tuning the range of conditions at which they form adhesions and activate transcriptional regulation.

Bioengineered constructs combined with exercise enhance stem cell-mediated treatment of volumetric muscle loss

PubMed Central

Quarta, Marco; Cromie, Melinda; Chacon, Robert; Blonigan, Justin; Garcia, Victor; Akimenko, Igor; Hamer, Mark; Paine, Patrick; Stok, Merel; Shrager, Joseph B.; Rando, Thomas A.

2017-01-01

Volumetric muscle loss (VML) is associated with loss of skeletal muscle function, and current treatments show limited efficacy. Here we show that bioconstructs suffused with genetically-labelled muscle stem cells (MuSCs) and other muscle resident cells (MRCs) are effective to treat VML injuries in mice. Imaging of bioconstructs implanted in damaged muscles indicates MuSCs survival and growth, and ex vivo analyses show force restoration of treated muscles. Histological analysis highlights myofibre formation, neovascularisation, but insufficient innervation. Both innervation and in vivo force production are enhanced when implantation of bioconstructs is followed by an exercise regimen. Significant improvements are also observed when bioconstructs are used to treat chronic VML injury models. Finally, we demonstrate that bioconstructs made with human MuSCs and MRCs can generate functional muscle tissue in our VML model. These data suggest that stem cell-based therapies aimed to engineer tissue in vivo may be effective to treat acute and chronic VML. PMID:28631758

Assembly and positioning of actomyosin rings by contractility and planar cell polarity

PubMed Central

Sehring, Ivonne M; Recho, Pierre; Denker, Elsa; Kourakis, Matthew; Mathiesen, Birthe; Hannezo, Edouard; Dong, Bo; Jiang, Di

2015-01-01

The actomyosin cytoskeleton is a primary force-generating mechanism in morphogenesis, thus a robust spatial control of cytoskeletal positioning is essential. In this report, we demonstrate that actomyosin contractility and planar cell polarity (PCP) interact in post-mitotic Ciona notochord cells to self-assemble and reposition actomyosin rings, which play an essential role for cell elongation. Intriguingly, rings always form at the cells′ anterior edge before migrating towards the center as contractility increases, reflecting a novel dynamical property of the cortex. Our drug and genetic manipulations uncover a tug-of-war between contractility, which localizes cortical flows toward the equator and PCP, which tries to reposition them. We develop a simple model of the physical forces underlying this tug-of-war, which quantitatively reproduces our results. We thus propose a quantitative framework for dissecting the relative contribution of contractility and PCP to the self-assembly and repositioning of cytoskeletal structures, which should be applicable to other morphogenetic events. DOI: http://dx.doi.org/10.7554/eLife.09206.001 PMID:26486861

Circulating Tumor Cells (CTCs): Emerging Technologies for Detection, Diagnosis and Treatment

NASA Astrophysics Data System (ADS)

McCarty, Owen

2010-03-01

Circulating tumor cell enumeration and characterization have the potential of providing real-time access to epithelial cancers in patients. This fluid phase biopsy of solid phase tumors is crucial to the development of quantitative diagnostic aiding personalized medicine. Cancer is a highly heterogeneous disease over space and time. Our goal is to generate a mechanistic, yet comprehensive view of both the `FORCE-journey' of a cancer cell during the metastatic phase, and a `TIME-journey' of the disease as it progresses. The approach will correlate the `FORCE' and `TIME' journey with both the bio-clinical aspects and the genomics of this complex problem. Presented will be results from a case study in lung cancer patients for which CTC analysis is compared with clinical progression. Morphologic and molecular characterization at the single cell level will be discussed in the context of the data set and in the context of individual patient management. Preliminary data will be shown to guide a future research agenda to investigate the fluid phase of solid tumors.

Force reflecting hand controller for manipulator teleoperation

NASA Technical Reports Server (NTRS)

Bryfogle, Mark D.

1991-01-01

A force reflecting hand controller based upon a six degree of freedom fully parallel mechanism, often termed a Stewart Platform, has been designed, constructed, and tested as an integrated system with a slave robot manipulator test bed. A force reflecting hand controller comprises a kinesthetic device capable of transmitting position and orientation commands to a slave robot manipulator while simultaneously representing the environmental interaction forces of the slave manipulator back to the operator through actuators driving the hand controller mechanism. The Stewart Platform was chosen as a novel approach to improve force reflecting teleoperation because of its inherently high ratio of load generation capability to system mass content and the correspondingly high dynamic bandwidth. An additional novelty of the program was to implement closed loop force and torque control about the hand controller mechanism by equipping the handgrip with a six degree of freedom force and torque measuring cell. The mechanical, electrical, computer, and control systems are discussed and system tests are presented.

Emergent mechanics of biological structures

PubMed Central

Dumont, Sophie; Prakash, Manu

2014-01-01

Mechanical force organizes life at all scales, from molecules to cells and tissues. Although we have made remarkable progress unraveling the mechanics of life's individual building blocks, our understanding of how they give rise to the mechanics of larger-scale biological structures is still poor. Unlike the engineered macroscopic structures that we commonly build, biological structures are dynamic and self-organize: they sculpt themselves and change their own architecture, and they have structural building blocks that generate force and constantly come on and off. A description of such structures defies current traditional mechanical frameworks. It requires approaches that account for active force-generating parts and for the formation of spatial and temporal patterns utilizing a diverse array of building blocks. In this Perspective, we term this framework “emergent mechanics.” Through examples at molecular, cellular, and tissue scales, we highlight challenges and opportunities in quantitatively understanding the emergent mechanics of biological structures and the need for new conceptual frameworks and experimental tools on the way ahead. PMID:25368421

Conductivity affects nanosecond electrical pulse induced pressure transient formation

NASA Astrophysics Data System (ADS)

Roth, Caleb C.; Barnes, Ronald A.; Ibey, Bennett L.; Beier, Hope T.; Glickman, Randolph D.

2016-03-01

Nanoporation occurs in cells exposed to high amplitude short duration (< 1μs) electrical pulses. The biophysical mechanism(s) responsible for nanoporation is unknown although several theories exist. Current theories focus exclusively on the electrical field, citing electrostriction, water dipole alignment and/or electrodeformation as the primary mechanisms for pore formation. Our group has shown that mechanical forces of substantial magnitude are also generated during nsEP exposures. We hypothesize that these mechanical forces may contribute to pore formation. In this paper, we report that alteration of the conductivity of the exposure solution also altered the level of mechanical forces generated during a nsEP exposure. By reducing the conductivity of the exposure solutions, we found that we could completely eliminate any pressure transients normally created by nsEP exposure. The data collected for this proceeding does not definitively show that the pressure transients previously identified contribute to nanoporation; however; it indicates that conductivity influences both survival and pressure transient formation.

Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimada, Hidenori; Hashimoto, Yoshiya; Nakada, Akira

2012-01-13

Highlights: Black-Right-Pointing-Pointer Very rapid generation of human iPS cells under optimized conditions. Black-Right-Pointing-Pointer Five chemical inhibitors under hypoxia boosted reprogramming. Black-Right-Pointing-Pointer We performed genome-wide DNA methylation analysis. -- Abstract: Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain largemore » amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1 month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of iPS cell technology to biomedical research.« less

Hydrostatic pressure and shear stress affect endothelin-1 and nitric oxide release by endothelial cells in bioreactors.

PubMed

Vozzi, Federico; Bianchi, Francesca; Ahluwalia, Arti; Domenici, Claudio

2014-01-01

Abundant experimental evidence demonstrates that endothelial cells are sensitive to flow; however, the effect of fluid pressure or pressure gradients that are used to drive viscous flow is not well understood. There are two principal physical forces exerted on the blood vessel wall by the passage of intra-luminal blood: pressure and shear. To analyze the effects of pressure and shear independently, these two stresses were applied to cultured cells in two different types of bioreactors: a pressure-controlled bioreactor and a laminar flow bioreactor, in which controlled levels of pressure or shear stress, respectively, can be generated. Using these bioreactor systems, endothelin-1 (ET-1) and nitric oxide (NO) release from human umbilical vein endothelial cells were measured under various shear stress and pressure conditions. Compared to the controls, a decrease of ET-1 production by the cells cultured in both bioreactors was observed, whereas NO synthesis was up-regulated in cells under shear stress, but was not modulated by hydrostatic pressure. These results show that the two hemodynamic forces acting on blood vessels affect endothelial cell function in different ways, and that both should be considered when planning in vitro experiments in the presence of flow. Understanding the individual and synergic effects of the two forces could provide important insights into physiological and pathological processes involved in vascular remodeling and adaptation. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Trespassing cancer cells: 'fingerprinting' invasive protrusions reveals metastatic culprits.

PubMed

Klemke, Richard L

2012-10-01

Metastatic cancer cells produce invasive membrane protrusions called invadopodia and pseudopodia, which play a central role in driving cancer cell dissemination in the body. Malignant cells use these structures to attach to and degrade extracellular matrix proteins, generate force for cell locomotion, and to penetrate the vasculature. Recent work using unique subcellular fractionation methodologies combined with spatial genomic, proteomic, and phosphoproteomic profiling has provided insight into the invadopodiome and pseudopodiome signaling networks that control the protrusion of invasive membranes. Here I highlight how these powerful spatial 'omics' approaches reveal important signatures of metastatic cancer cells and possible new therapeutic targets aimed at treating metastatic disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

Investigation of the characteristics of a stacked direct borohydride fuel cell for portable applications

NASA Astrophysics Data System (ADS)

Kim, Cheolhwan; Kim, Kyu-Jung; Ha, Man Yeong

To investigate the possibility of the portable application of a direct borohydride fuel cell (DBFC), weight reduction of the stack and high stacking of the cells are investigated for practical running conditions. For weight reduction, carbon graphite is adopted as the bipolar plate material even though it has disadvantages in tight stacking, which results in stacking loss from insufficient material strength. For high stacking, it is essential to have a uniform fuel distribution among cells and channels to maintain equal electric load on each cell. In particular, the design of the anode channel is important because active hydrogen generation causes non-uniformity in the fuel flow-field of the cells and channels. To reduce the disadvantages of stacking force margin and fuel maldistribution, an O-ring type-sealing system with an internal manifold and a parallel anode channel design is adopted, and the characteristics of a single and a five-cell fuel cell stack are analyzed. By adopting carbon graphite, the stack weight can be reduced by 4.2 times with 12% of performance degradation from the insufficient stacking force. When cells are stacked, the performance exceeds the single-cell performance because of the stack temperature increase from the reduction of the radiation area from the narrow stacking of cells.

Characterization and evaluation of femtosecond laser-induced sub-micron periodic structures generated on titanium to improve osseointegration of implants

NASA Astrophysics Data System (ADS)

Lee, Bryan E. J.; Exir, Hourieh; Weck, Arnaud; Grandfield, Kathryn

2018-05-01

Reproducible and controllable methods of modifying titanium surfaces for dental and orthopaedic applications are of interest to prevent poor implant outcomes by improving osseointegration. This study made use of a femtosecond laser to generate laser-induced periodic surface structures with periodicities of 300, 620 and 760 nm on titanium substrates. The reproducible rippled patterns showed consistent submicron scale roughness and relatively hydrophobic surfaces as measured by atomic force microscopy and contact angle, respectively. Transmission electron microscopy and Auger electron spectroscopy identified a thicker oxide layer on ablated surfaces compared to controls. In vitro testing was conducted using osteosarcoma Saos-2 cells. Cell metabolism on the laser-ablated surfaces was comparable to controls and alkaline phosphatase activity was notably increased at late time points for the 620 and 760 nm surfaces compared to controls. Cells showed a more elongated shape on laser-ablated surfaces compared to controls and showed perpendicular alignment to the periodic structures. This work has demonstrated the feasibility of generating submicron features on an implant material with the ability to influence cell response and improve implant outcomes.

Mechanical force-mediated pathological cartilage thinning is regulated by necroptosis and apoptosis.

PubMed

Zhang, C; Lin, S; Li, T; Jiang, Y; Huang, Z; Wen, J; Cheng, W; Li, H

2017-08-01

This study aimed to identify the mechanisms underlying mandibular chondrocyte cell death and cartilage thinning in response to mechanical force. An in vivo model (compressive mechanical force) and an in vitro model (TNF-α+cycloheximide) were used to induce mandibular chondrocyte necroptosis. Hematoxylin and eosin staining and transmission electron microscopy were used to assess histological and subcellular changes in mandibular chondrocyte. Immunohistochemistry, western blotting, and real-time PCR were performed to evaluate changes in necroptotic protein markers. Cell activity, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were examined in vitro. The expression of RIP1, RIP3 and Caspase-8 in mandibular chondrocytes significantly increased after 4 days of compressive mechanical force. Furthermore, the inhibition of necroptosis by Necrostatin-1 (Nec-1) or the inhibition of apoptosis by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD) partially restored mechanical force-mediated mandibular cartilage thinning and chondrocyte death. Moreover, a synergistic effect on cell death inhibition and mandibular cartilage thickness restoration were found when treated with Nec-1+Z-VAD. The results of the in vitro model were in line with the in vivo ones, indicating that the changes in MMP and ROS generation contributed to mandibular chondrocyte apoptosis and necroptosis. In addition to apoptosis, necroptosis also plays critical roles in pathological changes in mandibular cartilage after compressive mechanical force stimulation, implying RIP1, a master protein that mediates both necroptosis and apoptosis, as a potential therapeutic target in temporal mandibular osteoarthritis. Copyright © 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers.

PubMed

Roche, Stuart M; Gumucio, Jonathan P; Brooks, Susan V; Mendias, Christopher L; Claflin, Dennis R

2015-06-16

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Push-me-pull-you: how microtubules organize the cell interior

PubMed Central

2008-01-01

Dynamic organization of the cell interior, which is crucial for cell function, largely depends on the microtubule cytoskeleton. Microtubules move and position organelles by pushing, pulling, or sliding. Pushing forces can be generated by microtubule polymerization, whereas pulling typically involves microtubule depolymerization or molecular motors, or both. Sliding between a microtubule and another microtubule, an organelle, or the cell cortex is also powered by molecular motors. Although numerous examples of microtubule-based pushing and pulling in living cells have been observed, it is not clear why different cell types and processes employ different mechanisms. This review introduces a classification of microtubule-based positioning strategies and discusses the efficacy of pushing and pulling. The positioning mechanisms based on microtubule pushing are efficient for movements over small distances, and for centering of organelles in symmetric geometries. Mechanisms based on pulling, on the other hand, are typically more elaborate, but are necessary when the distances to be covered by the organelles are large, and when the geometry is asymmetric and complex. Thus, taking into account cell geometry and the length scale of the movements helps to identify general principles of the intracellular layout based on microtubule forces. PMID:18404264

Contractility of the cell rear drives invasion of breast tumor cells in 3D Matrigel

PubMed Central

Poincloux, Renaud; Collin, Olivier; Lizárraga, Floria; Romao, Maryse; Debray, Marcel; Piel, Matthieu; Chavrier, Philippe

2011-01-01

Cancer cells use different modes of migration, including integrin-dependent mesenchymal migration of elongated cells along elements of the 3D matrix as opposed to low-adhesion-, contraction-based amoeboid motility of rounded cells. We report that MDA-MB-231 human breast adenocarcinoma cells invade 3D Matrigel with a characteristic rounded morphology and with F-actin and myosin-IIa accumulating at the cell rear in a uropod-like structure. MDA-MB-231 cells display neither lamellipodia nor bleb extensions at the leading edge and do not require Arp2/3 complex activity for 3D invasion in Matrigel. Accumulation of phospho-MLC and blebbing activity were restricted to the uropod as reporters of actomyosin contractility, and velocimetric analysis of fluorescent beads embedded within the 3D matrix showed that pulling forces exerted to the matrix are restricted to the side and rear of cells. Inhibition of actomyosin contractility or β1 integrin function interferes with uropod formation, matrix deformation, and invasion through Matrigel. These findings support a model whereby actomyosin-based uropod contractility generates traction forces on the β1 integrin adhesion system to drive cell propulsion within the 3D matrix, with no contribution of lamellipodia extension or blebbing to movement. PMID:21245302

Measurements of Elastic Moduli of Silicone Gel Substrates with a Microfluidic Device

PubMed Central

Gutierrez, Edgar; Groisman, Alex

2011-01-01

Thin layers of gels with mechanical properties mimicking animal tissues are widely used to study the rigidity sensing of adherent animal cells and to measure forces applied by cells to their substrate with traction force microscopy. The gels are usually based on polyacrylamide and their elastic modulus is measured with an atomic force microscope (AFM). Here we present a simple microfluidic device that generates high shear stresses in a laminar flow above a gel-coated substrate and apply the device to gels with elastic moduli in a range from 0.4 to 300 kPa that are all prepared by mixing two components of a transparent commercial silicone Sylgard 184. The elastic modulus is measured by tracking beads on the gel surface under a wide-field fluorescence microscope without any other specialized equipment. The measurements have small and simple to estimate errors and their results are confirmed by conventional tensile tests. A master curve is obtained relating the mixing ratios of the two components of Sylgard 184 with the resulting elastic moduli of the gels. The rigidity of the silicone gels is less susceptible to effects from drying, swelling, and aging than polyacrylamide gels and can be easily coated with fluorescent tracer particles and with molecules promoting cellular adhesion. This work can lead to broader use of silicone gels in the cell biology laboratory and to improved repeatability and accuracy of cell traction force microscopy and rigidity sensing experiments. PMID:21980487

Force sensor

DOEpatents

Grahn, A.R.

1993-05-11

A force sensor and related method for determining force components is described. The force sensor includes a deformable medium having a contact surface against which a force can be applied, a signal generator for generating signals that travel through the deformable medium to the contact surface, a signal receptor for receiving the signal reflected from the contact surface, a generation controller, a reception controller, and a force determination apparatus. The signal generator has one or more signal generation regions for generating the signals. The generation controller selects and activates the signal generation regions. The signal receptor has one or more signal reception regions for receiving signals and for generating detections signals in response thereto. The reception controller selects signal reception regions and detects the detection signals. The force determination apparatus measures signal transit time by timing activation and detection and, optionally, determines force components for selected cross-field intersections. The timer which times by activation and detection can be any means for measuring signal transit time. A cross-field intersection is defined by the overlap of a signal generation region and a signal reception region.

Force sensor

DOEpatents

Grahn, Allen R.

1993-01-01

A force sensor and related method for determining force components. The force sensor includes a deformable medium having a contact surface against which a force can be applied, a signal generator for generating signals that travel through the deformable medium to the contact surface, a signal receptor for receiving the signal reflected from the contact surface, a generation controller, a reception controller, and a force determination apparatus. The signal generator has one or more signal generation regions for generating the signals. The generation controller selects and activates the signal generation regions. The signal receptor has one or more signal reception regions for receiving signals and for generating detections signals in response thereto. The reception controller selects signal reception regions and detects the detection signals. The force determination apparatus measures signal transit time by timing activation and detection and, optionally, determines force components for selected cross-field intersections. The timer which times by activation and detection can be any means for measuring signal transit time. A cross-field intersection is defined by the overlap of a signal generation region and a signal reception region.

Microneedle-based analysis of the micromechanics of the metaphase spindle assembled in Xenopus laevis egg extracts

PubMed Central

Shimamoto, Yuta; Kapoor, Tarun M.

2014-01-01

SUMMARY To explain how micron-sized cellular structures generate and respond to forces we need to characterize their micromechanical properties. Here we provide a protocol to build and use a dual force-calibrated microneedle-based set-up to quantitatively analyze the micromechanics of a metaphase spindle assembled in Xenopus laevis egg extracts. This cell-free extract system allows for controlled biochemical perturbations of spindle components. We describe how the microneedles are prepared and how they can be used to apply and measure forces. A multi-mode imaging system allows tracking of microtubules, chromosomes and needle tips. This set-up can be used to analyze the viscoelastic properties of the spindle on time-scales ranging from minutes to sub-seconds. A typical experiment, along with data analysis, is also detailed. We anticipate that our protocol can be readily extended to analyze the micromechanics of other cellular structures assembled in cell-free extracts. The entire procedure can take 3-4 days. PMID:22538847

The Geophysical Fluid Flow Cell Experiment

NASA Technical Reports Server (NTRS)

Hart, J. E.; Ohlsen, D.; Kittleman, S.; Borhani, N.; Leslie, F.; Miller, T.

1999-01-01

The Geophysical Fluid Flow Cell (GFFC) experiment performed visualizations of thermal convection in a rotating differentially heated spherical shell of fluid. In these experiments dielectric polarization forces are used to generate a radially directed buoyancy force. This enables the laboratory simulation of a number of geophysically and astrophysically important situations in which sphericity and rotation both impose strong constraints on global scale fluid motions. During USML-2 a large set of experiments with spherically symmetric heating were carried out. These enabled the determination of critical points for the transition to various forms of nonaxisymmetric convection and, for highly turbulent flows, the transition latitudes separating the different modes of motion. This paper presents a first analysis of these experiments as well as data on the general performance of the instrument during the USML-2 flight.

