Vinculin-GFP-Plasmid war ein Geschenk von Kenneth Yamada, National Institutes of Health, National Institute of Dental und kraniofaziale Research, Bethesda, Maryland, und Aktin-mRFP Plasmid war ein Geschenk von Michael Davidson, Florida State University, Tallahassee, Florida. Diese Arbeit wurde von der NSF Career Award # 0747334 und AHA-National SDG # 0835205N zu Andreea Trache unterstützt.

1. Übersicht über die Integrierte Mikroskop-System Das Mikroskop-System, das für diese Studien verwendet wird, hat in detail 1 beschrieben worden. Kurz gesagt, ist eine umgekehrte Olympus IX-81-Mikroskop mit TIRF Befestigung (Olympus, Center Valley, PA) in Kombination mit CSU-22 Yokogawa Abtastkopf (Yokogawa Electric Inc, Japan). Ein Bioscope SZ Rasterkraftmikroskop (Veeco Instruments Inc, Santa Barbara, CA) ist auf der Oberseite des optischen inverse Mikroskop montiert, und das gesamte System ist auf der Spitze eines Forschungs-grade optischen Tisch (Newport, Irvine, CA) platziert. Das konfokale Montage von der Spinnerei-Festplatten-Scanner, externe Filterrad und seine EMCCD Kamera (Roper Scientific-Photometrics, Tucson, AZ) zusammengesetzt sind fest mit einer Bodenplatte aus Aluminium auf einem Silizium-Pad erforderlich zur Schwingungsdämpfung angebracht verbunden. Das konfokale Scanner verbindet mit dem Mikroskop durch einen Flansch mit einem profilierten Kragen, der aus dem Mikroskop bei AFM-Messungen können entkoppelt werden, so dass keine Vibrationen von der Spinnerei-Disk-Scanner wird die AFM-Messungen beeinflussen. Ein Argon-Krypton-Laser (Spectra Physics, Mountain View, CA) als Anregungsquelle für TIRF und konfokale Bildgebung Modi verwendet, und kann alternativ in zwei optische Fasern, die den Laser liefern die TIRF-Anlage oder der Abtastkopf gekoppelt werden, bzw. . Zwei Computer die Steuerung des Systems: Slidebook Software steuert die optische Bildgebung und Nanoscope Software steuert die AFM. 2. Cell Culture Lebende Zellen, die GFP-Konstrukte gezielt gegen bestimmte Proteine ​​sollten in einem Glasboden Zellkulturschale platziert 24 Stunden vor der mechanische Stimulation Experiment sein. Am Tag des Experiments ersetzen Zellkulturmedium mit Phenolrot-freiem Medium. Die Zellkulturschale auf der AFM Bühne mit einer magnetischen Kragen befestigt. 3. AFM-Spitze Vorbereitung und Mikroskop Set-up Die AFM-Spitze mit einem 2-Mikron angepasst biotinylierten Glasperlen fünfmal mit DPBS gewaschen und dann inkubiert 5 min mit Avidin (1 mg / ml), erneut gewaschen mit DPBS fünfmal gewaschen und danach 5 min mit 1 mg / mL biotinylierten Fibronektin. Die Spitze wird noch fünfmal am Ende 2 gewaschen werden, montiert auf dem AFM-Scanner, und dann die schützende Silizium Rock wird rund um die Halter platziert werden. Set dem Mikroskop für Video-Anzeigen auf der AFM-Spitze in der Mitte des Sichtfeldes auszurichten. Richten Sie die optischen Hebel, indem der Laserstrahl am Ende des Auslegers, und ändern Sie das Ziel, eine hohe Vergrößerung Ziel richtige für Einzel-cell imaging. Die Federkonstante der Spitze hat vor dem Start des Experiments bestimmt werden. Dieser Parameter wird unter Verwendung der thermischen tune-Methode in den Nanoscope Software. Wechseln Sie von Videokamera auf EMCCD Kamera für Fluoreszenz Imaging ermöglichen. Dann wählen Sie eine Zelle und bringen die AFM-Spitze in Kontakt mit dieser bestimmten Zelle. Nach 20 min Wartezeit, die für eine starke fokale Adhäsion an der AFM Berührungspunkt mit der Zelloberfläche Form ist, wird die mechanische Stimulation zu starten. 4. Mechanische Stimulation des Live Cell TIRF Bildgebung 3-4 wird die Methode der Wahl für die Visualisierung und Quantifizierung von fokalen Adhäsionen, und Spinning-Disk-konfokale Abbildung 5 wird für die Visualisierung des Aktin-Zytoskeletts während der Zellkörper verwendet werden. Die AFM 6 wird in contact mode Imaging unter Flüssigkeit verwendet werden. Ein TIRF oder konfokalen Bild der Zelle wird vor Beginn der mechanische Stimulation Verfahren erworben werden. Die mechanische Stimulation wird durch die funktionalisierte AFM-Sonde, die nach oben wird in diskreten Schritten verschoben werden alle 3-5 min über einen Zeitraum von Zeit (1-1.5 Stunden) induziert werden. Die Zelle reaktive-Reaktion wird kontinuierlich in Nanoscope Software als eine Reihe von hoch Bilder, jede Linie im Bild entspricht einer Zeiteinheit von 1 Sekunde aufgezeichnet werden. Während den Erwerb der AFM-Daten, überwacht der Betreiber jederzeit die z-Position der AFM-Spitze, um das richtige Timing für den nächsten Zug zu ermitteln. Nach jeder mechanische Stimulation ist es möglich, eine TIRF oder konfokalen Bild der Zelle in Slidebook Software erwerben, während er kontinuierlich den Erwerb AFM-Daten. Die optischen Bilder können off-line verarbeitet werden, um Filme, die Aktin-Zytoskelett und fokalen Adhäsionen Reorganisation zeigen zu schaffen. Darüber hinaus kann jedes Bild quantitativ verarbeitet werden, um Zellfläche, spezifische Protein-Bereich zu ermitteln und analysieren die allgemeine Morphologie der Zellen. Am Ende des Experiments die AFM-Spitze aus der Zelle und AFM-Spitze Empfindlichkeit zurückgenommen wird, wird in der Nanoscope Software gemessen werden. Das Experiment ist sehr Lärm empfindlich! Alle reden oder Mikroskop zu berühren bei der ExExperiment sollte vermieden werden. Zur Minimierung der Lärm in der AFM-Messungen sollte der Spinn-disk konfokalen vom Mikroskop entkoppelt werden. Die Wartezeit auf eine starke fokale Adhäsion zwischen Zelloberfläche und die AFM-Spitze bilden kann je nach verwendetem Zelltyp und der Matrix beschichtet an der Spitze. 5. Repräsentative Ergebnisse <img src="/files/ftp_upload/2072/2072fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Gleiche glatten Gefäßmuskelzellen (VSMC) mit Aktin-mRFP und Vinculin-GFP transfiziert wurde durch abgebildet: (A) AFM an der apikalen Zelloberfläche; (B) TIRF am basalen Zelloberfläche, und (C) Spinning-Disk-konfokale Mikroskopie in der gesamten Zellkörper. Das konfokale Bild wird als maximale Projektion eines 3-D-Stack gezeigt. Gute Überschneidungen zwischen den AFM-und TIRF oder Spinnen-Disk-konfokale Bilder sind in (D) und (E), jeweils dargestellt. Bildgröße beträgt 64 x 50 micron. <img src="/files/ftp_upload/2072/2072fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Ein VSMC mit Aktin-mRFP transfiziert wurde mechanisch durch die AFM angeregt und abgebildet durch Spinnen-disk konfokale Mikroskopie Die Pfeile zeigen Aktinfasern dass:. (A) polymerisieren / verdicken, (b) Depolymerisation / verschwinden, oder (c) bündeln durch an Aktin Umstrukturierung. Weiß gestrichelte Linien stellen die AFM-Spitze über die Zelle. Bildgröße 91 x 91 micron.