Special Test Methods for Batteries

NASA Technical Reports Server (NTRS)

Gross, S.

1984-01-01

Various methods are described for measuring heat generation in primary and secondary batteries as well as the specific heat of batteries and cell thermal conductance. Problems associated with determining heat generation in large batteries are examined. Special attention is given to monitoring temperature gradients in nickel cadmium cells, the use of auxiliary electrodes for conducting tests on battery charge control, evaluating the linear sweep of current from charge to discharge, and determining zero current voltage. The fast transient behavior of batteries in the microsecond range, and the electrical conductance of nickel sinters in the thickness direction are also considered. Mechanical problems experienced in the vibration of Ni-Cd batteries and tests to simulate cyclic fatigue of the steel table connecting the plates to the comb are considered. Methods of defining the distribution of forces when cells are compressed during battery packaging are also explored.

Special test methods for batteries

NASA Astrophysics Data System (ADS)

Gross, S.

1984-09-01

Various methods are described for measuring heat generation in primary and secondary batteries as well as the specific heat of batteries and cell thermal conductance. Problems associated with determining heat generation in large batteries are examined. Special attention is given to monitoring temperature gradients in nickel cadmium cells, the use of auxiliary electrodes for conducting tests on battery charge control, evaluating the linear sweep of current from charge to discharge, and determining zero current voltage. The fast transient behavior of batteries in the microsecond range, and the electrical conductance of nickel sinters in the thickness direction are also considered. Mechanical problems experienced in the vibration of Ni-Cd batteries and tests to simulate cyclic fatigue of the steel table connecting the plates to the comb are considered. Methods of defining the distribution of forces when cells are compressed during battery packaging are also explored.

Effects of Combined Shear and Thermal Forces on Destruction of Microbacterium lacticum

PubMed Central

Bulut, S.; Waites, W. M.; Mitchell, J. R.

1999-01-01

A twin-screw extruder and a rotational rheometer were used to generate shear forces in concentrated gelatin inoculated with a heat-resistant isolate of a vegetative bacterial species, Microbacterium lacticum. Shear forces in the extruder were mainly controlled by varying the water feed rate. The water content of the extrudates changed between 19 and 45% (wet weight basis). Higher shear forces generated at low water contents and the calculated die wall shear stress correlated strongly with bacterial destruction. No surviving microorganisms could be detected at the highest wall shear stress of 409 kPa, giving log reduction of 5.3 (minimum detection level, 2 × 104 CFU/sample). The mean residence time of the microorganism in the extruder was 49 to 58 s, and the maximum temperature measured in the end of the die was 73°C. The D75°C of the microorganism in gelatin at 65% water content was 20 min. It is concluded that the physical forces generated in the reverse screw element and the extruder die rather than heat played a major part in cell destruction. In a rotational rheometer, after shearing of a mix of microorganisms with gelatin at 65% (wt/wt) moisture content for 4 min at a shear stress of 2.8 kPa and a temperature of 75°C, the number of surviving microorganisms in the sheared sample was 5.2 × 106 CFU/g of sample compared with 1.4 × 108 CFU/g of sample in the nonsheared control. The relative effectiveness of physical forces in the killing of bacteria and destruction of starch granules is discussed. PMID:10508076

Mechanosensory Genes Pkd1 and Pkd2 Contribute to the Planar Polarization of Brain Ventricular Epithelium

PubMed Central

Herranz-Pérez, Vicente; Nakatani, Jin; Boletta, Alessandra; García-Verdugo, José Manuel

2015-01-01

Directional beating of ependymal (E) cells' cilia in the walls of the ventricles in the brain is essential for proper CSF flow. E cells display two forms of planar cell polarity (PCP): rotational polarity of individual cilium and translational polarity (asymmetric positioning of cilia in the apical area). The orientation of individual E cells varies according to their location in the ventricular wall (location-specific PCP). It has been hypothesized that hydrodynamic forces on the apical surface of radial glia cells (RGCs), the embryonic precursors of E cells, could guide location-specific PCP in the ventricular epithelium. However, the detection mechanisms for these hydrodynamic forces have not been identified. Here, we show that the mechanosensory proteins polycystic kidney disease 1 (Pkd1) and Pkd2 are present in primary cilia of RGCs. Ablation of Pkd1 or Pkd2 in Nestin-Cre;Pkd1flox/flox or Nestin-Cre;Pkd2flox/flox mice, affected PCP development in RGCs and E cells. Early shear forces on the ventricular epithelium may activate Pkd1 and Pkd2 in primary cilia of RGCs to properly polarize RGCs and E cells. Consistently, Pkd1, Pkd2, or primary cilia on RGCs were required for the proper asymmetric localization of the PCP protein Vangl2 in E cells' apical area. Analyses of single- and double-heterozygous mutants for Pkd1 and/or Vangl2 suggest that these genes function in the same pathway to establish E cells' PCP. We conclude that Pkd1 and Pkd2 mechanosensory proteins contribute to the development of brain PCP and prevention of hydrocephalus. SIGNIFICANCE STATEMENT This study identifies key molecules in the development of planar cell polarity (PCP) in the brain and prevention of hydrocephalus. Multiciliated ependymal (E) cells within the brain ventricular epithelium generate CSF flow through ciliary beating. E cells display location-specific PCP in the orientation and asymmetric positioning of their cilia. Defects in this PCP can result in hydrocephalus. Hydrodynamic forces on radial glial cells (RGCs), the embryonic progenitors of E cells, have been suggested to guide PCP. We show that the mechanosensory proteins Pkd1 and Pkd2 localize to primary cilia in RGCs, and their ablation disrupts the development of PCP in E cells. Early shear forces on RGCs may activate Pkd1 and Pkd2 in RGCs' primary cilia to properly orient E cells. This study identifies key molecules in the development of brain PCP and prevention of hydrocephalus. PMID:26245976

Traction in smooth muscle cells varies with cell spreading

NASA Technical Reports Server (NTRS)

Tolic-Norrelykke, Iva Marija; Wang, Ning

2005-01-01

Changes in cell shape regulate cell growth, differentiation, and apoptosis. It has been suggested that the regulation of cell function by the cell shape is a result of the tension in the cytoskeleton and the distortion of the cell. Here we explore the association between cell-generated mechanical forces and the cell morphology. We hypothesized that the cell contractile force is associated with the degree of cell spreading, in particular with the cell length. We measured traction fields of single human airway smooth muscle cells plated on a polyacrylamide gel, in which fluorescent microbeads were embedded to serve as markers of gel deformation. The traction exerted by the cells at the cell-substrate interface was determined from the measured deformation of the gel. The traction was measured before and after treatment with the contractile agonist histamine, or the relaxing agonist isoproterenol. The relative increase in traction induced by histamine was negatively correlated with the baseline traction. On the contrary, the relative decrease in traction due to isoproterenol was independent of the baseline traction, but it was associated with cell shape: traction decreased more in elongated than in round cells. Maximum cell width, mean cell width, and projected area of the cell were the parameters most tightly coupled to both baseline and histamine-induced traction in this study. Wide and well-spread cells exerted larger traction than slim cells. These results suggest that cell contractility is controlled by cell spreading.

Cryo-EM Structure of the Mechanotransduction Channel NOMPC

PubMed Central

Jin, Peng; Bulkley, David; Guo, Yanmeng; Zhang, Wei; Guo, Zhenhao; Huynh, Walter; Wu, Shenping; Meltzer, Shan; Cheng, Tong; Jan, Lily Yeh; Jan, Yuh-Nung; Cheng, Yifan

2017-01-01

Mechanosensory transduction for senses such as proprioception, touch, balance, acceleration, hearing and pain relies on mechanotransduction channels, which convert mechanical stimuli into electrical signals in specialized sensory cells1. How force gates mechanotransduction channels is a central question in the field, for which there are two major models. One is the membrane-tension model: force applied to the membrane generates a change in membrane tension that is sufficient to gate the channel, as in the case of bacterial MscL channel and certain eukaryotic potassium channels2-5. The other is the tether model: force is transmitted via a tether to gate the channel. Recent study suggests that NOMPC, a mechanotransduction channel that mediates hearing and touch sensation in Drosophila, is gated by tethering of its ankyrin repeat (AR) domain to microtubules of the cytoskeleton6. Thus, a goal of studying NOMPC is to reveal the underlying mechanism of force induced gating, which could serve as a paradigm of the tether model. NOMPC, a Transient Receptor Potential (TRP) channel and the founding member of the TRPN sub-family7, fulfills all the criteria for a bona fide mechanotransduction channel1,8, and is important for a variety of mechanosensation-related behaviors such as locomotion, touch and sound sensation across different species including C. elegans9, Drosophila8,10-11 and zebrafish12. NOMPC has 29 ARs, the largest number among TRP channels. They are implicated as tether to convey force from cytoskeleton to the channel, thus to mediate mechanosensation6,13-15. A key question is how the long AR domain is organized as a tether that can trigger channel gating. Here we present a de novo atomic structure of NOMPC determined by single particle electron cryo-microscopy (cryo-EM), and discuss how its architecture could provide a means to convey mechanical force to generating an electrical signal within a cell. PMID:28658211

Electric and Magnetic Manipulation of Biological Systems

NASA Astrophysics Data System (ADS)

Lee, H.; Hunt, T. P.; Liu, Y.; Ham, D.; Westervelt, R. M.

2005-06-01

New types of biological cell manipulation systems, a micropost matrix, a microelectromagnet matrix, and a microcoil array, were developed. The micropost matrix consists of post-shaped electrodes embedded in an insulating layer. With a separate ac voltage applied to each electrode, the micropost matrix generates dielectrophoretic force to trap and move individual biological cells. The microelectromagnet matrix consists of two arrays of straight wires aligned perpendicular to each other, that are covered with insulating layers. By independently controlling the current in each wire, the microelectromagnet matrix creates versatile magnetic fields to manipulate individual biological cells attached to magnetic beads. The microcoil array is a set of coils implemented in a foundry using a standard silicon fabrication technology. Current sources to the coils, and control circuits are integrated on a single chip, making the device self-contained. Versatile manipulation of biological cells was demonstrated using these devices by generating optimized electric or magnetic field patterns. A single yeast cell was trapped and positioned with microscopic resolution, and multiple yeast cells were trapped and independently moved along the separate paths for cell-sorting.

Self-Rotation of Cells in an Irrotational AC E-Field in an Opto-Electrokinetics Chip

PubMed Central

Chau, Long-Ho; Liang, Wenfeng; Cheung, Florence Wing Ki; Liu, Wing Keung; Li, Wen Jung; Chen, Shih-Chi; Lee, Gwo-Bin

2013-01-01

The use of optical dielectrophoresis (ODEP) to manipulate microparticles and biological cells has become increasingly popular due to its tremendous flexibility in providing reconfigurable electrode patterns and flow channels. ODEP enables the parallel and free manipulation of small particles on a photoconductive surface on which light is projected, thus eliminating the need for complex electrode design and fabrication processes. In this paper, we demonstrate that mouse cells comprising melan-a cells, RAW 267.4 macrophage cells, peripheral white blood cells and lymphocytes, can be manipulated in an opto-electrokinetics (OEK) device with appropriate DEP parameters. Our OEK device generates a non-rotating electric field and exerts a localized DEP force on optical electrodes. Hitherto, we are the first group to report that among all the cells investigated, melan-a cells, lymphocytes and white blood cells were found to undergo self-rotation in the device in the presence of a DEP force. The rotational speed of the cells depended on the voltage and frequency applied and the cells' distance from the optical center. We discuss a possible mechanism for explaining this new observation of induced self-rotation based on the physical properties of cells. We believe that this rotation phenomenon can be used to identify cell type and to elucidate the dielectric and physical properties of cells. PMID:23320067

Molecular dynamics analysis of conformational change of paramyxovirus F protein during the initial steps of membrane fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin-Garcia, Fernando; Mendieta-Moreno, Jesus Ignacio; Mendieta, Jesus

2012-03-30

Highlights: Black-Right-Pointing-Pointer Initial conformational change of paramyxovirus F protein is caused only by mechanical forces. Black-Right-Pointing-Pointer HRA region undergoes a structural change from a beta + alpha conformation to an extended coil and then to an all-alpha conformation. Black-Right-Pointing-Pointer HRS domains of F protein form three single {alpha}-helices prior to generation of the coiled coil. -- Abstract: The fusion of paramyxovirus to the cell membrane is mediated by fusion protein (F protein) present in the virus envelope, which undergoes a dramatic conformational change during the process. Unlike hemagglutinin in orthomyxovirus, this change is not mediated by an alteration of environmentalmore » pH, and its cause remains unknown. Steered molecular dynamics analysis leads us to suggest that the conformational modification is mediated only by stretching mechanical forces once the transmembrane fusion peptide of the protein is anchored to the cell membrane. Such elongating forces will generate major secondary structure rearrangement in the heptad repeat A region of the F protein; from {beta}-sheet conformation to an elongated coil and then spontaneously to an {alpha}-helix. In addition, it is proposed that the heptad repeat A region adopts a final three-helix coiled coil and that this structure appears after the formation of individual helices in each monomer.« less

Self-repairing symmetry in jellyfish through mechanically driven reorganization

PubMed Central

Abrams, Michael J.; Basinger, Ty; Yuan, William; Guo, Chin-Lin; Goentoro, Lea

2015-01-01

What happens when an animal is injured and loses important structures? Some animals simply heal the wound, whereas others are able to regenerate lost parts. In this study, we report a previously unidentified strategy of self-repair, where moon jellyfish respond to injuries by reorganizing existing parts, and rebuilding essential body symmetry, without regenerating what is lost. Specifically, in response to arm amputation, the young jellyfish of Aurelia aurita rearrange their remaining arms, recenter their manubria, and rebuild their muscular networks, all completed within 12 hours to 4 days. We call this process symmetrization. We find that symmetrization is not driven by external cues, cell proliferation, cell death, and proceeded even when foreign arms were grafted on. Instead, we find that forces generated by the muscular network are essential. Inhibiting pulsation using muscle relaxants completely, and reversibly, blocked symmetrization. Furthermore, we observed that decreasing pulse frequency using muscle relaxants slowed symmetrization, whereas increasing pulse frequency by lowering the magnesium concentration in seawater accelerated symmetrization. A mathematical model that describes the compressive forces from the muscle contraction, within the context of the elastic response from the mesoglea and the ephyra geometry, can recapitulate the recovery of global symmetry. Thus, self-repair in Aurelia proceeds through the reorganization of existing parts, and is driven by forces generated by its own propulsion machinery. We find evidence for symmetrization across species of jellyfish (Chrysaora pacifica, Mastigias sp., and Cotylorhiza tuberculata). PMID:26080418

ROCK inhibition promotes microtentacles that enhance reattachment of breast cancer cells

PubMed Central

Bhandary, Lekhana; Whipple, Rebecca A.; Vitolo, Michele I.; Charpentier, Monica S.; Boggs, Amanda E.; Chakrabarti, Kristi R.; Thompson, Keyata N.; Martin, Stuart S.

2015-01-01

The presence of circulating tumor cells (CTCs) in blood predicts poor patient outcome and CTC frequency is correlated with higher risk of metastasis. Recently discovered, novel microtubule-based structures, microtentacles, can enhance reattachment of CTCs to the vasculature. Microtentacles are highly dynamic membrane protrusions formed in detached cells and occur when physical forces generated by the outwardly expanding microtubules overcome the contractile force of the actin cortex. Rho-associated kinase (ROCK) is a major regulator of actomyosin contractility and Rho/ROCK over-activation is implicated in tumor metastasis. ROCK inhibitors are gaining popularity as potential cancer therapeutics based on their success in reducing adherent tumor cell migration and invasion. However, the effect of ROCK inhibition on detached cells in circulation is largely unknown. In this study, we use breast tumor cells in suspension to mimic detached CTCs and show that destabilizing the actin cortex through ROCK inhibition in suspended cells promotes the formation of microtentacles and enhances reattachment of cells from suspension. Conversely, increasing actomyosin contraction by Rho over-activation reduces microtentacle frequency and reattachment. Although ROCK inhibitors may be effective in reducing adherent tumor cell behavior, our results indicate that they could inadvertently increase metastatic potential of non-adherent CTCs by increasing their reattachment efficacy. PMID:25749040

Multistage Force Amplification of Piezoelectric Stacks

NASA Technical Reports Server (NTRS)

Xu, Tian-Bing (Inventor); Siochi, Emilie J. (Inventor); Zuo, Lei (Inventor); Jiang, Xiaoning (Inventor); Kang, Jin Ho (Inventor)

2015-01-01

Embodiments of the disclosure include an apparatus and methods for using a piezoelectric device, that includes an outer flextensional casing, a first cell and a last cell serially coupled to each other and coupled to the outer flextensional casing such that each cell having a flextensional cell structure and each cell receives an input force and provides an output force that is amplified based on the input force. The apparatus further includes a piezoelectric stack coupled to each cell such that the piezoelectric stack of each cell provides piezoelectric energy based on the output force for each cell. Further, the last cell receives an input force that is the output force from the first cell and the last cell provides an output apparatus force In addition, the piezoelectric energy harvested is based on the output apparatus force. Moreover, the apparatus provides displacement based on the output apparatus force.

Left-right asymmetry is formed in individual cells by intrinsic cell chirality.

PubMed

Hatori, Ryo; Ando, Tadashi; Sasamura, Takeshi; Nakazawa, Naotaka; Nakamura, Mitsutoshi; Taniguchi, Kiichiro; Hozumi, Shunya; Kikuta, Junichi; Ishii, Masaru; Matsuno, Kenji

2014-08-01

Many animals show left-right (LR) asymmetric morphology. The mechanisms of LR asymmetric development are evolutionarily divergent, and they remain elusive in invertebrates. Various organs in Drosophila melanogaster show stereotypic LR asymmetry, including the embryonic gut. The Drosophila embryonic hindgut twists 90° left-handedly, thereby generating directional LR asymmetry. We recently revealed that the hindgut epithelial cell is chiral in shape and other properties; this is termed planar cell chirality (PCC). We previously showed by computer modeling that PCC is sufficient to induce the hindgut rotation. In addition, both the PCC and the direction of hindgut twisting are reversed in Myosin31DF (Myo31DF) mutants. Myo31DF encodes Drosophila MyosinID, an actin-based motor protein, whose molecular functions in LR asymmetric development are largely unknown. Here, to understand how PCC directs the asymmetric cell-shape, we analyzed PCC in genetic mosaics composed of cells homozygous for mutant Myo31DF, some of which also overexpressed wild-type Myo31DF. Wild-type cell-shape chirality only formed in the Myo31DF-overexpressing cells, suggesting that cell-shape chirality was established in each cell and reflects intrinsic PCC. A computer model recapitulating the development of this genetic mosaic suggested that mechanical interactions between cells are required for the cell-shape behavior seen in vivo. Our mosaic analysis also suggested that during hindgut rotation in vivo, wild-type Myo31DF suppresses the elongation of cell boundaries, supporting the idea that cell-shape chirality is an intrinsic property determined in each cell. However, the amount and distribution of F-actin and Myosin II, which are known to help generate the contraction force on cell boundaries, did not show differences between Myo31DF mutant cells and wild-type cells, suggesting that the static amount and distribution of these proteins are not involved in the suppression of cell-boundary elongation. Taken together, our results suggest that cell-shape chirality is intrinsically formed in each cell, and that mechanical force from intercellular interactions contributes to its formation and/or maintenance. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue.

PubMed

Jacob, Fabian; Yonis, Amina Y; Cuello, Friederike; Luther, Pradeep; Schulze, Thomas; Eder, Alexandra; Streichert, Thomas; Mannhardt, Ingra; Hirt, Marc N; Schaaf, Sebastian; Stenzig, Justus; Force, Thomas; Eschenhagen, Thomas; Hansen, Arne

2016-01-01

Left ventricular dysfunction is a frequent and potentially severe side effect of many tyrosine kinase inhibitors (TKI). The mode of toxicity is not identified, but may include impairment of mitochondrial or sarcomeric function, autophagy or angiogenesis, either as an on-target or off-target mechanism. We studied concentration-response curves and time courses for nine TKIs in three-dimensional, force generating engineered heart tissue (EHT) from neonatal rat heart cells. We detected a concentration- and time-dependent decline in contractile force for gefitinib, lapatinib, sunitinib, imatinib, sorafenib, vandetanib and lestaurtinib and no decline in contractile force for erlotinib and dasatinib after 96 hours of incubation. The decline in contractile force was associated with an impairment of autophagy (LC3 Western blot) and appearance of autophagolysosomes (transmission electron microscopy). This study demonstrates the feasibility to study TKI-mediated force effects in EHTs and identifies an association between a decline in contractility and inhibition of autophagic flux.

Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue

PubMed Central

Cuello, Friederike; Luther, Pradeep; Schulze, Thomas; Eder, Alexandra; Streichert, Thomas; Mannhardt, Ingra; Hirt, Marc N.; Schaaf, Sebastian; Stenzig, Justus; Force, Thomas

2016-01-01

Introduction Left ventricular dysfunction is a frequent and potentially severe side effect of many tyrosine kinase inhibitors (TKI). The mode of toxicity is not identified, but may include impairment of mitochondrial or sarcomeric function, autophagy or angiogenesis, either as an on-target or off-target mechanism. Methods and Results We studied concentration-response curves and time courses for nine TKIs in three-dimensional, force generating engineered heart tissue (EHT) from neonatal rat heart cells. We detected a concentration- and time-dependent decline in contractile force for gefitinib, lapatinib, sunitinib, imatinib, sorafenib, vandetanib and lestaurtinib and no decline in contractile force for erlotinib and dasatinib after 96 hours of incubation. The decline in contractile force was associated with an impairment of autophagy (LC3 Western blot) and appearance of autophagolysosomes (transmission electron microscopy). Conclusion This study demonstrates the feasibility to study TKI-mediated force effects in EHTs and identifies an association between a decline in contractility and inhibition of autophagic flux. PMID:26840448

Mechanical control of cyclic AMP signalling and gene transcription through integrins

NASA Technical Reports Server (NTRS)

Meyer, C. J.; Alenghat, F. J.; Rim, P.; Fong, J. H.; Fabry, B.; Ingber, D. E.

2000-01-01

This study was carried out to discriminate between two alternative hypotheses as to how cells sense mechanical forces and transduce them into changes in gene transcription. Do cells sense mechanical signals through generalized membrane distortion or through specific transmembrane receptors, such as integrins? Here we show that mechanical stresses applied to the cell surface alter the cyclic AMP signalling cascade and downstream gene transcription by modulating local release of signals generated by activated integrin receptors in a G-protein-dependent manner, whereas distortion of integrins in the absence of receptor occupancy has no effect.

Electrochemical methods for generation of a biological proton motive force

DOEpatents

Zeikus, Joseph Gregory [Okemos, MI; Shin, Hyoun S [Lansing, MI; Jain, Mahendra K [Lexington, KY

2008-12-02

Disclosed are methods using neutral red to mediate the interconversion of chemical and electrical energy. Electrically reduced neutral red has been found to promote cell growth and formation of reduced products by reversibly increasing the ratio of the reduced:oxidized forms of NAD(H) or NADP(H). Electrically reduced neutral red is able to serve as the sole source of reducing power for microbial cell growth. Neutral red is also able to promote conversion of chemical energy to electrical energy by facilitating the transfer of electrons from microbial reducing power to a fuel cell cathode.

Electrochemical methods for generation of a biological proton motive force and pyridine nucleotide cofactor regeneration

DOEpatents

Zeikus, Gregory J.; Shin, Hyoun S.; Jain, Mahendra K.

2002-01-01

Disclosed are methods using neutral red to mediate the interconversion of chemical and electrical energy. Electrically reduced neutral red has been found to promote cell growth and formation of reduced products by reversibly increasing the ratio of the reduced:oxidized forms of NAD(H) or NADP(H). Electrically reduced neutral red is able to serve as the sole source of reducing power for microbial, cell growth. Neutral red is also able to promote conversion of chemical energy to electrical energy by facilitating the transfer of electrons from microbial reducing power to a fuel cell cathode.

Electrochemical methods for generation of a biological proton motive force and pyridine nucleotide cofactor regeneration

DOEpatents

Zeikus, Joseph G.; Park, Doo

2001-01-01

Disclosed are methods using neutral red to mediate the interconversion of chemical and electrical energy. Electrically reduced neutral red has been found to promote cell growth and formation of reduced products by reversibly increasing the ratio of the reduced:oxidized forms of NAD(H) or NADP(H). Electrically reduced neutral red is able to serve as the sole source of reducing power for microbial cell growth. Neutral red is also able to promote conversion of chemical energy to electrical energy by facilitating the transfer of electrons from microbial reducing power to a fuel cell cathode.

Involvement of suppressors of cytokine signaling in toll-like receptor-mediated block of dendritic cell differentiation.

PubMed

Bartz, Holger; Avalos, Nicole M; Baetz, Andrea; Heeg, Klaus; Dalpke, Alexander H

2006-12-15

Dendritic cells (DCs) are important sentinels within innate immunity, monitoring the presence of infectious microorganisms. They operate in 2 different maturation stages, with transition from immature to mature DCs being induced by activation of toll-like receptors (TLRs). However, TLRs are also expressed on precursor cells of DCs. Here we analyzed the effects of TLR stimulation during the process of granulocyte-macrophage-colony-stimulating factor (GM-CSF)-mediated in vitro generation of immature DCs from precursor cells. We show that TLR triggering deviated phenotypic and functional differentiation from CD14+ monocytes to CD1a+ DCs. Similar results were obtained when differentiation of murine myeloid DCs from bone marrow cells was analyzed. The inhibitory effects were independent of soluble factors. TLR stimulation in DC precursor cells induced proteins of the suppressor of cytokine signaling family (SOCS), which correlated with loss of sensitivity to GM-CSF. Overexpression of SOCS-1 abolished GM-CSF signal transduction. Moreover, forced SOCS-1 expression in DC precursors mimicked the inhibitory effects on DC generation observed for TLR stimulation. The results indicate that TLR stimulation during the period of DC generation interferes with and deviates DC differentiation and that these effects are mediated particularly by SOCS-1.

[The theory of postmortem rigidity: the history and an original concept].

PubMed

Kil'diushov, E M; Tumanov, É V; Sokolova, Z Iu

2012-01-01

The original theory of postmortem rigidity has been developed and substantiated based on the concept of postmortem muscular contracture. It is postulated that the unrestricted growth of Ca2+ concentration in myoplasm of contractile cells during the immediate postmortal period brings the actin-myosine complex to the force generation state without subsequent relaxation.

Photothermal and photoacoustic processes of laser activated nano-thermolysis of cells

NASA Astrophysics Data System (ADS)

Lapotko, Dmitri; Lukianova, Ekaterina; Mitskevich, Pavel; Smolnikova, Victoria; Potapnev, Michail; Konopleva, Marina; Andreeff, Michael; Oraevsky, Alexander

2007-02-01

Laser Activated Nano-Thermolysis was recently proposed for selective damage of individual target (cancer) cells by pulsed laser induced microbubbles around superheated clusters of optically absorbing nanoparticles (NP). One of the clinical applications of this technology is the elimination of residual tumor cells from human blood and bone marrow. Clinical standards for the safety and efficacy of such procedure require the development and verification of highly selective and controllable mechanisms of cell killing. Our previous experiments showed that laser-induced microbubble is the main damaging factor in the case cell irradiation by short laser pulses above the threshold. Our current aim was to study the cell damage mechanisms and analyze selectivity and efficacy of cell damage as a function of NP parameters, NP-cell interaction conditions, and conditions of bubble generation around NP and NP clusters in cells. Generation of laser-induced bubbles around gold NP with diameters 10-250 nm was studied in Acute Myeloblast Leukemia (AML) cultures, normal stem and model K562 human cells. Short laser pulses (10 ns, 532 nm) were applied to those cells in vitro and the processes in cells were investigated with photothermal, fluorescent and atomic force microscopies and also with fluorescence flow cytometry. We have found that the best selectivity of cell damage is achieved by (1) forming large clusters of optically absorbing NP in target cells and (2) irradiating the cells with single laser pulses with the lowest fluence that can generate microbubble only around large clusters but not around single NP. Laser microbubbles with the lifetime from 20 ns to 2000 ns generated in individual cells caused damage and lysis of the cellular membrane and consequently cell death. Laser microbubbles did not damage normal cells around the damaged target (tumor) cell. Laser irradiation with equal fluence did not cause any damage of cells without accumulated NP clusters.

On the mechanochemical theory of biological pattern formation with application to vasculogenesis.

PubMed

Murray, James D

2003-02-01

We first describe the Murray-Oster mechanical theory of pattern formation, the biological basis of which is experimentally well documented. The model quantifies the interaction of cells and the extracellular matrix via the cell-generated forces. The model framework is described in quantitative detail. Vascular endothelial cells, when cultured on gelled basement membrane matrix, rapidly aggregate into clusters while deforming the matrix into a network of cord-like structures tessellating the planar culture. We apply the mechanical theory of pattern formation to this culture system and show that neither strain-biased anisotropic cell traction nor cell migration are necessary for pattern formation: isotropic, strain-stimulated cell traction is sufficient to form the observed patterns. Predictions from the model were confirmed experimentally.

The Removal of Human Breast Cancer Cells from Hematopoietic CD34+ Stem Cells by Dielectrophoretic Field-Flow-Fractionation

PubMed Central

HUANG, YING; YANG, JUN; WANG, XIAO-BO; BECKER, FREDERICK F.; GASCOYNE, PETER R.C.

2009-01-01

Dielectrophoretic field-flow-fractionation (DEP-FFF) was used to purge human breast cancer MDA-435 cells from hematopoietic CD34+ stem cells. An array of interdigitated microelectrodes lining the bottom surface of a thin chamber was used to generate dielectrophoretic forces that levitated the cell mixture in a fluid flow profile. CD34+ stem cells were levitated higher, were carried faster by the fluid flow, and exited the separation chamber earlier than the cancer cells. Using on-line flow cytometry, efficient separation of the cell mixture was observed in less than 12 min, and CD34+ stem cell fractions with a purity >99.2% were obtained. The method of DEP-FFF is potentially applicable to many biomedical cell separation problems, including microfluidic-scale diagnosis and preparative-scale purification of cell subpopulations. PMID:10791899

Hydrodynamic Contributions to Amoeboid Cell Motility

NASA Astrophysics Data System (ADS)

Lewis, Owen; Guy, Robert

2011-11-01

Understanding the methods by which cells move is a fundamental problem in modern biology. Recent evidence has shown that the fluid dynamics of cytoplasm can play a vital role in cellular motility. The slime mold Physarum polycephalum provides an excellent model organism for the study of amoeboid motion. In this research, we use both analytic and computational models to investigate intracellular fluid flow in a simple model of Physarum. In both models, of we are specifically interested in stresses generated by cytoplasmic flow which act in the direction of cellular motility. In our numerical model, the Immersed Boundary Method is used to account for such stresses. We investigate the relationship between contraction waves, low waves and locomotive forces, and attempt characterize conditions necessary to generate directed motion.

Steady-state force-velocity relation in the ATP-dependent sliding movement of myosin-coated beads on actin cables in vitro studied with a centrifuge microscope.

PubMed Central

Oiwa, K; Chaen, S; Kamitsubo, E; Shimmen, T; Sugi, H

1990-01-01

To eliminate the gap between the biochemistry of actomyosin in solution and the physiology of contracting muscle, we developed an in vitro force-movement assay system in which the steady-state force-velocity relation in the actin-myosin interaction can be studied. The assay system consists of the internodal cells of an alga, Nitellopsis obtusa, containing well-organized actin filament arrays (actin cables); tosyl-activated polystyrene beads (diameter, 2.8 microns; specific gravity, 1.3) coated with skeletal muscle myosin; and a centrifuge microscope equipped with a stroboscopic light source and a video system. The internodal cell preparation was mounted on the rotor of the centrifuge microscope, so that centrifugal forces were applied to the myosin-coated beads moving along the actin cables in the presence of ATP. Under constant centrifugal forces directed opposite to the bead movement ("positive" loads), the beads continued to move with constant velocities, which decreased with increasing centrifugal forces. The steady-state force-velocity curve thus obtained was analogous to the double-hyperbolic force-velocity curve of single muscle fibers. The unloaded velocity of bead movement was 1.6-3.6 microns/s (20-23 degrees C), while the maximum "isometric" force generated by the myosin molecules on the bead was 1.9-39 pN. If, on the other hand, the beads were subjected to constant centrifugal forces in the direction of bead movement ("negative" loads), the bead also moved with constant velocities. Unexpectedly, the velocity of bead movement did not increase with increasing negative loads but first decreased by 20-60% and then increased towards the initial unloaded velocity until the beads were eventually detached from the actin cables. Images PMID:2236007

Steady-state force-velocity relation in the ATP-dependent sliding movement of myosin-coated beads on actin cables in vitro studied with a centrifuge microscope.

PubMed

Oiwa, K; Chaen, S; Kamitsubo, E; Shimmen, T; Sugi, H

1990-10-01

To eliminate the gap between the biochemistry of actomyosin in solution and the physiology of contracting muscle, we developed an in vitro force-movement assay system in which the steady-state force-velocity relation in the actin-myosin interaction can be studied. The assay system consists of the internodal cells of an alga, Nitellopsis obtusa, containing well-organized actin filament arrays (actin cables); tosyl-activated polystyrene beads (diameter, 2.8 microns; specific gravity, 1.3) coated with skeletal muscle myosin; and a centrifuge microscope equipped with a stroboscopic light source and a video system. The internodal cell preparation was mounted on the rotor of the centrifuge microscope, so that centrifugal forces were applied to the myosin-coated beads moving along the actin cables in the presence of ATP. Under constant centrifugal forces directed opposite to the bead movement ("positive" loads), the beads continued to move with constant velocities, which decreased with increasing centrifugal forces. The steady-state force-velocity curve thus obtained was analogous to the double-hyperbolic force-velocity curve of single muscle fibers. The unloaded velocity of bead movement was 1.6-3.6 microns/s (20-23 degrees C), while the maximum "isometric" force generated by the myosin molecules on the bead was 1.9-39 pN. If, on the other hand, the beads were subjected to constant centrifugal forces in the direction of bead movement ("negative" loads), the bead also moved with constant velocities. Unexpectedly, the velocity of bead movement did not increase with increasing negative loads but first decreased by 20-60% and then increased towards the initial unloaded velocity until the beads were eventually detached from the actin cables.

Local traction force in the proximal leading process triggers nuclear translocation during neuronal migration.

PubMed

Umeshima, Hiroki; Nomura, Ken-Ichi; Yoshikawa, Shuhei; Hörning, Marcel; Tanaka, Motomu; Sakuma, Shinya; Arai, Fumihito; Kaneko, Makoto; Kengaku, Mineko

2018-04-05

Somal translocation in long bipolar neurons is regulated by actomyosin contractile forces, yet the precise spatiotemporal sites of force generation are unknown. Here we investigate the force dynamics generated during somal translocation using traction force microscopy. Neurons with a short leading process generated a traction force in the growth cone and counteracting forces in the leading and trailing processes. In contrast, neurons with a long leading process generated a force dipole with opposing traction forces in the proximal leading process during nuclear translocation. Transient accumulation of actin filaments was observed at the dipole center of the two opposing forces, which was abolished by inhibition of myosin II activity. A swelling in the leading process emerged and generated a traction force that pulled the nucleus when nuclear translocation was physically hampered. The traction force in the leading process swelling was uncoupled from somal translocation in neurons expressing a dominant negative mutant of the KASH protein, which disrupts the interaction between cytoskeletal components and the nuclear envelope. Our results suggest that the leading process is the site of generation of actomyosin-dependent traction force in long bipolar neurons, and that the traction force is transmitted to the nucleus via KASH proteins. Copyright © 2018 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.

Cellular basis of gastrulation in the sand dollar Scaphechinus mirabilis.

PubMed

Kominami, T; Takata, H

2000-12-01

The processes of gastrulation in the sand dollar Scaphechinus mirabilis are quite different from those in regular echinoids. In this study, we explored the cellular basis of gastrulation in this species with several methods. Cell-tracing experiments revealed that the prospective endodermal cells were convoluted throughout the invagination processes. Histological observation showed that the ectodermal layer remained thickened, and the vegetal cells retained an elongated shape until the last step of invagination. Further, most of the vegetal ectodermal cells were skewed or distorted. Wedge-shaped cells were common in the vegetal ectoderm, especially at the subequatorial region. In these embryos, unlike the embryos of regular echinoids, secondary mesenchyme cells did not seem to exert the force to pull up the archenteron toward the inner surface of the apical plate. In fact, the archenteron cells were not stretched along the axis of elongation and were in close contact with each other. Here we found that gastrulation was completely blocked when the embryos were attached to a glass dish coated with poly-L-lysine, in which the movement of the ectodermal layer was inhibited. These results suggest that a force generated by the thickened ectoderm, rather than rearrangement of the archenteron cells, may play a key role in the archenteron elongation in S. mirabilis embryos.

Magnetic Beads Enhance Adhesion of NIH 3T3 Fibroblasts: A Proof-of-Principle In Vitro Study for Implant-Mediated Long-Term Drug Delivery to the Inner Ear

PubMed Central

Aliuos, Pooyan; Schulze, Jennifer; Schomaker, Markus; Reuter, Günter; Stolle, Stefan R. O.; Werner, Darja; Ripken, Tammo; Lenarz, Thomas; Warnecke, Athanasia

2016-01-01

Introduction Long-term drug delivery to the inner ear may be achieved by functionalizing cochlear implant (CI) electrodes with cells providing neuroprotective factors. However, effective strategies in order to coat implant surfaces with cells need to be developed. Our vision is to make benefit of electromagnetic field attracting forces generated by CI electrodes to bind BDNF-secreting cells that are labelled with magnetic beads (MB) onto the electrode surfaces. Thus, the effect of MB-labelling on cell viability and BDNF production were investigated. Materials and Methods Murine NIH 3T3 fibroblasts—genetically modified to produce BDNF—were labelled with MB. Results Atomic force and bright field microscopy illustrated the internalization of MB by fibroblasts after 24 h of cultivation. Labelling cells with MB did not expose cytotoxic effects on fibroblasts and allowed adhesion on magnetic surfaces with sufficient BDNF release. Discussion Our data demonstrate a novel approach for mediating enhanced long-term adhesion of BDNF-secreting fibroblasts on model electrode surfaces for cell-based drug delivery applications in vitro and in vivo. This therapeutic strategy, once transferred to cells suitable for clinical application, may allow the biological modifications of CI surfaces with cells releasing neurotrophic or other factors of interest. PMID:26918945

Mechanical design in embryos: mechanical signalling, robustness and developmental defects.

PubMed

Davidson, Lance A

2017-05-19

Embryos are shaped by the precise application of force against the resistant structures of multicellular tissues. Forces may be generated, guided and resisted by cells, extracellular matrix, interstitial fluids, and how they are organized and bound within the tissue's architecture. In this review, we summarize our current thoughts on the multiple roles of mechanics in direct shaping, mechanical signalling and robustness of development. Genetic programmes of development interact with environmental cues to direct the composition of the early embryo and endow cells with active force production. Biophysical advances now provide experimental tools to measure mechanical resistance and collective forces during morphogenesis and are allowing integration of this field with studies of signalling and patterning during development. We focus this review on concepts that highlight this integration, and how the unique contributions of mechanical cues and gradients might be tested side by side with conventional signalling systems. We conclude with speculation on the integration of large-scale programmes of development, and how mechanical responses may ensure robust development and serve as constraints on programmes of tissue self-assembly.This article is part of the themed issue 'Systems morphodynamics: understanding the development of tissue hardware'. © 2017 The Author(s).

Oscillatory magnetic tweezers based on ferromagnetic beads and simple coaxial coils

NASA Astrophysics Data System (ADS)

Trepat, Xavier; Grabulosa, Mireia; Buscemi, Lara; Rico, Fèlix; Fabry, Ben; Fredberg, Jeffrey J.; Farré, Ramon

2003-09-01

We report the design and validation of simple magnetic tweezers for oscillating ferromagnetic beads in the piconewton and nanometer scales. The system is based on a single pair of coaxial coils operating in two sequential modes: permanent magnetization of the beads through a large and brief pulse of magnetic field and generation of magnetic gradients to produce uniaxial oscillatory forces. By using this two step method, the magnetic moment of the beads remains constant during measurements. Therefore, the applied force can be computed and varies linearly with the driving signal. No feedback control is required to produce well defined force oscillations over a wide bandwidth. The design of the coils was optimized to obtain high magnetic fields (280 mT) and gradients (2 T/m) with high homogeneity (5% variation) within the sample. The magnetic tweezers were implemented in an inverted optical microscope with a videomicroscopy-based multiparticle tracking system. The apparatus was validated with 4.5 μm magnetite beads obtaining forces up to ˜2 pN and subnanometer resolution. The applicability of the device includes microrheology of biopolymer and cell cytoplasm, molecular mechanics, and mechanotransduction in living cells.