<ol>
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Zytoskelett 7-10 und 11-16 fokalen Adhäsionen sind dynamische Strukturen, die, montieren verteilen und umdrehen, wie Zellen wandern oder reagieren auf mechanische Kraft 17. Sie haben wichtige Funktionen bei der Regulation des Zellwachstums, das Überleben und die Genexpression. Das Wissen ihrer spezifischen Verteilung und Dynamik ermöglicht ein besseres Verständnis, wie Zellen mit der Matrix und untereinander zu kommunizieren. Das neue Mikroskop-System ist...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
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<td>AFM tip</td>
<td>&nbsp;</td>
<td>Novascan Technologies</td>
<td>&nbsp;</td>
<td>2 micron Borosilicate bead, 0.01N/m, Biotin Surface</td>
</tr>
<tr>
<td>avidin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>A9275</td>
<td>1mg/mL in DPBS</td>
</tr>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>F4759</td>
<td>1 mg/mL</td>
</tr>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21600-051</td>
<td>&nbsp;</td>
<tr>
<td>Amaxa transfection kit for Smooth Muscle Cells</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>VPI-1004</td>
<td>&nbsp;</td>
<tr>
<td>Mattek glass bottom dishes</td>
<td>&nbsp;</td>
<td>MatTek Corporation</td>
<td>P60G-1.5-30-F</td>
<td>Sterile 60mm Falcon dishes with glass bottom coverslip no. 1.5</td>
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live cell response to mechanical stimulation studied by integrated optical and atomic force microscopy