Comparative study of human-induced pluripotent stem cells derived from bone marrow cells, hair keratinocytes, and skin fibroblasts.

PubMed

Streckfuss-Bömeke, Katrin; Wolf, Frieder; Azizian, Azadeh; Stauske, Michael; Tiburcy, Malte; Wagner, Stefan; Hübscher, Daniela; Dressel, Ralf; Chen, Simin; Jende, Jörg; Wulf, Gerald; Lorenz, Verena; Schön, Michael P; Maier, Lars S; Zimmermann, Wolfram H; Hasenfuss, Gerd; Guan, Kaomei

2013-09-01

Induced pluripotent stem cells (iPSCs) provide a unique opportunity for the generation of patient-specific cells for use in disease modelling, drug screening, and regenerative medicine. The aim of this study was to compare human-induced pluripotent stem cells (hiPSCs) derived from different somatic cell sources regarding their generation efficiency and cardiac differentiation potential, and functionalities of cardiomyocytes. We generated hiPSCs from hair keratinocytes, bone marrow mesenchymal stem cells (MSCs), and skin fibroblasts by using two different virus systems. We show that MSCs and fibroblasts are more easily reprogrammed than keratinocytes. This corresponds to higher methylation levels of minimal promoter regions of the OCT4 and NANOG genes in keratinocytes than in MSCs and fibroblasts. The success rate and reprogramming efficiency was significantly higher by using the STEMCCA system than the OSNL system. All analysed hiPSCs are pluripotent and show phenotypical characteristics similar to human embryonic stem cells. We studied the cardiac differentiation efficiency of generated hiPSC lines (n = 24) and found that MSC-derived hiPSCs exhibited a significantly higher efficiency to spontaneously differentiate into beating cardiomyocytes when compared with keratinocyte-, and fibroblast-derived hiPSCs. There was no significant difference in the functionalities of the cardiomyocytes derived from hiPSCs with different origins, showing the presence of pacemaker-, atrial-, ventricular- and Purkinje-like cardiomyocytes, and exhibiting rhythmic Ca2+ transients and Ca2+ sparks in hiPSC-derived cardiomyocytes. Furthermore, spontaneously and synchronously beating and force-developing engineered heart tissues were generated. Human-induced pluripotent stem cells can be reprogrammed from all three somatic cell types, but with different efficiency. All analysed iPSCs can differentiate into cardiomyocytes, and the functionalities of cardiomyocytes derived from different cell origins are similar. However, MSC-derived hiPSCs revealed a higher cardiac differentiation efficiency than keratinocyte- and fibroblast-derived hiPSCs.

Multiscale modeling of bacterial colonies: how pili mediate the dynamics of single cells and cellular aggregates

NASA Astrophysics Data System (ADS)

Pönisch, Wolfram; Weber, Christoph A.; Juckeland, Guido; Biais, Nicolas; Zaburdaev, Vasily

2017-01-01

Neisseria gonorrhoeae is the causative agent of one of the most common sexually transmitted diseases, gonorrhea. Over the past two decades there has been an alarming increase of reported gonorrhea cases where the bacteria were resistant to the most commonly used antibiotics thus prompting for alternative antimicrobial treatment strategies. The crucial step in this and many other bacterial infections is the formation of microcolonies, agglomerates consisting of up to several thousands of cells. The attachment and motility of cells on solid substrates as well as the cell-cell interactions are primarily mediated by type IV pili, long polymeric filaments protruding from the surface of cells. While the crucial role of pili in the assembly of microcolonies has been well recognized, the exact mechanisms of how they govern the formation and dynamics of microcolonies are still poorly understood. Here, we present a computational model of individual cells with explicit pili dynamics, force generation and pili-pili interactions. We employ the model to study a wide range of biological processes, such as the motility of individual cells on a surface, the heterogeneous cell motility within the large cell aggregates, and the merging dynamics and the self-assembly of microcolonies. The results of numerical simulations highlight the central role of pili generated forces in the formation of bacterial colonies and are in agreement with the available experimental observations. The model can quantify the behavior of multicellular bacterial colonies on biologically relevant temporal and spatial scales and can be easily adjusted to include the geometry and pili characteristics of various bacterial species. Ultimately, the combination of the microbiological experimental approach with the in silico model of bacterial colonies might provide new qualitative and quantitative insights on the development of bacterial infections and thus pave the way to new antimicrobial treatments.

Quantitative Analysis of Filament Branch Orientation in Listeria Actin Comet Tails.

PubMed

Jasnin, Marion; Crevenna, Alvaro H

2016-02-23

Several bacterial and viral pathogens hijack the host actin cytoskeleton machinery to facilitate spread and infection. In particular, Listeria uses Arp2/3-mediated actin filament nucleation at the bacterial surface to generate a branched network that will help propel the bacteria. However, the mechanism of force generation remains elusive due to the lack of high-resolution three-dimensional structural data on the spatial organization of the actin mother and daughter (i.e., branch) filaments within this network. Here, we have explored the three-dimensional structure of Listeria actin tails in Xenopus laevis egg extracts using cryo-electron tomography. We found that the architecture of Listeria actin tails is shared between those formed in cells and in cell extracts. Both contained nanoscopic bundles along the plane of the substrate, where the bacterium lies, and upright filaments (also called Z filaments), both oriented tangentially to the bacterial cell wall. Here, we were able to identify actin filament intersections, which likely correspond to branches, within the tails. A quantitative analysis of putative Arp2/3-mediated branches in the actin network showed that mother filaments lie on the plane of the substrate, whereas daughter filaments have random deviations out of this plane. Moreover, the analysis revealed that branches are randomly oriented with respect to the bacterial surface. Therefore, the actin filament network does not push directly toward the surface but rather accumulates, building up stress around the Listeria surface. Our results favor a mechanism of force generation for Listeria movement where the stress is released into propulsive motion. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The zipper mechanism in phagocytosis: energetic requirements and variability in phagocytic cup shape

PubMed Central

2010-01-01

Background Phagocytosis is the fundamental cellular process by which eukaryotic cells bind and engulf particles by their cell membrane. Particle engulfment involves particle recognition by cell-surface receptors, signaling and remodeling of the actin cytoskeleton to guide the membrane around the particle in a zipper-like fashion. Despite the signaling complexity, phagocytosis also depends strongly on biophysical parameters, such as particle shape, and the need for actin-driven force generation remains poorly understood. Results Here, we propose a novel, three-dimensional and stochastic biophysical model of phagocytosis, and study the engulfment of particles of various sizes and shapes, including spiral and rod-shaped particles reminiscent of bacteria. Highly curved shapes are not taken up, in line with recent experimental results. Furthermore, we surprisingly find that even without actin-driven force generation, engulfment proceeds in a large regime of parameter values, albeit more slowly and with highly variable phagocytic cups. We experimentally confirm these predictions using fibroblasts, transfected with immunoreceptor FcγRIIa for engulfment of immunoglobulin G-opsonized particles. Specifically, we compare the wild-type receptor with a mutant receptor, unable to signal to the actin cytoskeleton. Based on the reconstruction of phagocytic cups from imaging data, we indeed show that cells are able to engulf small particles even without support from biological actin-driven processes. Conclusions This suggests that biochemical pathways render the evolutionary ancient process of phagocytic highly robust, allowing cells to engulf even very large particles. The particle-shape dependence of phagocytosis makes a systematic investigation of host-pathogen interactions and an efficient design of a vehicle for drug delivery possible. PMID:21059234

Effect of salicylate on outer hair cell plasma membrane viscoelasticity: studies using optical tweezers

NASA Astrophysics Data System (ADS)

Ermilov, Sergey A.; Brownell, William E.; Anvari, Bahman

2004-06-01

The plasma membrane (PM) of mammalian outer hair cells (OHCs) generates mechanical forces in response to changes in the transmembrane electrical potential. The resulting change in the cell length is known as electromotility. Salicylate (Sal), the anionic, amphipathic derivative of aspirin induces reversible hearing loss and decreases electromotile response of the OHCs. Sal may change the local curvature and mechanical properties of the PM, eventually resulting in reduced electromotility or it may compete with intracellular monovalent anions, particularly Cl-, which are essential for electromotility. In this work we have used optical tweezers to study the effects of Sal on viscoelastic properties of the OHC PM when separated from the underlying composite structures of the cell wall. In this procedure, an optically trapped microsphere is brought in contact with PM and subsequently pulled away to form a tether. We measured the force exerted on the tether as a function of time during the process of tether growth at different pulling rates. Effective tether viscosity, steady-state tethering force extrapolated to zero pulling rate, and the time constant for tether growth were estimated from the measurements of the instantaneous tethering force. The time constant for the tether growth measured for the OHC basal end decreased 1.65 times after addition of 10 mM Sal, which may result from an interaction between Sal and cholesterol, which is more prevalent in the PM of OHC basal end. The time constants for the tether growth calculated for the OHC lateral wall and control human embryonic kidney cells as well as the other calculated viscoelastic parameters remained the same after Sal perfusion, favoring the hypothesis of competitive inhibition of electromotility by salicylate.

Quantitative modeling of forces in electromagnetic tweezers

NASA Astrophysics Data System (ADS)

Bijamov, Alex; Shubitidze, Fridon; Oliver, Piercen M.; Vezenov, Dmitri V.

2010-11-01

This paper discusses numerical simulations of the magnetic field produced by an electromagnet for generation of forces on superparamagnetic microspheres used in manipulation of single molecules or cells. Single molecule force spectroscopy based on magnetic tweezers can be used in applications that require parallel readout of biopolymer stretching or biomolecular binding. The magnetic tweezers exert forces on the surface-immobilized macromolecule by pulling a magnetic bead attached to the free end of the molecule in the direction of the field gradient. In a typical force spectroscopy experiment, the pulling forces can range between subpiconewton to tens of piconewtons. In order to effectively provide such forces, an understanding of the source of the magnetic field is required as the first step in the design of force spectroscopy systems. In this study, we use a numerical technique, the method of auxiliary sources, to investigate the influence of electromagnet geometry and material parameters of the magnetic core on the magnetic forces pulling the target beads in the area of interest. The close proximity of the area of interest to the magnet body results in deviations from intuitive relations between magnet size and pulling force, as well as in the force decay with distance. We discuss the benefits and drawbacks of various geometric modifications affecting the magnitude and spatial distribution of forces achievable with an electromagnet.

Epsin deficiency impairs endocytosis by stalling the actin-dependent invagination of endocytic clathrin-coated pits

PubMed Central

Messa, Mirko; Fernández-Busnadiego, Rubén; Sun, Elizabeth Wen; Chen, Hong; Czapla, Heather; Wrasman, Kristie; Wu, Yumei; Ko, Genevieve; Ross, Theodora; Wendland, Beverly; De Camilli, Pietro

2014-01-01

Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function(s) in clathrin coat dynamics remain(s) elusive. To elucidate such function(s), we generated embryonic fibroblasts from conditional epsin triple KO mice. Triple KO cells displayed a dramatic cell division defect. Additionally, a robust impairment in clathrin-mediated endocytosis was observed, with an accumulation of early and U-shaped pits. This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton, which we confirmed in a cell-free assay of endocytosis. Our results indicate that a key evolutionary conserved function of epsin, in addition to other roles that include, as we show here, a low affinity interaction with SNAREs, is to help generate the force that leads to invagination and then fission of clathrin-coated pits. DOI: http://dx.doi.org/10.7554/eLife.03311.001 PMID:25122462

Yap is required for ependymal integrity and is suppressed in LPA-induced hydrocephalus

PubMed Central

Park, Raehee; Moon, Uk Yeol; Park, Jun Young; Hughes, Lucinda J.; Johnson, Randy L.; Cho, Seo-Hee; Kim, Seonhee

2016-01-01

Timely generation and normal maturation of ependymal cells along the aqueduct are critical for preventing physical blockage between the third and fourth ventricles and the development of fetal non-communicating hydrocephalus. Our study identifies Yap, the downstream effector of the evolutionarily conserved Hippo pathway, as a central regulator for generating developmentally controlled ependymal cells along the ventricular lining of the aqueduct. Yap function is necessary for proper proliferation of progenitors and apical attachment of ependymal precursor cells. Importantly, an injury signal initiated by lysophosphatidic acid (LPA), an upstream regulator of Yap that can cause fetal haemorrhagic hydrocephalus, deregulates Yap in the developing aqueduct. LPA exposure leads to the loss of N-cadherin concentrations at the apical endfeet, which can be partially restored by forced Yap expression and more efficiently by phosphomimetic Yap. These results reveal a novel function of Yap in retaining tissue junctions during normal development and after fetal brain injury. PMID:26754915

Single-Cell Force Spectroscopy of Probiotic Bacteria

PubMed Central

Beaussart, Audrey; El-Kirat-Chatel, Sofiane; Herman, Philippe; Alsteens, David; Mahillon, Jacques; Hols, Pascal; Dufrêne, Yves F.

2013-01-01

Single-cell force spectroscopy is a powerful atomic force microscopy modality in which a single living cell is attached to the atomic force microscopy cantilever to quantify the forces that drive cell-cell and cell-substrate interactions. Although various single-cell force spectroscopy protocols are well established for animal cells, application of the method to individual bacterial cells remains challenging, mainly owing to the lack of appropriate methods for the controlled attachment of single live cells on cantilevers. We present a nondestructive protocol for single-bacterial cell force spectroscopy, which combines the use of colloidal probe cantilevers and of a bioinspired polydopamine wet adhesive. Living cells from the probiotic species Lactobacillus plantarum are picked up with a polydopamine-coated colloidal probe, enabling us to quantify the adhesion forces between single bacteria and biotic (lectin monolayer) or abiotic (hydrophobic monolayer) surfaces. These minimally invasive single-cell experiments provide novel, to our knowledge, insight into the specific and nonspecific forces driving the adhesion of L. plantarum, and represent a generic platform for studying the molecular mechanisms of cell adhesion in probiotic and pathogenic bacteria. PMID:23663831

Integration of actomyosin contractility with cell-cell adhesion during dorsal closure.

PubMed

Duque, Julia; Gorfinkiel, Nicole

2016-12-15

In this work, we combine genetic perturbation, time-lapse imaging and quantitative image analysis to investigate how pulsatile actomyosin contractility drives cell oscillations, apical cell contraction and tissue closure during morphogenesis of the amnioserosa, the main force-generating tissue during the dorsal closure in Drosophila We show that Myosin activity determines the oscillatory and contractile behaviour of amnioserosa cells. Reducing Myosin activity prevents cell shape oscillations and reduces cell contractility. By contrast, increasing Myosin activity increases the amplitude of cell shape oscillations and the time cells spend in the contracted phase relative to the expanded phase during an oscillatory cycle, promoting cell contractility and tissue closure. Furthermore, we show that in AS cells, Rok controls Myosin foci formation and Mbs regulates not only Myosin phosphorylation but also adhesion dynamics through control of Moesin phosphorylation, showing that Mbs coordinates actomyosin contractility with cell-cell adhesion during amnioserosa morphogenesis. © 2016. Published by The Company of Biologists Ltd.

Penium margaritaceum as a model organism for cell wall analysis of expanding plant cells.

PubMed

Rydahl, Maja G; Fangel, Jonatan U; Mikkelsen, Maria Dalgaard; Johansen, I Elisabeth; Andreas, Amanda; Harholt, Jesper; Ulvskov, Peter; Jørgensen, Bodil; Domozych, David S; Willats, William G T

2015-01-01

The growth of a plant cell encompasses a complex set of subcellular components interacting in a highly coordinated fashion. Ultimately, these activities create specific cell wall structural domains that regulate the prime force of expansion, internally generated turgor pressure. The precise organization of the polymeric networks of the cell wall around the protoplast also contributes to the direction of growth, the shape of the cell, and the proper positioning of the cell in a tissue. In essence, plant cell expansion represents the foundation of development. Most studies of plant cell expansion have focused primarily upon late divergent multicellular land plants and specialized cell types (e.g., pollen tubes, root hairs). Here, we describe a unicellular green alga, Penium margaritaceum (Penium), which can serve as a valuable model organism for understanding cell expansion and the underlying mechanics of the cell wall in a single plant cell.

Chimeric Protein Complexes in Hybrid Species Generate Novel Phenotypes

PubMed Central

Piatkowska, Elzbieta M.; Naseeb, Samina; Knight, David; Delneri, Daniela

2013-01-01

Hybridization between species is an important mechanism for the origin of novel lineages and adaptation to new environments. Increased allelic variation and modification of the transcriptional network are the two recognized forces currently deemed to be responsible for the phenotypic properties seen in hybrids. However, since the majority of the biological functions in a cell are carried out by protein complexes, inter-specific protein assemblies therefore represent another important source of natural variation upon which evolutionary forces can act. Here we studied the composition of six protein complexes in two different Saccharomyces “sensu stricto” hybrids, to understand whether chimeric interactions can be freely formed in the cell in spite of species-specific co-evolutionary forces, and whether the different types of complexes cause a change in hybrid fitness. The protein assemblies were isolated from the hybrids via affinity chromatography and identified via mass spectrometry. We found evidence of spontaneous chimericity for four of the six protein assemblies tested and we showed that different types of complexes can cause a variety of phenotypes in selected environments. In the case of TRP2/TRP3 complex, the effect of such chimeric formation resulted in the fitness advantage of the hybrid in an environment lacking tryptophan, while only one type of parental combination of the MBF complex allowed the hybrid to grow under respiratory conditions. These phenotypes were dependent on both genetic and environmental backgrounds. This study provides empirical evidence that chimeric protein complexes can freely assemble in cells and reveals a new mechanism to generate phenotypic novelty and plasticity in hybrids to complement the genomic innovation resulting from gene duplication. The ability to exchange orthologous members has also important implications for the adaptation and subsequent genome evolution of the hybrids in terms of pattern of gene loss. PMID:24137105

Epigenomic Views of Innate Lymphoid Cells.

PubMed

Sciumè, Giuseppe; Shih, Han-Yu; Mikami, Yohei; O'Shea, John J

2017-01-01

The discovery of innate lymphoid cells (ILCs) with selective production of cytokines typically attributed to subsets of T helper cells forces immunologists to reassess the mechanisms by which selective effector functions arise. The parallelism between ILCs and T cells extends beyond these two cell types and comprises other innate-like T lymphocytes. Beyond the recognition of specialized effector functionalities in diverse lymphocytes, features typical of T cells, such as plasticity and memory, are also relevant for innate lymphocytes. Herein, we review what we have learned in terms of the molecular mechanisms underlying these shared functions, focusing on insights provided by next generation sequencing technologies. We review data on the role of lineage-defining- and signal-dependent transcription factors (TFs). ILC regulomes emerge developmentally whereas the much of the open chromatin regions of T cells are generated acutely, in an activation-dependent manner. And yet, these regions of open chromatin in T cells and ILCs have remarkable overlaps, suggesting that though accessibility is acquired by distinct modes, the end result is that convergent signaling pathways may be involved. Although much is left to be learned, substantial progress has been made in understanding how TFs and epigenomic status contribute to ILC biology in terms of differentiation, specification, and plasticity.

Epigenomic Views of Innate Lymphoid Cells

PubMed Central

Sciumè, Giuseppe; Shih, Han-Yu; Mikami, Yohei; O’Shea, John J.

2017-01-01

The discovery of innate lymphoid cells (ILCs) with selective production of cytokines typically attributed to subsets of T helper cells forces immunologists to reassess the mechanisms by which selective effector functions arise. The parallelism between ILCs and T cells extends beyond these two cell types and comprises other innate-like T lymphocytes. Beyond the recognition of specialized effector functionalities in diverse lymphocytes, features typical of T cells, such as plasticity and memory, are also relevant for innate lymphocytes. Herein, we review what we have learned in terms of the molecular mechanisms underlying these shared functions, focusing on insights provided by next generation sequencing technologies. We review data on the role of lineage-defining- and signal-dependent transcription factors (TFs). ILC regulomes emerge developmentally whereas the much of the open chromatin regions of T cells are generated acutely, in an activation-dependent manner. And yet, these regions of open chromatin in T cells and ILCs have remarkable overlaps, suggesting that though accessibility is acquired by distinct modes, the end result is that convergent signaling pathways may be involved. Although much is left to be learned, substantial progress has been made in understanding how TFs and epigenomic status contribute to ILC biology in terms of differentiation, specification, and plasticity. PMID:29250060

Aggregate formation and suspension culture of human pluripotent stem cells and differentiated progeny.

PubMed

Hookway, Tracy A; Butts, Jessica C; Lee, Emily; Tang, Hengli; McDevitt, Todd C

2016-05-15

Culture of human pluripotent stem cells (hPSC) as in vitro multicellular aggregates has been increasingly used as a method to model early embryonic development. Three-dimensional assemblies of hPSCs facilitate interactions between cells and their microenvironment to promote morphogenesis, analogous to the multicellular organization that accompanies embryogenesis. In this paper, we describe a method for reproducibly generating and maintaining populations of homogeneous three-dimensional hPSC aggregates using forced aggregation and rotary orbital suspension culture. We propose solutions to several challenges associated with the consistent formation and extended culture of cell spheroids generated from hPSCs and their differentiated progeny. Further, we provide examples to demonstrate how aggregation can be used as a tool to select specific subpopulations of cells to create homotypic spheroids, or as a means to introduce multiple cell types to create heterotypic tissue constructs. Finally, we demonstrate that the aggregation and rotary suspension method can be used to support culture and maintenance of hPSC-derived cell populations representing each of the three germ layers, underscoring the utility of this platform for culturing many different cell types. Copyright © 2015 Elsevier Inc. All rights reserved.

Mitochondrial pyruvate carrier function determines cell stemness and metabolic reprogramming in cancer cells

PubMed Central

Li, Xiaoran; Kan, Quancheng; Fan, Zhirui; Li, Yaqing; Ji, Yasai; Zhao, Jing; Zhang, Mingzhi; Grigalavicius, Mantas; Berge, Viktor; Goscinski, Mariusz Adam; M. Nesland, Jahn; Suo, Zhenhe

2017-01-01

One of the remarkable features of cancer cells is aerobic glycolysis, a phenomenon known as the “Warburg Effect”, in which cells rely preferentially on glycolysis instead of oxidative phosphorylation (OXPHOS) as the main energy source even in the presence of high oxygen tension. Cells with dysfunctional mitochondria are unable to generate sufficient ATP from mitochondrial OXPHOS, and then are forced to rely on glycolysis for ATP generation. Here we report our results in a prostate cancer cell line in which the mitochondrial pyruvate carrier 1 (MPC1) gene was knockout. It was discovered that the MPC1 gene knockout cells revealed a metabolism reprogramming to aerobic glycolysis with reduced ATP production, and the cells became more migratory and resistant to both chemotherapy and radiotherapy. In addition, the MPC1 knockout cells expressed significantly higher levels of the stemness markers Nanog, Hif1α, Notch1, CD44 and ALDH. To further verify the correlation of MPC gene function and cell stemness/metabolic reprogramming, MPC inhibitor UK5099 was applied in two ovarian cancer cell lines and similar results were obtained. Taken together, our results reveal that functional MPC may determine the fate of metabolic program and the stemness status of cancer cells in vitro. PMID:28624784

Load Adaptation of Lamellipodial Actin Networks.

PubMed

Mueller, Jan; Szep, Gregory; Nemethova, Maria; de Vries, Ingrid; Lieber, Arnon D; Winkler, Christoph; Kruse, Karsten; Small, J Victor; Schmeiser, Christian; Keren, Kinneret; Hauschild, Robert; Sixt, Michael

2017-09-21

Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load. Copyright © 2017 Elsevier Inc. All rights reserved.

The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rossier, Olivier; Giannone, Grégory; CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux

Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements andmore » interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells.« less

The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking.

PubMed

Rossier, Olivier; Giannone, Grégory

2016-04-10

Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells. Copyright © 2015. Published by Elsevier Inc.

A resilient formin-derived cortical actin meshwork in the rear drives actomyosin-based motility in 2D confinement

PubMed Central

Ramalingam, Nagendran; Franke, Christof; Jaschinski, Evelin; Winterhoff, Moritz; Lu, Yao; Brühmann, Stefan; Junemann, Alexander; Meier, Helena; Noegel, Angelika A.; Weber, Igor; Zhao, Hongxia; Merkel, Rudolf; Schleicher, Michael; Faix, Jan

2015-01-01

Cell migration is driven by the establishment of disparity between the cortical properties of the softer front and the more rigid rear allowing front extension and actomyosin-based rear contraction. However, how the cortical actin meshwork in the rear is generated remains elusive. Here we identify the mDia1-like formin A (ForA) from Dictyostelium discoideum that generates a subset of filaments as the basis of a resilient cortical actin sheath in the rear. Mechanical resistance of this actin compartment is accomplished by actin crosslinkers and IQGAP-related proteins, and is mandatory to withstand the increased contractile forces in response to mechanical stress by impeding unproductive blebbing in the rear, allowing efficient cell migration in two-dimensional-confined environments. Consistently, ForA supresses the formation of lateral protrusions, rapidly relocalizes to new prospective ends in repolarizing cells and is required for cortical integrity. Finally, we show that ForA utilizes the phosphoinositide gradients in polarized cells for subcellular targeting. PMID:26415699

Microtubules and motor proteins: Mechanically regulated self-organization in vivo

NASA Astrophysics Data System (ADS)

Vogel, S. K.; Pavin, N.; Maghelli, N.; Jülicher, F.; Tolić-Nørrelykke, I. M.

2009-11-01

A key aspect of life is sexual reproduction, which requires concerted movement. For successful mixing of the genetic material, molecular motors move the nucleus back and forth inside the cell. How motors work together to produce these large-scale movements, however, remains a mystery. To answer this question, we studied nuclear movement in fission yeast, which is driven by motor proteins pulling on microtubules. We show that motor proteins dynamically redistribute from one part of the cell to the other, generating asymmetric patterns of motors and, consequently, of forces that generate movement. By combining quantitative live cell imaging and laser ablation with a theoretical model, we find that this dynamic motor redistribution occurs purely as a result of changes in the mechanical strain sensed by the motor proteins. Our work therefore demonstrates that spatio-temporal pattern formation within a cell can occur as a result of mechanical cues (Vogel et al., 2009), which differs from conventional molecular signaling, as well as from self-organization based on a combination of biochemical reactions and diffusion.

Loading, electromyograph, and motion during exercise

NASA Technical Reports Server (NTRS)

Todd, Beth A.

1993-01-01

A bicycle ergometer system has been developed to determine forces acting in specific muscles and muscle groups for both cycling and isometric exercise. The bicycle has been instrumented with encoders, accelerometers, and load cells. A harnessing system has been developed to keep subjects in place during isometric exercise. EMG data will also be collected with electrodes attached to various muscles on the subject's leg. Data has been collected for static loading and will be collected for cycling in both an earth-based laboratory and on the KC-135. Once the data is analyzed, the forces will be entered into finite element models of bones of the lower extremities. A finite element model of the tibia-fibula has been generated from the experimental subject's MRI data. The linear elastic isoparametric brick elements representing the bones are connected by linear elastic isoparametric shell elements placed at the locations of ligaments. Models will be generated for the calcaneus and the femur. Material properties for the various tissues will be taken from the literature. The experimentally determined muscle forces will be applied to the models to determine the stress distribution which is created in the bones.

Cell separation technique in dilectrophoretic chip with bulk electrode

NASA Astrophysics Data System (ADS)

Iliescu, Ciprian; Tay, Francis E. H.; Xu, Guolin; Yu, Liming

2006-01-01

This paper presents a new technique for separation of two cell populations in a dielectrophoretic chip with bulk silicon electrode. A characteristic of the dielectrophoretic chip is its "sandwich" structure: glass/silicon/glass that generates a unique definition of the microfluidic channel with conductive walls (silicon) and isolating floor and ceiling (glass). The structure confers the opportunity to use the electrodes not only to generate a gradient of the electric field but also to generate a gradient of velocity of the fluid inside the channel. This interesting combination gives rise to a new solution for dielectrophoretic separation of two cell populations. The separation method consists of four steps. First, the microchannel is field with the cells mixture. Second, the cells are trapped in different locations of the microfluidic channel, the cell population which exhibits positive dielectrophoresis is trapped in the area where the distance between the electrodes is the minimum whilst, the other population that exhibit negative dielectrophoresis is trapped where the distance between electrodes is the maximum. In the next step, increasing the flow in the microchannel will result in an increased hydrodynamic force that sweeps the cells trapped by positive dielectrophoresis out of the chip. In the last step, the electric field is removed and the second population is sweep out and collected at the outlet. The device was tested for separation of dead yeast cells from live yeast cells. The paper presents analytical aspects of the separation method a comparative study between different electrode profiles and experimental results.

How muscle fiber lengths and velocities affect muscle force generation as humans walk and run at different speeds

PubMed Central

Arnold, Edith M.; Hamner, Samuel R.; Seth, Ajay; Millard, Matthew; Delp, Scott L.

2013-01-01

SUMMARY The lengths and velocities of muscle fibers have a dramatic effect on muscle force generation. It is unknown, however, whether the lengths and velocities of lower limb muscle fibers substantially affect the ability of muscles to generate force during walking and running. We examined this issue by developing simulations of muscle–tendon dynamics to calculate the lengths and velocities of muscle fibers from electromyographic recordings of 11 lower limb muscles and kinematic measurements of the hip, knee and ankle made as five subjects walked at speeds of 1.0–1.75 m s−1 and ran at speeds of 2.0–5.0 m s−1. We analyzed the simulated fiber lengths, fiber velocities and forces to evaluate the influence of force–length and force–velocity properties on force generation at different walking and running speeds. The simulations revealed that force generation ability (i.e. the force generated per unit of activation) of eight of the 11 muscles was significantly affected by walking or running speed. Soleus force generation ability decreased with increasing walking speed, but the transition from walking to running increased the force generation ability by reducing fiber velocities. Our results demonstrate the influence of soleus muscle architecture on the walk-to-run transition and the effects of muscle–tendon compliance on the plantarflexors' ability to generate ankle moment and power. The study presents data that permit lower limb muscles to be studied in unprecedented detail by relating muscle fiber dynamics and force generation to the mechanical demands of walking and running. PMID:23470656

Estimation of actomyosin active force maintained by tropomyosin and troponin complex under vertical forces in the in vitro motility assay system

PubMed Central

Ishii, Shuya; Kawai, Masataka; Ishiwata, Shin'ichi

2018-01-01

The interaction between actin filaments and myosin molecular motors is a power source of a variety of cellular functions including cell division, cell motility, and muscular contraction. In vitro motility assay examines actin filaments interacting with myosin molecules that are adhered to a substrate (e.g., glass surface). This assay has been the standard method of studying the molecular mechanisms of contraction under an optical microscope. While the force generation has been measured through an optically trapped bead to which an actin filament is attached, a force vector vertical to the glass surface has been largely ignored with the in vitro motility assay. The vertical vector is created by the gap (distance) between the trapped bead and the glass surface. In this report, we propose a method to estimate the angle between the actin filament and the glass surface by optically determining the gap size. This determination requires a motorized stage in a standard epi-fluorescence microscope equipped with optical tweezers. This facile method is applied to force measurements using both pure actin filaments, and thin filaments reconstituted from actin, tropomyosin and troponin. We find that the angle-corrected force per unit filament length in the active condition (pCa = 5.0) decreases as the angle between the filament and the glass surface increases; i.e. as the force in the vertical direction increases. At the same time, we demonstrate that the force on reconstituted thin filaments is approximately 1.5 times larger than that on pure actin filaments. The range of angles we tested was between 11° and 36° with the estimated measurement error less than 6°. These results suggest the ability of cytoplasmic tropomyosin isoforms maintaining actomyosin active force to stabilize cytoskeletal architecture. PMID:29420610

Cell mechanics: a dialogue.

PubMed

Tao, Jiaxiang; Li, Yizeng; Vig, Dhruv K; Sun, Sean X

2017-03-01

Under the microscope, eukaryotic animal cells can adopt a variety of different shapes and sizes. These cells also move and deform, and the physical mechanisms driving these movements and shape changes are important in fundamental cell biology, tissue mechanics, as well as disease biology. This article reviews some of the basic mechanical concepts in cells, emphasizing continuum mechanics description of cytoskeletal networks and hydrodynamic flows across the cell membrane. We discuss how cells can generate movement and shape changes by controlling mass fluxes at the cell boundary. These mass fluxes can come from polymerization/depolymerization of actin cytoskeleton, as well as osmotic and hydraulic pressure-driven flow of water across the cell membrane. By combining hydraulic pressure control with force balance conditions at the cell surface, we discuss a quantitative mechanism of cell shape and volume control. The broad consequences of this model on cell mechanosensation and tissue mechanics are outlined.

Cell mechanics: a dialogue

PubMed Central

Tao, Jiaxiang; Li, Yizeng; Vig, Dhruv K; Sun, Sean X

2017-01-01

Under the microscope, eukaryotic animal cells can adopt a variety of different shapes and sizes. These cells also move and deform, and the physical mechanisms driving these movements and shape changes are important in fundamental cell biology, tissue mechanics, as well as disease biology. This article reviews some of the basic mechanical concepts in cells, emphasizing continuum mechanics description of cytoskeletal networks and hydrodynamic flows across the cell membrane. We discuss how cells can generate movement and shape changes by controlling mass fluxes at the cell boundary. These mass fluxes can come from polymerization/depolymerization of actin cytoskeleton, as well as osmotic and hydraulic pressure-driven flow of water across the cell membrane. By combining hydraulic pressure control with force balance conditions at the cell surface, we discuss a quantitative mechanism of cell shape and volume control. The broad consequences of this model on cell mechanosensation and tissue mechanics are outlined. PMID:28129208

Cell mechanics: a dialogue

NASA Astrophysics Data System (ADS)

Tao, Jiaxiang; Li, Yizeng; Vig, Dhruv K.; Sun, Sean X.

2017-03-01

Under the microscope, eukaryotic animal cells can adopt a variety of different shapes and sizes. These cells also move and deform, and the physical mechanisms driving these movements and shape changes are important in fundamental cell biology, tissue mechanics, as well as disease biology. This article reviews some of the basic mechanical concepts in cells, emphasizing continuum mechanics description of cytoskeletal networks and hydrodynamic flows across the cell membrane. We discuss how cells can generate movement and shape changes by controlling mass fluxes at the cell boundary. These mass fluxes can come from polymerization/depolymerization of actin cytoskeleton, as well as osmotic and hydraulic pressure-driven flow of water across the cell membrane. By combining hydraulic pressure control with force balance conditions at the cell surface, we discuss a quantitative mechanism of cell shape and volume control. The broad consequences of this model on cell mechanosensation and tissue mechanics are outlined.

TIC-Tox: A preliminary discussion on identifying the forcing agents of DBP-mediated toxicity of disinfected water.

PubMed

Plewa, Michael J; Wagner, Elizabeth D; Richardson, Susan D

2017-08-01

The disinfection of drinking water is a major public health achievement; however, an unintended consequence of disinfection is the generation of disinfection by-products (DBPs). Many of the identified DBPs exhibit in vitro and in vivo toxicity, generate a diversity of adverse biological effects, and may be hazards to the public health and the environment. Only a few DBPs are regulated by several national and international agencies and it is not clear if these regulated DBPs are the forcing agents that drive the observed toxicity and their associated health effects. In this study, we combine analytical chemical and biological data to resolve the forcing agents associated with mammalian cell cytotoxicity of drinking water samples from three cities. These data suggest that the trihalomethanes (THMs) and haloacetic acids may be a small component of the overall cytotoxicity of the organic material isolated from disinfected drinking water. Chemical classes of nitrogen-containing DBPs, such as the haloacetonitriles and haloacetamides, appear to be the major forcing agents of toxicity in these samples. These findings may have important implications for the design of epidemiological studies that primarily rely on the levels of THMs to define DBP exposure among populations. The TIC-Tox approach constitutes a beginning step in the process of identifying the forcing agents of toxicity in disinfected water. Copyright © 2017. Published by Elsevier B.V.

Single particle train ordering in microchannel based on inertial and vortex effects

NASA Astrophysics Data System (ADS)

Fan, Liang-Liang; Yan, Qing; Zhe, Jiang; Zhao, Liang

2018-06-01

A new microfluidic device for microparticle focusing and ordering in a single particle train is reported. The particle focusing and ordering are based on inertial and vortex effects in a microchannel with a series of suddenly contracted and widely expanded structures on one side. In the suddenly contracted regions, particles located near the contracted structures are subjected to a strong wall-effect lift force and momentum-change-induced inertial force due to the highly curved trajectory, migrating to the straight wall. A horizontal vortex is generated downstream of the contracted structure, which prevents the particle from getting close to the wall. In the widely expanded regions, the streamline is curved and no vortex is generated. The shear-gradient lift force and the momentum-change-induced inertial force are dominant for particle lateral migration, driving particles towards the wall of the expanded structures. Eventually, particles are focused and ordered in a single particle train by the combination effects of the inertial forces and the vortex. In comparison with other single-stream particle focusing methods, this device requires no sheath flow, is easy for fabrication and operation, and can work over a wide range of Reynolds numbers from 19.1–142.9. The highly ordered particle chain could be potentially utilized in a variety of lab-chip applications, including micro-flow cytometer, imaging and droplet-based cell entrapment.

Microtubule dynamics in cell division: exploring living cells with polarized light microscopy.

PubMed

Inoué, Shinya

2008-01-01

This Perspective is an account of my early experience while I studied the dynamic organization and behavior of the mitotic spindle and its submicroscopic filaments using polarized light microscopy. The birefringence of spindle filaments in normally dividing plant and animal cells, and those treated by various agents, revealed (a) the reality of spindle fibers and fibrils in healthy living cells; (b) the labile, dynamic nature of the molecular filaments making up the spindle fibers; (c) the mode of fibrogenesis and action of orienting centers; and (d) force-generating properties based on the disassembly and assembly of the fibrils. These studies, which were carried out directly on living cells using improved polarizing microscopes, in fact predicted the reversible assembly properties of microtubules.

External push and internal pull forces recruit curvature sensing N-BAR domain proteins to the plasma membrane

PubMed Central

Galic, Milos; Jeong, Sangmoo; Tsai, Feng-Chiao; Joubert, Lydia-Marie; Wu, Yi I.; Hahn, Klaus M.; Cui, Yi; Meyer, Tobias

2012-01-01

Many of the more than 20 mammalian proteins with N-BAR domains1-2 control cell architecture3 and endocytosis4-5 by associating with curved sections of the plasma membrane (PM)6. It is not well understood whether N-BAR proteins are recruited directly by processes that mechanically curve the PM or indirectly by PM-associated adaptor proteins that recruit proteins with N-BAR domains that then induce membrane curvature. Here, we show that externally-induced inward deformation of the PM by cone-shaped nanostructures (Nanocones) and internally-induced inward deformation by contracting actin cables both trigger recruitment of isolated N-BAR domains to the curved PM. Markedly, live-cell imaging in adherent cells showed selective recruitment of full length N-BAR proteins and isolated N-BAR domains to PM sub-regions above Nanocone stripes. Electron microscopy confirmed that N-BAR domains are recruited to local membrane sites curved by Nanocones. We further showed that N-BAR domains are periodically recruited to curved PM sites during local lamellipodia retraction in the front of migrating cells. Recruitment required Myosin II-generated force applied to PM connected actin cables. Together, our study shows that N-BAR domains can be directly recruited to the PM by external push or internal pull forces that locally curve the PM. PMID:22750946

Multipurpose high-pressure high-temperature diamond-anvil cell with a novel high-precision guiding system and a dual-mode pressurization device

NASA Astrophysics Data System (ADS)

Pippinger, Thomas; Miletich, Ronald; Burchard, Michael

2011-09-01

A novel diamond-anvil cell (DAC) design has been constructed and tested for in situ applications at high-pressure (HP) operations and has proved to be suitable even for HP sample environments at non-ambient temperature conditions. The innovative high-precision guiding mechanism, comparable to a dog clutch, consists of perpendicular planar sliding-plane elements and is integrated directly into the base body of the cylindrically shaped DAC. The combination of two force-generating devices, i.e., mechanical screws and an inflatable gas membrane, allows the user to choose independently between, and to apply individually, two different forcing mechanisms for pressure generation. Both mechanisms are basically independent of each other, but can also be operated simultaneously. The modularity of the DAC design allows for an easy exchange of functional core-element groups optimized not only for various analytical in situ methods but also for HP operation with or without high-temperature (HT) application. For HP-HT experiments a liquid cooling circuit inside the specific inner modular groups has been implemented to obtain a controlled and limited heat distribution within the outer DAC body.