Um zu verstehen, den Mechanismus, mit dem sich lebende Zellen Sinn mechanische Kräfte, und wie sie zu reagieren und an ihre Umgebung anpassen, eine neue Technologie in der Lage, Zellen Verhalten bei sub-zellulärer Ebene zu untersuchen mit hoher räumlicher und zeitlicher Auflösung entwickelt wurde. So wurde ein Rasterkraftmikroskop (AFM) mit Total Internal Reflection Fluorescence (TIRF) Mikroskopie und schnell drehenden Scheibe (FSD) konfokale Mikroskopie integriert. Das integrierte System ist breit anwendbar für eine Vielzahl von molekularen dynamischen Studien in anhaftendem lebenden Zellen, die eine direkte optische Bildgebung von Zell-Reaktionen auf mechanische Reize in Echtzeit. Signifikante Umlagerung des Aktin-Filamente und fokalen Adhäsionen war aufgrund der örtlichen mechanische Stimulation an der apikalen Zelloberfläche, dass Veränderungen in der Zellstruktur im gesamten Zellkörper induzierte gezeigt. Diese innovative Technik wird neue Informationen zum Verständnis lebender Zellen Umstrukturierung und Dynamik in Reaktion auf die mechanische Kraft. Ein detailliertes Protokoll sowie ein Vertreter Datensatz, zeigen Live-Zell-Antwort auf mechanische Reize präsentiert werden.

Watch this Scientific Journal Video about Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy at JoVE.com

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

Dieses Papier soll den Leser in den Betrieb eines integrierten atomic force-optische Bildgebung Mikroskop für die mechanische Stimulation von lebenden Zellen in Kultur zu unterrichten. Eine Schritt-für-Schritt-Protokoll vorgestellt. Eine repräsentative Datenmenge, die Live-Zell-Antwort auf mechanische Stimulation zeigt vorgestellt.

Live Cell Reaktion auf mechanische Stimulation von Integrated Optical und Atomic Force Microscopy Studium

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

live-cell-response-to-mechanical-stimulation-studied-integrated

Research

JoVE Journal

Biology

11.2K Aufrufe.  Texas A&M Health Science Center. Dieses Papier soll den Leser in den Betrieb eines integrierten atomic force-optische Bildgebung Mikroskop für die mechanische Stimulation von lebenden Zellen in Kultur zu unterrichten. Eine Schritt-für-Schritt-Protokoll vorgestellt. Eine repräsentative Datenmenge, die Live-Zell-Antwort auf mechanische Stimulation zeigt vorgestellt.

Serie: Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Phasenkontrast und Differential Interferenz Kontrast (DIC) Mikroskopie

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Forschung

Biologie

Presynaptically Silent Synapses Studied with Light Microscopy

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise damaging insults from becoming neurotoxic.  We have been studying a form of presynaptic plasticity that is interesting in part because it is expressed as a digital switching on and off of a presynaptic terminal s ability to release vesicles containing the neurotransmitter glutamate.  Here we demonstrate a protocol for visualizing the activity status of presynaptic terminals in dissociated cell cultures prepared from the rodent hippocampus.  The method relies on detecting active synapses using staining with a fixable form of the styryl dye FM1-43, commonly used to label synaptic vesicles.  This staining profile is compared with immunostaining of the same terminals with an antibody directed against the vesicular glutamate transporter 1 (vGluT-1), a stain designed to label all glutamate synapses regardless of activation status.   We find that depolarizing stimuli induce presynaptic silencing.  The population of synapses that is silent under baseline conditions can be activated by prolonged electrical silencing or by activation of cAMP signaling pathways.

Glutamatergic synapses can switch from an active mode to a silent mode. We demonstrate that presynaptic activity status in dissociated culture of rodent neurons is visualized using a fixable form of the FM1-43 dye to visualize active synapses and immunostaining with vGluT-1 antibody to visualize all glutamate synapses.

Präsynaptisch Stille Synapsen mit Lichtmikroskopie Studium

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

Micro-Mechanische Charakterisierung von Lungengewebe mit Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Probenvorbereitung für Single Virion Atomic Force Microscopy und Super-Resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In-vivo-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Messung der Steifigkeit von<em&gt; Ex Vivo</em&gt; Maus Aorten Mit Rasterkraftmikroskopie

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Glukoseaufnahme Messung und Response auf Insulin Stimulation in<em&gt; In vitro</em&gt; Kultivierte menschliche primäre Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Bewertung von Dictyostelium Discoideum Reaktion auf akute mechanische Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

Vorsprung-Kraft-Mikroskopie: Eine Methode, um Kräfte entwickelt von Zelle Vorsprünge zu quantifizieren

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

Charakterisierung mechanischer Eigenschaften der primären Zellwand in lebenden Pflanzenorganen mittels Rasterkraftmikroskopie

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...

Methods Article

Live Cell Reaktion auf mechanische Stimulation von Integrated Optical und Atomic Force Microscopy Studium