Microtubule Depolymerization as a Driver for Chromosome Motion

NASA Astrophysics Data System (ADS)

McIntosh, Richard

2014-03-01

Microtubules (MTs) are rigid polymers of the protein, tubulin, which function as intracellular struts. They are also tracks along which motor enzymes can run, carrying cargo to specific cellular locations. Most MTs are dynamic; they assemble and disassemble rapidly, particularly during cell division when the cell forms the ``mitotic spindle,'' a machine that organizes the duplicated chromosomes into a planar disk, then pulls the duplicate copies apart, moving them to opposite ends of the cell. This process is necessary for the daughter cells to have a full complement of DNA. The mitotic spindle is a labile framework that exerts several kinds of forces on the chromosomes to move them in well organized ways. It contains many motor enzymes that contribute to spindle formation, but genetic evidence shows that the motors that attach to chromosomes and might contribute to chromosome motion are dispensable for normal mitosis. Apparently MT dynamics can also serve as a motor and is an important source of force for chromosome motion. We have studied this process and find that MTs can be coupled to a load by specific spindle proteins so that MT depolymerization can exert substantial force. With the yeast protein, Dam1, a single MT can generate 30 pN, about 5-fold more than is generated by a motor enzyme like kinesin or myosin. The resulting motions are processive, so a depolymerizing MT can carry its load for many micrometers. However, Dam1 is found only in fungi. We have therefore sought other proteins that can serve as analogous couplers. Several MT-dependent motor enzymes can do the job in ways that do not require ATP, their normal source of energy. Some non-motor MT-associated proteins will also work, e.g., the kinetochore proteins NDC80 and CENP-F. Data will be presented that show the strengths and weaknesses of each coupler, allowing some generalization about how the mitotic machinery works. Supported by NIH GM033787.

Motor unit firing rate patterns during voluntary muscle force generation: a simulation study

NASA Astrophysics Data System (ADS)

Hu, Xiaogang; Rymer, William Z.; Suresh, Nina L.

2014-04-01

Objective. Muscle force is generated by a combination of motor unit (MU) recruitment and changes in the discharge rate of active MUs. There have been two basic MU recruitment and firing rate paradigms reported in the literature, which describe the control of the MUs during force generation. The first (termed the reverse ‘onion skin’ profile), exhibits lower firing rates for lower threshold units, with higher firing rates occurring in higher threshold units. The second (termed the ‘onion skin’ profile), exhibits an inverse arrangement, with lower threshold units reaching higher firing rates. Approach. Using a simulation of the MU activity in a hand muscle, this study examined the force generation capacity and the variability of the muscle force magnitude at different excitation levels of the MU pool under these two different MU control paradigms. We sought to determine which rate/recruitment scheme was more efficient for force generation, and which scheme gave rise to the lowest force variability. Main results. We found that the force output of both firing patterns leads to graded force output at low excitation levels, and that the force generation capacity of the two different paradigms diverged around 50% excitation. In the reverse ‘onion skin’ pattern, at 100% excitation, the force output reached up to 88% of maximum force, whereas for the ‘onion skin’ pattern, the force output only reached up to 54% of maximum force at 100% excitation. The force variability was lower at the low to moderate force levels under the ‘onion skin’ paradigm than with the reverse ‘onion skin’ firing patterns, but this effect was reversed at high force levels. Significance. This study captures the influence of MU recruitment and firing rate organization on muscle force properties, and our results suggest that the different firing organizations can be beneficial at different levels of voluntary muscle force generation and perhaps for different tasks.

Emergence of multicellular organisms with dynamic differentiation and spatial pattern.

PubMed

Furusawa, C; Kaneko, K

1998-01-01

The origin of multicellular organisms and the mechanism of development in cell societies are studied by choosing a model with intracellular biochemical dynamics allowing for oscillations, cell-cell interaction through diffusive chemicals on a two-dimensional grid, and state-dependent cell adhesion. Cells differentiate due to a dynamical instability, as described by our "isologous diversification" theory. A fixed spatial pattern of differentiated cells emerges, where spatial information is sustained by cell-cell interactions. This pattern is robust against perturbations. With an adequate cell adhesion force, active cells are release that form the seed of a new generation of multicellular organisms, accompanied by death of the original multicellular unit as a halting state. It is shown that the emergence of multicellular organisms with differentiation, regulation, and life cycle is not an accidental event, but a natural consequence in a system of replicating cells with growth.

Born to run: creating the muscle fiber.

PubMed

Schejter, Eyal D; Baylies, Mary K

2010-10-01

From the muscles that control the blink of your eye to those that allow you to walk, the basic architecture of muscle is the same: muscles consist of bundles of the unit muscle cell, the muscle fiber. The unique morphology of the individual muscle fiber is dictated by the functional demands necessary to generate and withstand the forces of contraction, which in turn leads to movement. Contractile muscle fibers are elongated, syncytial cells, which interact with both the nervous and skeletal systems to govern body motion. In this review, we focus on three key cell-cell and cell-matrix contact processes, that are necessary to create this exquisitely specialized cell: cell fusion, cell elongation, and establishment of a myotendinous junction. We address these processes by highlighting recent findings from the Drosophila model system. Copyright © 2010 Elsevier Ltd. All rights reserved.

Eukaryotic membrane tethers revisited using magnetic tweezers.

PubMed

Hosu, Basarab G; Sun, Mingzhai; Marga, Françoise; Grandbois, Michel; Forgacs, Gabor

2007-04-19

Membrane nanotubes, under physiological conditions, typically form en masse. We employed magnetic tweezers (MTW) to extract tethers from human brain tumor cells and compared their biophysical properties with tethers extracted after disruption of the cytoskeleton and from a strongly differing cell type, Chinese hamster ovary cells. In this method, the constant force produced with the MTW is transduced to cells through super-paramagnetic beads attached to the cell membrane. Multiple sudden jumps in bead velocity were manifest in the recorded bead displacement-time profiles. These discrete events were interpreted as successive ruptures of individual tethers. Observation with scanning electron microscopy supported the simultaneous existence of multiple tethers. The physical characteristics, in particular, the number and viscoelastic properties of the extracted tethers were determined from the analytic fit to bead trajectories, provided by a standard model of viscoelasticity. Comparison of tethers formed with MTW and atomic force microscopy (AFM), a technique where the cantilever-force transducer is moved at constant velocity, revealed significant differences in the two methods of tether formation. Our findings imply that extreme care must be used to interpret the outcome of tether pulling experiments performed with single molecular techniques (MTW, AFM, optical tweezers, etc). First, the different methods may be testing distinct membrane structures with distinct properties. Second, as soon as a true cell membrane (as opposed to that of a vesicle) can attach to a substrate, upon pulling on it, multiple nonspecific membrane tethers may be generated. Therefore, under physiological conditions, distinguishing between tethers formed through specific and nonspecific interactions is highly nontrivial if at all possible.

Eukaryotic membrane tethers revisited using magnetic tweezers

NASA Astrophysics Data System (ADS)

Hosu, Basarab G.; Sun, Mingzhai; Marga, Françoise; Grandbois, Michel; Forgacs, Gabor

2007-06-01

Membrane nanotubes, under physiological conditions, typically form en masse. We employed magnetic tweezers (MTW) to extract tethers from human brain tumor cells and compared their biophysical properties with tethers extracted after disruption of the cytoskeleton and from a strongly differing cell type, Chinese hamster ovary cells. In this method, the constant force produced with the MTW is transduced to cells through super-paramagnetic beads attached to the cell membrane. Multiple sudden jumps in bead velocity were manifest in the recorded bead displacement-time profiles. These discrete events were interpreted as successive ruptures of individual tethers. Observation with scanning electron microscopy supported the simultaneous existence of multiple tethers. The physical characteristics, in particular, the number and viscoelastic properties of the extracted tethers were determined from the analytic fit to bead trajectories, provided by a standard model of viscoelasticity. Comparison of tethers formed with MTW and atomic force microscopy (AFM), a technique where the cantilever-force transducer is moved at constant velocity, revealed significant differences in the two methods of tether formation. Our findings imply that extreme care must be used to interpret the outcome of tether pulling experiments performed with single molecular techniques (MTW, AFM, optical tweezers, etc). First, the different methods may be testing distinct membrane structures with distinct properties. Second, as soon as a true cell membrane (as opposed to that of a vesicle) can attach to a substrate, upon pulling on it, multiple nonspecific membrane tethers may be generated. Therefore, under physiological conditions, distinguishing between tethers formed through specific and nonspecific interactions is highly nontrivial if at all possible.

Regulation of the basement membrane by epithelia generated forces

NASA Astrophysics Data System (ADS)

Tanner, Kandice

2012-12-01

Tumor metastasis involves a progressive loss of tissue architecture and dissolution of structural boundaries between the epithelium and connective tissue. The basement membrane (BM), a specialized network of extracellular matrix proteins forms a barrier that physically restricts pre-invasive lesions such that they remain as local insults. The BM is not a static structure, but one that is constantly regenerated and remodeled in the adult organism. Matrix organization also regulates cell function. Thus alterations in the balance of synthesis, remodeling and proteolytic degradation of the extracellular matrix proteins may contribute to a loss of structural integrity. However, the de novo assembly and maintenance of the complex structural properties of in vivo basement membranes remain elusive. Here, this paper highlights the current understanding on the structural properties and the establishment of the BM, and discusses the potential role of self-generated forces in adult tissue remodeling and the maintenance of the BM as a malignancy suppressor.

Ultrasonic disintegration of biosolids for improved biodegradation.

PubMed

Nickel, Klaus; Neis, Uwe

2007-04-01

Biological cell lysis is known to be the rate-limiting step of anaerobic biosolids degradation. Shear forces generated by low frequency ultrasound can be used to disintegrate bacterial cells in sewage sludge. Thus, the quantity of dissolved organic substrate is increased. Consequently, the degradation rate and the biodegradability of organic biosolids mass are improved. Fundamental pilot-studies showed a significantly accelerated biosolids degradation with less digested sludge being produced and increased biogas production being attained. A full-scale ultrasound reactor system was developed for continuous operation under real life conditions on sewage treatment plants (STP).

Lightweight armor system

DOEpatents

Chu, Henry S; Langhorst, Benjamin R; Bakas, Michael P; Thinnes, Gary L

2013-02-26

The disclosure provides a shock absorbing layer comprised of one or more shock absorbing cells, where a shock absorbing cell is comprised of a cell interior volume containing a plurality of hydrogel particles and a free volume, and where the cell interior volume is surrounded by a containing layer. The containing layer has a permeability such that the hydrogel particles when swollen remain at least partially within the cell interior volume when subjected to a design shock pressure wave, allowing for force relaxation through hydrogel compression response. Additionally, the permeability allows for the flow of exuded free water, further dissipating wave energy. In an embodiment, a plurality of shock absorbing cells is combined with a penetration resistant material to mitigate the transmitted shock wave generated by an elastic precursor wave in the penetration resistant material.

W.R. Grove and the fuel cell

NASA Astrophysics Data System (ADS)

Meurig Thomas, John

2012-11-01

Though known these days largely because he invented the fuel cell, Grove also had many other distinguished achievements to his credit. His monumental book On the Correlation of Physical Forces contained all the arguments that led to the enunciation of the first law of thermodynamics. He was also an extremely versatile natural philosopher who was, in addition, well versed in classical literature, particularly the works of the eminent scholars of ancient Greece and Rome. This article touches upon these qualities, but is predominantly concerned with the emergence and modern aspects of the H2/O2 fuel cell and its potential for the clean generation of energy.

Rho-guanine nucleotide exchange factors during development

PubMed Central

Mulinari, Shai

2010-01-01

The development of multicellular organisms is associated with extensive rearrangements of tissues and cell sheets. The driving force for these rearrangements is generated mostly by the actin cytoskeleton. In order to permit the reproducible development of a specific body plan, dynamic reorganization of the actin cytoskeleton must be precisely coordinated in space and time. GTP-exchange factors that activate small GTPases of the Rho family play an important role in this process. Here we review the role of this class of cytoskeletal regulators during important developmental processes such as epithelial morphogenesis, cytokinesis, cell migration, cell polarity, neuronal growth cone extension and phagocytosis in different model systems. PMID:21686118

Correlation of fingertip shear force direction with somatosensory cortical activity in monkey

PubMed Central

Fortier-Poisson, Pascal; Langlais, Jean-Sébastien

2015-01-01

To examine the activity of somatosensory cortex (S1) neurons to self-generated shear forces on the index and thumb, two monkeys were trained to grasp a stationary metal tab with a key grip and exert forces without the fingers slipping in one of four orthogonal directions for 1 s. A majority (∼85%) of slowly adapting and rapidly adapting (RA) S1 neurons had activity modulated with shear force direction. The cells were recorded mainly in areas 1 and 2 of the S1, although some area 3b neurons also responded to shear direction or magnitude. The preferred shear vectors were distributed in every direction, with tuning arcs varying from 50° to 170°. Some RA neurons sensitive to dynamic shear force direction also responded to static shear force but within a narrower range, suggesting that the direction of the shear force may influence the adaptation rate. Other neurons were modulated with shear forces in diametrically opposite directions. The directional sensitivity of S1 cortical neurons is consistent with recordings from cutaneous afferents showing that shear direction, even without slip, is a powerful stimulus to S1 neurons. PMID:26467520

Coupled thermal, electrical, and fluid flow analyses of AMTEC converters, with illustrative application to OSC`s cell design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schock, A.; Noravian, H.; Or, C.

1997-12-31

This paper presents the background and introduction to the OSC AMTEC (Alkali Metal Thermal-to-Electrical Conversion) studies, which were conducted for the Department of energy (DOE) and NASA`s jet Propulsion Laboratory (JPL). After describing the basic principle of AMTEC, the paper describes and explains the operation of multi-tube vapor/vapor cells, which have been under development by AMPS (Advance Modular Power Systems, Inc.) for the Air Force Phillips Laboratory (AFPL) and JPL for possible application to the Europa Orbiter, Pluto Express, and other space missions. It then describes a novel OSC-generated methodology for analyzing the performance of such cells. This methodology consistsmore » of an iterative procedure for the coupled solution of the interdependent thermal, electrical, and fluid flow differential and integral equations governing the performance of AMTEC cells and generators, taking proper account of the non-linear axial variations of temperature, pressure, open-circuit voltage, inter-electrode voltages, current density, axial current, sodium mass flow rate, and power density. The paper illustrates that analytical procedure by applying it to OSC`s latest cell design and by presenting detailed analytical results for that design. The OSC-developed analytic methodology constitutes a unique and powerful tool for accurate parametric analyses and design optimizations of the multi-tube AMTEC cells and of radioisotope power systems. This is illustrated in two companion papers in these proceedings. The first of those papers applies the OSC-derived program to determine the effect of various design parameters on the performance of single AMTEC cells with adiabatic side walls, culminating in an OSC-recommended revised cell design. And the second describes a number of OSC-generated AMTEC generator designs consisting of 2 and 3 GPHS heat source modules, 16 multi-tube converter cells, and a hybrid insulation design, and presents the results of applying the above analysis program to determine the applicability of those generators to possible future missions under consideration by NASA.« less

Bacterial adhesion force quantification by fluidic force microscopy

NASA Astrophysics Data System (ADS)

Potthoff, Eva; Ossola, Dario; Zambelli, Tomaso; Vorholt, Julia A.

2015-02-01

Quantification of detachment forces between bacteria and substrates facilitates the understanding of the bacterial adhesion process that affects cell physiology and survival. Here, we present a method that allows for serial, single bacterial cell force spectroscopy by combining the force control of atomic force microscopy with microfluidics. Reversible bacterial cell immobilization under physiological conditions on the pyramidal tip of a microchanneled cantilever is achieved by underpressure. Using the fluidic force microscopy technology (FluidFM), we achieve immobilization forces greater than those of state-of-the-art cell-cantilever binding as demonstrated by the detachment of Escherichia coli from polydopamine with recorded forces between 4 and 8 nN for many cells. The contact time and setpoint dependence of the adhesion forces of E. coli and Streptococcus pyogenes, as well as the sequential detachment of bacteria out of a chain, are shown, revealing distinct force patterns in the detachment curves. This study demonstrates the potential of the FluidFM technology for quantitative bacterial adhesion measurements of cell-substrate and cell-cell interactions that are relevant in biofilms and infection biology.Quantification of detachment forces between bacteria and substrates facilitates the understanding of the bacterial adhesion process that affects cell physiology and survival. Here, we present a method that allows for serial, single bacterial cell force spectroscopy by combining the force control of atomic force microscopy with microfluidics. Reversible bacterial cell immobilization under physiological conditions on the pyramidal tip of a microchanneled cantilever is achieved by underpressure. Using the fluidic force microscopy technology (FluidFM), we achieve immobilization forces greater than those of state-of-the-art cell-cantilever binding as demonstrated by the detachment of Escherichia coli from polydopamine with recorded forces between 4 and 8 nN for many cells. The contact time and setpoint dependence of the adhesion forces of E. coli and Streptococcus pyogenes, as well as the sequential detachment of bacteria out of a chain, are shown, revealing distinct force patterns in the detachment curves. This study demonstrates the potential of the FluidFM technology for quantitative bacterial adhesion measurements of cell-substrate and cell-cell interactions that are relevant in biofilms and infection biology. Electronic supplementary information (ESI) available: Video S1. Detachment of a S. pyogenes cell chain from glass substrate. The cantilever is approached on the outermost adherent cell of a chain and four bacteria were then sequentially detached. The sequential cell detachment suddenly stopped after four bacteria. This possibly occurred because bacteria-glass interactions became too strong or the maximal probe retraction was reached. The cells spontaneously detached from the cantilever flipping back on the surface. Fig. S1. (A) Adhesion force-distance and (B) adhesion force-detaching work correlation of E.coli on PLL for setpoints of 1 and 10 nN. Circle: 1 nN setpoint, square: 10 nN. See DOI: 10.1039/c4nr06495j

The spindle protein CHICA mediates localization of the chromokinesin Kid to the mitotic spindle.

PubMed

Santamaria, Anna; Nagel, Susanna; Sillje, Herman H W; Nigg, Erich A

2008-05-20

Microtubule-based motor proteins provide essential forces for bipolar organization of spindle microtubules and chromosome movement, prerequisites of chromosome segregation during the cell cycle. Here, we describe the functional characterization of a novel spindle protein, termed "CHICA," that was originally identified in a proteomic survey of the human spindle apparatus [1]. We show that CHICA localizes to the mitotic spindle and is both upregulated and phosphorylated during mitosis. CHICA-depleted cells form shorter spindles and fail to organize a proper metaphase plate, highly reminiscent of the phenotype observed upon depletion of the chromokinesin Kid, a key mediator of polar ejection forces [2-6]. We further show that CHICA coimmunoprecipitates with Kid and is required for the spindle localization of Kid without affecting its chromosome association. Moreover, upon depletion of either CHICA or Kid (or both proteins simultaneously), chromosomes collapse onto the poles of monastrol-induced monopolar spindles. We conclude that CHICA represents a novel interaction partner of the chromokinesin Kid that is required for the generation of polar ejection forces and chromosome congression.

Mechanics and contraction dynamics of single platelets and implications for clot stiffening

NASA Astrophysics Data System (ADS)

Lam, Wilbur A.; Chaudhuri, Ovijit; Crow, Ailey; Webster, Kevin D.; Li, Tai-De; Kita, Ashley; Huang, James; Fletcher, Daniel A.

2011-01-01

Platelets interact with fibrin polymers to form blood clots at sites of vascular injury. Bulk studies have shown clots to be active materials, with platelet contraction driving the retraction and stiffening of clots. However, neither the dynamics of single-platelet contraction nor the strength and elasticity of individual platelets, both of which are important for understanding clot material properties, have been directly measured. Here we use atomic force microscopy to measure the mechanics and dynamics of single platelets. We find that platelets contract nearly instantaneously when activated by contact with fibrinogen and complete contraction within 15 min. Individual platelets can generate an average maximum contractile force of 29 nN and form adhesions stronger than 70 nN. Our measurements show that when exposed to stiffer microenvironments, platelets generated higher stall forces, which indicates that platelets may be able to contract heterogeneous clots more uniformly. The high elasticity of individual platelets, measured to be 10 kPa after contraction, combined with their high contractile forces, indicates that clots may be stiffened through direct reinforcement by platelets as well as by strain stiffening of fibrin under tension due to platelet contraction. These results show how the mechanosensitivity and mechanics of single cells can be used to dynamically alter the material properties of physiologic systems.

3D engineered cardiac tissue models of human heart disease: learning more from our mice.

PubMed

Ralphe, J Carter; de Lange, Willem J

2013-02-01

Mouse engineered cardiac tissue constructs (mECTs) are a new tool available to study human forms of genetic heart disease within the laboratory. The cultured strips of cardiac cells generate physiologic calcium transients and twitch force, and respond to electrical pacing and adrenergic stimulation. The mECT can be made using cells from existing mouse models of cardiac disease, providing a robust readout of contractile performance and allowing a rapid assessment of genotype-phenotype correlations and responses to therapies. mECT represents an efficient and economical extension to the existing tools for studying cardiac physiology. Human ECTs generated from iPSCMs represent the next logical step for this technology and offer significant promise of an integrated, fully human, cardiac tissue model. Copyright © 2013. Published by Elsevier Inc.

Hydrodynamic Contributions to Amoeboid Cell Motility

NASA Astrophysics Data System (ADS)

Lewis, Owen; Guy, Robert

2012-11-01

Understanding the methods by which cells move is a fundamental problem in modern biology. Recent evidence has shown that the fluid dynamics of cytoplasm can play a vital role in cellular motility. The slime mold Physarum polycephalum provides an excellent model organism for the study of amoeboid motion. In this research, we use a simply analytic model in conjuction with computational experiments to investigate intracellular fluid flow in a simple model of Physarum. Of particlar interest are stresses generated by cytoplasmic flow which may be used to aid in cellular motility. In our numerical model, the Immersed Boundary Method is used to account for such stresses. We investigate the relationship between contraction waves, flow waves, adhesion, and locomotive forces in an attempt to characterize conditions necessary to generate directed motion.

Temporal variations in early developmental decisions: an engine of forebrain evolution.

PubMed

Bielen, H; Pal, S; Tole, S; Houart, C

2017-02-01

Tight control of developmental timing is pivotal to many major processes in developmental biology, such as patterning, fate specification, cell cycle dynamics, cell migration and connectivity. Temporal change in these ontogenetic sequences is known as heterochrony, a major force in the evolution of body plans and organogenesis. In the last 5 years, studies in fish and rodents indicate that heterochrony in signaling during early development generates diversity in forebrain size and complexity. Here, we summarize these findings and propose that, additionally to spatio-temporal tuning of neurogenesis, temporal and quantitative modulation of signaling events drive pivotal changes in shape, size and complexity of the forebrain across evolution, participating to the generation of diversity in animal behavior and emergence of cognition. Copyright © 2017 Elsevier Ltd. All rights reserved.

Estimation of sonodynamic treatment region with sonochemiluminescence in gel phantom

NASA Astrophysics Data System (ADS)

Mashiko, Daisaku; Nishitaka, Shinya; Iwasaki, Ryosuke; Lafond, Maxime; Yoshizawa, Shin; Umemura, Shin-ichiro

2018-07-01

Sonodynamic treatment is a non-invasive cancer treatment using ultrasound through the generation of reactive oxygen species (ROS) by acoustic cavitation. High-intensity focused ultrasound (HIFU) can generate cavitation bubbles using highly negative pressure in its focal region. When cavitation bubbles are forced to collapse, they generate ROS, which can attack cancer cells, typically assisted by a sonodynamically active antitumor agent. For sonodynamic treatment, both localization and efficiency of generating ROS are important. To improve them, the region of ROS generation was quantitatively estimated in this study using a polyacrylamide gel containing luminol as the target exposed to “Trigger HIFU”, consisting of a highly intense short “trigger pulse” to generate a cavitation cloud followed by a moderate-intensity long “sustaining burst” to keep the cavitation bubbles oscillating. It was found to be important for efficient ROS generation that the focal region of the trigger pulse should be immediately exposed to the sustaining burst.

Motility of catalytic nanoparticles through self-generated forces.

PubMed

Paxton, Walter F; Sen, Ayusman; Mallouk, Thomas E

2005-11-04

Small-scale synthetic motors capable of generating their own motive forces by exploiting the chemical free energy of their environment represent an important step in developing practical nanomachines. Catalytic particles are capable of generating concentration and other gradients that can be used to self-propel small objects. However, the autonomous movement of catalytic nanoparticles by self-generated forces is a relatively unexplored area in colloid and interfacial chemistry. This paper explores the potential of catalytically self-generated forces for propulsion of small objects through fluids.

Consecutive monitoring of lifelong production of conidia by individual conidiophores of Blumeria graminis f. sp. hordei on barley leaves by digital microscopic techniques with electrostatic micromanipulation.

PubMed

Moriura, Nobuyuki; Matsuda, Yoshinori; Oichi, Wataru; Nakashima, Shinya; Hirai, Tatsuo; Sameshima, Takeshi; Nonomura, Teruo; Kakutani, Koji; Kusakari, Shin-Ichi; Higashi, Katsuhide; Toyoda, Hideyoshi

2006-01-01

Conidial formation and secession by living conidiophores of Blumeria graminis f. sp. hordei on barley leaves were consecutively monitored using a high-fidelity digital microscopic technique combined with electrostatic micromanipulation to trap the released conidia. Conidial chains formed on conidiophores through a series of septum-mediated division and growth of generative cells. Apical conidial cells on the conidiophores were abstricted after the conidial chains developed ten conidial cells. The conidia were electrically conductive, and a positive charge was induced in the cells by a negatively polarized insulator probe (ebonite). The electrostatic force between the conidia and the insulator was used to attract the abstricted conidia from the conidiophores on leaves. This conidium movement from the targeted conidiophore to the rod was directly viewed under the digital microscope, and the length of the interval between conidial septation and secession, the total number of the conidia produced by a single conidiophore, and the modes of conidiogenesis were clarified. During the stage of conidial secession, the generative cells pushed new conidial cells upwards by repeated division and growth. The successive release of two apical conidia was synchronized with the successive septation and growth of a generative cell. The release ceased after 4-5 conidia were released without division and growth of the generative cell. Thus, the life of an individual conidiophore (from the erection of the conidiophore to the release of the final conidium) was shown to be 107 h and to produce an average of 33 conidia. To our knowledge, this is the first report on the direct estimation of life-long conidial production by a powdery mildew on host leaves.

Coupled thermal, electrical, and fluid flow analyses of AMTEC multitube cell with adiabatic side wall

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schock, A.; Or, C.; Noravian, H.

1997-01-01

The paper describes a novel OSC-generated methodology for analyzing the performance of multitube AMTEC (Alkali Metal Thermal-to-Electrical Conversion) cells, which are under development by AMPS (Advanced Modular Power Systems, Inc.) for the Air Force Phillips Laboratory (AFPL) and NASA{close_quote}s Jet Propulsion Laboratory (JPL), for possible application to the Pluto Express and other space missions. The OSC study was supported by the Department of Energy (DOE), and was strongly encouraged by JPL, AFPL, and AMPS. It resulted in an iterative procedure for the coupled solution of the interdependent thermal, electrical, and fluid flow differential and integral equations governing the performance ofmore » AMTEC cells and generators. The paper clarifies the OSC procedure by presenting detailed results of its application to an illustrative example of a converter cell with an adiabatic side wall, including the non-linear axial variation of temperature, pressure, open-circuit voltage, interelectrode voltage, current density, axial current, sodium mass flow, and power density. The next paper in these proceedings describes parametric results obtained by applying the same procedure to variations of the baseline adiabatic converter design, culminating in an OSC-recommended revised cell design. A subsequent paper in these proceedings extends the procedure to analyze a variety of OSC-designed radioisotope-heated generators employing non-adiabatic multitube AMTEC cells. {copyright} {ital 1997 American Institute of Physics.}« less

Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*

PubMed Central

Tondeleir, Davina; Lambrechts, Anja; Müller, Matthias; Jonckheere, Veronique; Doll, Thierry; Vandamme, Drieke; Bakkali, Karima; Waterschoot, Davy; Lemaistre, Marianne; Debeir, Olivier; Decaestecker, Christine; Hinz, Boris; Staes, An; Timmerman, Evy; Colaert, Niklaas; Gevaert, Kris; Vandekerckhove, Joël; Ampe, Christophe

2012-01-01

Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation. PMID:22448045

A Simple Force-Motion Relation for Migrating Cells Revealed by Multipole Analysis of Traction Stress

PubMed Central

Tanimoto, Hirokazu; Sano, Masaki

2014-01-01

For biophysical understanding of cell motility, the relationship between mechanical force and cell migration must be uncovered, but it remains elusive. Since cells migrate at small scale in dissipative circumstances, the inertia force is negligible and all forces should cancel out. This implies that one must quantify the spatial pattern of the force instead of just the summation to elucidate the force-motion relation. Here, we introduced multipole analysis to quantify the traction stress dynamics of migrating cells. We measured the traction stress of Dictyostelium discoideum cells and investigated the lowest two moments, the force dipole and quadrupole moments, which reflect rotational and front-rear asymmetries of the stress field. We derived a simple force-motion relation in which cells migrate along the force dipole axis with a direction determined by the force quadrupole. Furthermore, as a complementary approach, we also investigated fine structures in the stress field that show front-rear asymmetric kinetics consistent with the multipole analysis. The tight force-motion relation enables us to predict cell migration only from the traction stress patterns. PMID:24411233

Loss of electrostatic cell-surface repulsion mediates myelin membrane adhesion and compaction in the central nervous system.

PubMed

Bakhti, Mostafa; Snaidero, Nicolas; Schneider, David; Aggarwal, Shweta; Möbius, Wiebke; Janshoff, Andreas; Eckhardt, Matthias; Nave, Klaus-Armin; Simons, Mikael

2013-02-19

During the development of the central nervous system (CNS), oligodendrocytes wrap their plasma membrane around axons to form a multilayered stack of tightly attached membranes. Although intracellular myelin compaction and the role of myelin basic protein has been investigated, the forces that mediate the close interaction of myelin membranes at their external surfaces are poorly understood. Such extensive bilayer-bilayer interactions are usually prevented by repulsive forces generated by the glycocalyx, a dense and confluent layer of large and negatively charged oligosaccharides. Here we investigate the molecular mechanisms underlying myelin adhesion and compaction in the CNS. We revisit the role of the proteolipid protein and analyze the contribution of oligosaccharides using cellular assays, biophysical tools, and transgenic mice. We observe that differentiation of oligodendrocytes is accompanied by a striking down-regulation of components of their glycocalyx. Both in vitro and in vivo experiments indicate that the adhesive properties of the proteolipid protein, along with the reduction of sialic acid residues from the cell surface, orchestrate myelin membrane adhesion and compaction in the CNS. We suggest that loss of electrostatic cell-surface repulsion uncovers weak and unspecific attractive forces in the bilayer that bring the extracellular surfaces of a membrane into close contact over long distances.

CellNetVis: a web tool for visualization of biological networks using force-directed layout constrained by cellular components.

PubMed

Heberle, Henry; Carazzolle, Marcelo Falsarella; Telles, Guilherme P; Meirelles, Gabriela Vaz; Minghim, Rosane

2017-09-13

The advent of "omics" science has brought new perspectives in contemporary biology through the high-throughput analyses of molecular interactions, providing new clues in protein/gene function and in the organization of biological pathways. Biomolecular interaction networks, or graphs, are simple abstract representations where the components of a cell (e.g. proteins, metabolites etc.) are represented by nodes and their interactions are represented by edges. An appropriate visualization of data is crucial for understanding such networks, since pathways are related to functions that occur in specific regions of the cell. The force-directed layout is an important and widely used technique to draw networks according to their topologies. Placing the networks into cellular compartments helps to quickly identify where network elements are located and, more specifically, concentrated. Currently, only a few tools provide the capability of visually organizing networks by cellular compartments. Most of them cannot handle large and dense networks. Even for small networks with hundreds of nodes the available tools are not able to reposition the network while the user is interacting, limiting the visual exploration capability. Here we propose CellNetVis, a web tool to easily display biological networks in a cell diagram employing a constrained force-directed layout algorithm. The tool is freely available and open-source. It was originally designed for networks generated by the Integrated Interactome System and can be used with networks from others databases, like InnateDB. CellNetVis has demonstrated to be applicable for dynamic investigation of complex networks over a consistent representation of a cell on the Web, with capabilities not matched elsewhere.

Solo, a RhoA-targeting guanine nucleotide exchange factor, is critical for hemidesmosome formation and acinar development in epithelial cells.

PubMed

Fujiwara, Sachiko; Matsui, Tsubasa S; Ohashi, Kazumasa; Deguchi, Shinji; Mizuno, Kensaku

2018-01-01

Cell-substrate adhesions are essential for various physiological processes, including embryonic development and maintenance of organ functions. Hemidesmosomes (HDs) are multiprotein complexes that attach epithelial cells to the basement membrane. Formation and remodeling of HDs are dependent on the surrounding mechanical environment; however, the upstream signaling mechanisms are not well understood. We recently reported that Solo (also known as ARHGEF40), a guanine nucleotide exchange factor targeting RhoA, binds to keratin8/18 (K8/K18) intermediate filaments, and that their interaction is important for force-induced actin and keratin cytoskeletal reorganization. In this study, we show that Solo co-precipitates with an HD protein, β4-integrin. Co-precipitation assays revealed that the central region (amino acids 330-1057) of Solo binds to the C-terminal region (1451-1752) of β4-integrin. Knockdown of Solo significantly suppressed HD formation in MCF10A mammary epithelial cells. Similarly, knockdown of K18 or treatment with Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed HD formation. As Solo knockdown or Y-27632 treatment is known to disorganize K8/K18 filaments, these results suggest that Solo is involved in HD formation by regulating K8/K18 filament organization via the RhoA-ROCK signaling pathway. We also showed that knockdown of Solo impairs acinar formation in MCF10A cells cultured in 3D Matrigel. In addition, Solo accumulated at the site of traction force generation in 2D-cultured MCF10A cells. Taken together, these results suggest that Solo plays a crucial role in HD formation and acinar development in epithelial cells by regulating mechanical force-induced RhoA activation and keratin filament organization.

Solo, a RhoA-targeting guanine nucleotide exchange factor, is critical for hemidesmosome formation and acinar development in epithelial cells

PubMed Central

Matsui, Tsubasa S.; Ohashi, Kazumasa; Deguchi, Shinji; Mizuno, Kensaku

2018-01-01

Cell-substrate adhesions are essential for various physiological processes, including embryonic development and maintenance of organ functions. Hemidesmosomes (HDs) are multiprotein complexes that attach epithelial cells to the basement membrane. Formation and remodeling of HDs are dependent on the surrounding mechanical environment; however, the upstream signaling mechanisms are not well understood. We recently reported that Solo (also known as ARHGEF40), a guanine nucleotide exchange factor targeting RhoA, binds to keratin8/18 (K8/K18) intermediate filaments, and that their interaction is important for force-induced actin and keratin cytoskeletal reorganization. In this study, we show that Solo co-precipitates with an HD protein, β4-integrin. Co-precipitation assays revealed that the central region (amino acids 330–1057) of Solo binds to the C-terminal region (1451–1752) of β4-integrin. Knockdown of Solo significantly suppressed HD formation in MCF10A mammary epithelial cells. Similarly, knockdown of K18 or treatment with Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed HD formation. As Solo knockdown or Y-27632 treatment is known to disorganize K8/K18 filaments, these results suggest that Solo is involved in HD formation by regulating K8/K18 filament organization via the RhoA-ROCK signaling pathway. We also showed that knockdown of Solo impairs acinar formation in MCF10A cells cultured in 3D Matrigel. In addition, Solo accumulated at the site of traction force generation in 2D-cultured MCF10A cells. Taken together, these results suggest that Solo plays a crucial role in HD formation and acinar development in epithelial cells by regulating mechanical force-induced RhoA activation and keratin filament organization. PMID:29672603

Sensor apparatus using an electrochemical cell

DOEpatents

Thakur, Mrinal

2003-07-01

A method for sensing mechanical quantities such as force, stress, strain, pressure and acceleration is disclosed. This technology is based on a change in the electrochemically generated voltage (electromotive force) with application of force, stress, strain, pressure or acceleration. The change in the voltage is due to a change in the internal resistance of the electrochemical cell with a change in the relative position or orientation of the electrodes (anode and cathode) in the cell. The signal to be detected (e.g. force, stress, strain, pressure or acceleration) is applied to one of the electrodes to cause a change in the relative position or orientation between the electrodes. Various materials, solid, semisolid, gel, paste or liquid can be utilized as the electrolyte. The electrolyte must be an ion conductor. The examples of solid electrolytes include specific polymer conductors, polymer composites, ion conducting glasses and ceramics. The electrodes are made of conductors such as metals with dissimilar electro negativities. Significantly enhanced sensitivities, up to three orders of magnitude higher than that of comparable commercial sensors, are obtained. The materials are substantially less expensive than commercially used materials for mechanical sensors. An apparatus for sensing such mechanical quantities using materials such as doped 1,4 cis-polyisopropene and nafion. The 1,4 cis-polyisopropene may be doped with lithium perchlorate or iodine. The output voltage signal increases with an increase of the sensing area for a given stress. The device can be used as an intruder alarm, among other applications.

Aerodynamic effects of flexibility in flapping wings.

PubMed

Zhao, Liang; Huang, Qingfeng; Deng, Xinyan; Sane, Sanjay P

2010-03-06

Recent work on the aerodynamics of flapping flight reveals fundamental differences in the mechanisms of aerodynamic force generation between fixed and flapping wings. When fixed wings translate at high angles of attack, they periodically generate and shed leading and trailing edge vortices as reflected in their fluctuating aerodynamic force traces and associated flow visualization. In contrast, wings flapping at high angles of attack generate stable leading edge vorticity, which persists throughout the duration of the stroke and enhances mean aerodynamic forces. Here, we show that aerodynamic forces can be controlled by altering the trailing edge flexibility of a flapping wing. We used a dynamically scaled mechanical model of flapping flight (Re approximately 2000) to measure the aerodynamic forces on flapping wings of variable flexural stiffness (EI). For low to medium angles of attack, as flexibility of the wing increases, its ability to generate aerodynamic forces decreases monotonically but its lift-to-drag ratios remain approximately constant. The instantaneous force traces reveal no major differences in the underlying modes of force generation for flexible and rigid wings, but the magnitude of force, the angle of net force vector and centre of pressure all vary systematically with wing flexibility. Even a rudimentary framework of wing veins is sufficient to restore the ability of flexible wings to generate forces at near-rigid values. Thus, the magnitude of force generation can be controlled by modulating the trailing edge flexibility and thereby controlling the magnitude of the leading edge vorticity. To characterize this, we have generated a detailed database of aerodynamic forces as a function of several variables including material properties, kinematics, aerodynamic forces and centre of pressure, which can also be used to help validate computational models of aeroelastic flapping wings. These experiments will also be useful for wing design for small robotic insects and, to a limited extent, in understanding the aerodynamics of flapping insect wings.

Aerodynamic effects of flexibility in flapping wings

PubMed Central

Zhao, Liang; Huang, Qingfeng; Deng, Xinyan; Sane, Sanjay P.

2010-01-01

Recent work on the aerodynamics of flapping flight reveals fundamental differences in the mechanisms of aerodynamic force generation between fixed and flapping wings. When fixed wings translate at high angles of attack, they periodically generate and shed leading and trailing edge vortices as reflected in their fluctuating aerodynamic force traces and associated flow visualization. In contrast, wings flapping at high angles of attack generate stable leading edge vorticity, which persists throughout the duration of the stroke and enhances mean aerodynamic forces. Here, we show that aerodynamic forces can be controlled by altering the trailing edge flexibility of a flapping wing. We used a dynamically scaled mechanical model of flapping flight (Re ≈ 2000) to measure the aerodynamic forces on flapping wings of variable flexural stiffness (EI). For low to medium angles of attack, as flexibility of the wing increases, its ability to generate aerodynamic forces decreases monotonically but its lift-to-drag ratios remain approximately constant. The instantaneous force traces reveal no major differences in the underlying modes of force generation for flexible and rigid wings, but the magnitude of force, the angle of net force vector and centre of pressure all vary systematically with wing flexibility. Even a rudimentary framework of wing veins is sufficient to restore the ability of flexible wings to generate forces at near-rigid values. Thus, the magnitude of force generation can be controlled by modulating the trailing edge flexibility and thereby controlling the magnitude of the leading edge vorticity. To characterize this, we have generated a detailed database of aerodynamic forces as a function of several variables including material properties, kinematics, aerodynamic forces and centre of pressure, which can also be used to help validate computational models of aeroelastic flapping wings. These experiments will also be useful for wing design for small robotic insects and, to a limited extent, in understanding the aerodynamics of flapping insect wings. PMID:19692394

Low-noise humidity controller for imaging water mediated processes in atomic force microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaponenko, I., E-mail: iaroslav.gaponenko@unige.ch; Gamperle, L.; Herberg, K.

2016-06-15

We demonstrate the construction of a novel low-noise continuous flow humidity controller and its integration with a commercial variable-temperature atomic force microscope fluid cell, allowing precise control of humidity and temperature at the sample during nanoscale measurements. Based on wet and dry gas mixing, the design allows a high mechanical stability to be achieved by means of an ultrasonic atomiser for the generation of water-saturated gas, improving upon previous bubbler-based architectures. Water content in the flow is measured both at the inflow and outflow of the fluid cell, enabling the monitoring of water condensation and icing, and allowing controlled variationmore » of the sample temperature independently of the humidity. To benchmark the performance of the controller, the results of detailed noise studies and time-based imaging of the formation of ice layers on highly oriented pyrolytic graphite are shown.« less

Comprehensive Study of Microgel Electrode for On-Chip Electrophoretic Cell Sorting

NASA Astrophysics Data System (ADS)

Akihiro Hattori,; Kenji Yasuda,

2010-06-01

We have developed an on-chip cell sorting system and microgel electrode for applying electrostatic force in microfluidic pathways in the chip. The advantages of agarose electrodes are 1) current-driven electrostatic force generation, 2) stability against pH change and chemicals, and 3) no bubble formation caused by electrolysis. We examined the carrier ion type and concentration dependence of microgel electrode impedance, and found that CoCl2 has less than 1/10 of the impedance from NaCl, and the reduction of the impedance of NaCl gel electrode was plateaued at 0.5 M. The structure control of the microgel electrode exploiting the surface tension of sol-state agarose was also introduced. The addition of 1% (w/v) trehalose into the microgel electrode allowed the frozen storage of the microgel electrode chip. The experimental results demonstrate the potential of our system and microgel electrode for practical applications in microfluidic chips.

Electromagnetic needles with submicron pole tip radii for nanomanipulation of biomolecules and living cells

NASA Astrophysics Data System (ADS)

Matthews, Benjamin D.; LaVan, David A.; Overby, Darryl R.; Karavitis, John; Ingber, Donald E.

2004-10-01

We describe the design and fabrication of a temperature-controlled electromagnetic microneedle (EMN) to generate custom magnetic field gradients for biomedical and biophysical applications. An electropolishing technique was developed to sharpen the EMN pole tip to any desired radius between 100 nm and 20 μm. The EMN can be used to apply strong static or dynamic forces (>50nN) to micrometer- or nanometer-sized magnetic beads without producing significant heating or needle movement. Large tip radii (20 μm) allow magnetic force application to multiple magnetic beads over a large area, while small radii (0.1-6 μm) can be used to selectively pull or capture single magnetic beads from within a large population of similar particles. The customizable EMN is thus well suited for micro- and nanomanipulation of magnetic particles linked to biomolecules or living cells.

A multi-responsive water-driven actuator with instant and powerful performance for versatile applications

NASA Astrophysics Data System (ADS)

Mu, Jiuke; Hou, Chengyi; Zhu, Bingjie; Wang, Hongzhi; Li, Yaogang; Zhang, Qinghong

2015-03-01

Mechanical actuators driven by water that respond to multiple stimuli, exhibit fast responses and large deformations, and generate high stress have potential in artificial muscles, motors, and generators. Meeting all these requirements in a single device remains a challenge. We report a graphene monolayer paper that undergoes reversible deformation. Its graphene oxide cells wrinkle and extend in response to water desorption and absorption, respectively. Its fast (~0.3 s), powerful (>100 MPa output stress, 7.5 × 105 N kg-1 unit mass force), and controllable actuation can be triggered by moisture, heat, and light. The graphene monolayer paper has potential in artificial muscles, robotic hands, and electromagnetic-free generators.

A simple hanging drop cell culture protocol for generation of 3D spheroids.

PubMed

Foty, Ramsey

2011-05-06

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels or in biomaterial scaffolds. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model. Copyright © 2011 Journal of Visualized Experiments

High-permeability functionalized silicone magnetic microspheres with low autofluorescence for biomedical applications

PubMed Central

Evans, Benjamin A.; Ronecker, Julia C.; Han, David T.; Glass, Daniel R.; Train, Tonya L.; Deatsch, Alison E.

2017-01-01

Functionalized magnetic microspheres are widely used for cell separations, isolation of proteins and other biomolecules, in vitro diagnostics, tissue engineering, and microscale force spectroscopy. We present here the synthesis and characterization of a silicone magnetic microsphere which can be produced in diameters ranging from 0.5 to 50 μm via emulsion polymerization of a silicone ferrofluid precursor. This bottom-up approach to synthesis ensures a uniform magnetic concentration across all sizes, leading to significant advances in magnetic force generation. We demonstrate that in a size range of 5–20 μm, these spheres supply a full order of magnitude greater magnetic force than leading commercial products. In addition, the unique silicone matrix exhibits autofluorescence two orders of magnitude lower than polystyrene microspheres. Finally, we demonstrate the ability to chemically functionalize our silicone microspheres using a standard EDC reaction, and show that our folate-functionalized silicone microspheres specifically bind to targeted HeLa and Jurkat cells. These spheres show tremendous potential for replacing magnetic polystyrene spheres in applications which require either large magnetic forces or minimal autofluorescence, since they represent order-of-magnitude improvements in each. In addition, the unique silicone matrix and proven biocompatibility suggest that they may be useful for encapsulation and targeted delivery of lipophilic pharmaceuticals. PMID:26952493

Evidence of the late lignification of the G-layer in Simarouba tension wood, to assist understanding how non-G-layer species produce tensile stress.

PubMed

Roussel, Jean-Romain; Clair, Bruno

2015-12-01

To recover verticality after disturbance, angiosperm trees produce 'tension wood' allowing them to bend actively. The driving force of the tension has been shown to take place in the G-layer, a specific unlignified layer of the cell wall observed in most temperate species. However, in tropical rain forests, the G-layer is often absent and the mechanism generating the forces to reorient trees remains unclear. A study was carried out on tilted seedlings, saplings and adult Simarouba amara Aubl. trees-a species known to not produce a G-layer. Microscopic observations were done on sections of normal and tension wood after staining or observed under UV light to assess the presence/absence of lignin. We showed that S. amara produces a cell-wall layer with all of the characteristics typical of G-layers, but that this G-layer can be observed only as a temporary stage of the cell-wall development because it is masked by a late lignification. Being thin and lignified, tension wood fibres cannot be distinguished from normal wood fibres in the mature wood of adult trees. These observations indicate that the mechanism generating the high tensile stress in tension wood is likely to be the same as that in species with a typical G-layer and also in species where the G-layer cannot be observed in mature cells. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Live cell imaging of mitochondrial movement along actin cables in budding yeast.

PubMed

Fehrenbacher, Kammy L; Yang, Hyeong-Cheol; Gay, Anna Card; Huckaba, Thomas M; Pon, Liza A

2004-11-23

Mitochondrial inheritance is essential for cell division. In budding yeast, mitochondrial movement from mother to daughter requires (1) actin cables, F-actin bundles that undergo retrograde movement during elongation from buds into mother cells; (2) the mitochore, a mitochondrial protein complex implicated in linking mitochondria to actin cables; and (3) Arp2/3 complex-mediated force generation on mitochondria. We observed three new classes of mitochondrial motility: anterograde movement at velocities of 0.2-0.33 microm/s, retrograde movement at velocities of 0.26-0.51 microm/s, and no net anterograde or retrograde movement. In all cases, motile mitochondria were associated with actin cables undergoing retrograde flow at velocities of 0.18-0.62 microm/s. Destabilization of actin cables or mutations of the mitochore blocked all mitochondrial movements. In contrast, mutations in the Arp2/3 complex affected anterograde but not retrograde mitochondrial movements. Actin cables are required for movement of mitochondria, secretory vesicles, mRNA, and spindle alignment elements in yeast. We provide the first direct evidence that one of the proposed cargos use actin cables as tracks. In the case of mitochondrial inheritance, anterograde movement drives transfer of the organelle from mothers to buds, while retrograde movement contributes to retention of the organelle in mother cells. Interaction of mitochondria with actin cables is required for anterograde and retrograde movement. In contrast, force generation on mitochondria is required only for anterograde movement. Finally, we propose a novel mechanism in which actin cables serve as "conveyor belts" that drive retrograde organelle movement.

Imaging Cell Wall Architecture in Single Zinnia elegans Tracheary Elements1[OA

PubMed Central

Lacayo, Catherine I.; Malkin, Alexander J.; Holman, Hoi-Ying N.; Chen, Liang; Ding, Shi-You; Hwang, Mona S.; Thelen, Michael P.

2010-01-01

The chemical and structural organization of the plant cell wall was examined in Zinnia elegans tracheary elements (TEs), which specialize by developing prominent secondary wall thickenings underlying the primary wall during xylogenesis in vitro. Three imaging platforms were used in conjunction with chemical extraction of wall components to investigate the composition and structure of single Zinnia TEs. Using fluorescence microscopy with a green fluorescent protein-tagged Clostridium thermocellum family 3 carbohydrate-binding module specific for crystalline cellulose, we found that cellulose accessibility and binding in TEs increased significantly following an acidified chlorite treatment. Examination of chemical composition by synchrotron radiation-based Fourier-transform infrared spectromicroscopy indicated a loss of lignin and a modest loss of other polysaccharides in treated TEs. Atomic force microscopy was used to extensively characterize the topography of cell wall surfaces in TEs, revealing an outer granular matrix covering the underlying meshwork of cellulose fibrils. The internal organization of TEs was determined using secondary wall fragments generated by sonication. Atomic force microscopy revealed that the resulting rings, spirals, and reticulate structures were composed of fibrils arranged in parallel. Based on these combined results, we generated an architectural model of Zinnia TEs composed of three layers: an outermost granular layer, a middle primary wall composed of a meshwork of cellulose fibrils, and inner secondary wall thickenings containing parallel cellulose fibrils. In addition to insights in plant biology, studies using Zinnia TEs could prove especially productive in assessing cell wall responses to enzymatic and microbial degradation, thus aiding current efforts in lignocellulosic biofuel production. PMID:20592039

Mechanical control of mitotic progression in single animal cells

PubMed Central

Cattin, Cedric J.; Düggelin, Marcel; Martinez-Martin, David; Gerber, Christoph; Müller, Daniel J.; Stewart, Martin P.

2015-01-01

Despite the importance of mitotic cell rounding in tissue development and cell proliferation, there remains a paucity of approaches to investigate the mechanical robustness of cell rounding. Here we introduce ion beam-sculpted microcantilevers that enable precise force-feedback–controlled confinement of single cells while characterizing their progression through mitosis. We identify three force regimes according to the cell response: small forces (∼5 nN) that accelerate mitotic progression, intermediate forces where cells resist confinement (50–100 nN), and yield forces (>100 nN) where a significant decline in cell height impinges on microtubule spindle function, thereby inhibiting mitotic progression. Yield forces are coincident with a nonlinear drop in cell height potentiated by persistent blebbing and loss of cortical F-actin homogeneity. Our results suggest that a buildup of actomyosin-dependent cortical tension and intracellular pressure precedes mechanical failure, or herniation, of the cell cortex at the yield force. Thus, we reveal how the mechanical properties of mitotic cells and their response to external forces are linked to mitotic progression under conditions of mechanical confinement. PMID:26305930

Cerebrospinal fluid from subarachnoid haemorrhage patients causes excessive oxidative metabolism compared to vascular smooth muscle force generation.

PubMed

Pyne, G J; Cadoux-Hudson, T A; Clark, J F

2001-01-01

Cerebrospinal fluid (CSF) from subarachnoid haemorrhage (SAH) patients can stimulate vascular smooth muscle to generate force in vitro. CSF from SAH patients suffering from delayed ischaemic neurological deficits due to cerebral vasospasm can generate near maximal force in vitro and previous experiments have ascribed this generation of force to be a calcium mediated event. The intracellular calcium concentration has been demonstrated to rise during the vasospastic process. Calcium also stimulates oxidative metabolism as does adenosine diphosphate (ADP), the product of adenosine triphosphate (ATP) hydrolysis. Significant alteration in high energy metabolites such as ATP, ADP and phosphocreatine have also been demonstrated in various models of SAH mediated vasospasm. Vascular smooth muscle predominantly uses oxidative metabolism for force generation and reserves glycolytic metabolism for ion homeostasis. A decrease in oxidative metabolism during force generation would imply failing mitochondria and increased glycolytic high-energy phosphate supply. Increased oxidative metabolism would imply a decreased efficiency of the contractile apparatus or mitochondria. The aim of this study was to see if SAH CSF stimulation of porcine carotid artery oxidative metabolism was altered during force generation when compared with incremental calcium stimulation with potassium chloride depolarisation. CSF from patients (n = 10) who had subarachnoid haemorrhage stimulated force generation but with a significant 'right shift' in oxygen consumption. This 'right shift' is indicative of an increased energy cost for contractile work. These results suggest that vascular smooth muscle contractile apparatus, when stimulated by subarachnoid cerebrospinal fluid, is consuming excess adenosine triphosphate during force generation.

Effect of Slotted Anode on Gas Bubble Behaviors in Aluminum Reduction Cell

NASA Astrophysics Data System (ADS)

Sun, Meijia; Li, Baokuan; Li, Linmin; Wang, Qiang; Peng, Jianping; Wang, Yaowu; Cheung, Sherman C. P.

2017-12-01

In the aluminum reduction cells, gas bubbles are generated at the bottom of the anode which eventually reduces the effective current contact area and the system efficiency. To encourage the removal of gas bubbles, slotted anode has been proposed and increasingly adopted by some industrial aluminum reduction cells. Nonetheless, the exact gas bubble removal mechanisms are yet to be fully understood. A three-dimensional (3D) transient, multiphase flow mathematical model coupled with magnetohydrodynamics has been developed to investigate the effect of slotted anode on the gas bubble movement. The Eulerian volume of fluid approach is applied to track the electrolyte (bath)-molten aluminum (metal) interface. Meanwhile, the Lagrangian discrete particle model is employed to handle the dynamics of gas bubbles with considerations of the buoyancy force, drag force, virtual mass force, and pressure gradient force. The gas bubble coalescence process is also taken into account based on the O'Rourke's algorithm. The two-way coupling between discrete bubbles and fluids is achieved by the inter-phase momentum exchange. Numerical predictions are validated against the anode current variation in an industrial test. Comparing the results using slotted anode with the traditional one, the time-averaged gas bubble removal rate increases from 36 to 63 pct; confirming that the slotted anode provides more escaping ways and shortens the trajectories for gas bubbles. Furthermore, the slotted anode also reduces gas bubble's residence time and the probability of coalescence. Moreover, the bubble layer thickness in aluminum cell with slotted anode is reduced about 3.5 mm (17.4 pct), so the resistance can be cut down for the sake of energy saving and the metal surface fluctuation amplitude is significantly reduced for the stable operation due to the slighter perturbation with smaller bubbles.

N1N8-bis(gamma-glutamyl)spermidine cross-linking in epidermal-cell envelopes. Comparison of cross-link levels in normal and psoriatic cell envelopes.

PubMed Central

Martinet, N; Beninati, S; Nigra, T P; Folk, J E

1990-01-01

N1N8-Bis(gamma-glutamyl)spermidine was found in exhaustive proteolytic digests of isolated cell envelopes from human epidermis at levels comparable with those of epsilon-(gamma-glutamyl)lysine. Significantly higher than normal amounts of these compounds, particularly the bis(gamma-glutamyl)polyamine, were observed in envelopes from afflicted areas (scales) of psoriatic patients. These findings support the notions that N1N8-bis(gamma-glutamyl)spermidine, like epsilon-(gamma-glutamyl)lysine, functions in cell envelopes as an enzyme-generated protein cross-link and stabilizing force and that individuals with the chronic, recurrent skin disease, psoriasis, exhibit in involved epidermis abnormal cell-envelope-protein cross-linking. PMID:2241917

Prestressed F-actin networks cross-linked by hinged filamins replicate mechanical properties of cells

NASA Astrophysics Data System (ADS)

Gardel, M. L.; Nakamura, F.; Hartwig, J. H.; Crocker, J. C.; Stossel, T. P.; Weitz, D. A.

2006-02-01

We show that actin filaments, shortened to physiological lengths by gelsolin and cross-linked with recombinant human filamins (FLNs), exhibit dynamic elastic properties similar to those reported for live cells. To achieve elasticity values of comparable magnitude to those of cells, the in vitro network must be subjected to external prestress, which directly controls network elasticity. A molecular requirement for the strain-related behavior at physiological conditionsis a flexible hinge found in FLNa and some FLNb molecules. Basic physical properties of the in vitro filamin-F-actin network replicate the essential mechanical properties of living cells. This physical behavior could accommodate passive deformation and internal organelle trafficking at low strains yet resist externally or internally generated high shear forces. cytoskeleton | cell mechanics | nonlinear rheology

A simple force-motion relation for migrating cells revealed by multipole analysis of traction stress.

PubMed

Tanimoto, Hirokazu; Sano, Masaki

2014-01-07

For biophysical understanding of cell motility, the relationship between mechanical force and cell migration must be uncovered, but it remains elusive. Since cells migrate at small scale in dissipative circumstances, the inertia force is negligible and all forces should cancel out. This implies that one must quantify the spatial pattern of the force instead of just the summation to elucidate the force-motion relation. Here, we introduced multipole analysis to quantify the traction stress dynamics of migrating cells. We measured the traction stress of Dictyostelium discoideum cells and investigated the lowest two moments, the force dipole and quadrupole moments, which reflect rotational and front-rear asymmetries of the stress field. We derived a simple force-motion relation in which cells migrate along the force dipole axis with a direction determined by the force quadrupole. Furthermore, as a complementary approach, we also investigated fine structures in the stress field that show front-rear asymmetric kinetics consistent with the multipole analysis. The tight force-motion relation enables us to predict cell migration only from the traction stress patterns. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Mechanochemical Polarization of Contiguous Cell Walls Shapes Plant Pavement Cells.

PubMed

Majda, Mateusz; Grones, Peter; Sintorn, Ida-Maria; Vain, Thomas; Milani, Pascale; Krupinski, Pawel; Zagórska-Marek, Beata; Viotti, Corrado; Jönsson, Henrik; Mellerowicz, Ewa J; Hamant, Olivier; Robert, Stéphanie

2017-11-06

The epidermis of aerial plant organs is thought to be limiting for growth, because it acts as a continuous load-bearing layer, resisting tension. Leaf epidermis contains jigsaw puzzle piece-shaped pavement cells whose shape has been proposed to be a result of subcellular variations in expansion rate that induce local buckling events. Paradoxically, such local compressive buckling should not occur given the tensile stresses across the epidermis. Using computational modeling, we show that the simplest scenario to explain pavement cell shapes within an epidermis under tension must involve mechanical wall heterogeneities across and along the anticlinal pavement cell walls between adjacent cells. Combining genetics, atomic force microscopy, and immunolabeling, we demonstrate that contiguous cell walls indeed exhibit hybrid mechanochemical properties. Such biochemical wall heterogeneities precede wall bending. Altogether, this provides a possible mechanism for the generation of complex plant cell shapes. Copyright © 2017 Elsevier Inc. All rights reserved.

The G1 phase Cdks regulate the centrosome cycle and mediate oncogene-dependent centrosome amplification

PubMed Central

2011-01-01

Because centrosome amplification generates aneuploidy and since centrosome amplification is ubiquitous in human tumors, a strong case is made for centrosome amplification being a major force in tumor biogenesis. Various evidence showing that oncogenes and altered tumor suppressors lead to centrosome amplification and aneuploidy suggests that oncogenes and altered tumor suppressors are a major source of genomic instability in tumors, and that they generate those abnormal processes to initiate and sustain tumorigenesis. We discuss how altered tumor suppressors and oncogenes utilize the cell cycle regulatory machinery to signal centrosome amplification and aneuploidy. PMID:21272329

Contribution of actin filaments to the global compressive properties of fibroblasts.

PubMed

Ujihara, Yoshihiro; Nakamura, Masanori; Miyazaki, Hiroshi; Wada, Shigeo

2012-10-01

Actin filaments are often regarded as tension-bearing components. Here, we examined the effects of actin filaments on global compressive properties of cells experimentally and numerically. Fibroblasts were harvested from the patellar tendon of a mature Japanese white rabbit and treated with cytochalasin D to depolymerize the actin filaments. Intact cells and cells with disrupted actin filaments were subjected to the compressive tests. Each floating cell was held between the cantilever and compressive plates and compressed by moving the compressive plate with a linear actuator to obtain a load-deformation curve under quasi-static conditions. The experimental results demonstrated that the initial stiffness of a cell with disrupted actin filaments decreased by 51%. After the experiments, we simulated the compressive test of cells with/without bundles of actin filaments. A bundle of actin filaments was modeled as a tension-bearing component that generates a force based on Hooke's law only when it was elongated. By contrast, if it was shortened, it was assumed to exert no force. The computational results revealed that the alignment of bundles of actin filaments significantly affected the cell stiffness. In addition, the passive reorientation of bundles of actin filaments perpendicular to the compression induced an increase in the resistance to the vertical elongation of a cell and thereby increased the cell stiffness. These results clearly indicated that bundles of actin filaments contribute to the compressive properties of a cell, even if they are tension-bearing components. Copyright © 2012 Elsevier Ltd. All rights reserved.