Yazarlar ilk katkılarından dolayı bu eser ve Matthieu Sanchez ve Françoise Viala için video filme ve düzenleme ile yardım için Anna Labernadie, Guillaume Charrière ve Patrick Delobelle için minnettarız. Bu eser l'Agence Nationale de la Recherche (ANR14-CE11-0020-02), la Fondation pour la Recherche Médicale (FRM DEQ2016 0334894), INSERM planı kanser, Fondation Toulouse kanser ve insan sınır bilim programı (RGP0035/2016) tarafından desteklenmiştir.

1. Formvar kaplamalı ızgaralar hazırlanması
<ol><li>Elektron mikroskobu ızgaralar saf aseton ile temiz ve kuru onları filtre kağıt üzerinde. Sonra temiz bir mikroskop slayt saf etanol ile objektif kağıt ile silin ve toz bir üfleyici ile kaldırın.</li>
	<li>Bir etanol temizledim cam slayt film döküm cihazın alt kısmındaki Formvar çözeltilerine içeren huni dikey olarak yerleştirin. Huni üst kapak.</li>
	<li>Düzeyi üçte ikisi slayt ulaşıncaya kadar 100 mL Formvar çözeltisi (etilen ve %0,5) atomizer ampul ile pompa.</li>
	<li>Slayt 1 dk. için çözüm unutmayın.</li>
	<li>Aygıt bir akışı Formvar çözümünde drenaj valfi açın. Bir akış hızı 10 15 mL/s 30-80 nm Formvar film ortaya çıkarır. Film kalınlığı Formvar çözüm konsantrasyonu ve drenaj oranına göre belirlenir. Not: Drenaj oranı tarafından havalandırma basınç yoluyla hava-out Vana Vana yavaş yavaş açarak kontrol edilebilir. Daha hızlı drenaj, kalın film. Uygulamada, film kalınlığı Formvar akış hızı düşük tahmin etmek zordur çünkü biz Formvar filmlerin birkaç toplu hazırlamak ve onların kalınlığı AFM tarafından kontrol (adım 2 bakınız).</li>
	<li>Slayt odasından kaldırın ve kibar bir şekilde herhangi bir sıvı Formvar kaldırmak için filtre kağıdına kuru.</li>
	<li>20 mm x 50 mm dikdörtgen bir tıraş bıçağı ile Formvar filmden kesip.</li>
	<li>Bir ölçek temiz distile su ile doldurun ve yavaş yavaş slayt dikey olarak filmin yüzer suya dalma: film su yüzeyine float.</li>
	<li>Yer kuru aseton temizlenmiş ızgaralar üzerinde film, parlak ön yüz yukarıda.</li>
	<li>Yuvarlak cam coverslip (Ø 12 mm) birkaç ızgaralar yerleştirin. Bu coverslip film kalınlığı ölçmek için hizmet edecek (adım 2 bakınız).</li>
	<li>Bir mikroskop slayt sığdırmak için yeniden boyutlandırılmış bir beyaz sopa ile kapak. Bütün film için slayt bağlıdır kadar slayt dikey kayan Formvar film sınırında daldırma. Aşırı su filtre kağıdı ile slayt kaldırmak ve oda sıcaklığında gecede kurumaya bırakın.</li>
</ol>2. Film kalınlığı ölçümü
<ol><li>Coverslip çevresinde Formvar slayttan ayırmak için cımbız ile kesti.</li>
	<li>Coverslip altında kılavuzunun bir kalemle işaretlemek.</li>
	<li>Coverslip ayırmak için Formvar işaretli kılavuz çevresinde kesin. Bu nedenle film kalınlığı ölçmek için izin verir kalan Formvar sayfasının içinde bir delik olacak. Coverslip PBS ile kapak ve bir cam slayt mikroskop üzerinde yerleştirin.</li>
	<li>Bir silikon nitrür prensibine göre altın şerit bahar ipucu tarafından kapsanmayan emin AFM cam blok bağlama. Not: Duyarlılığı ve bahar sabiti prensibine göre daha önce PBS içinde kalibre varsa.</li>
	<li>Cam blok AFM modülü üzerine monte ve modül mikroskop üzerine yerleştirin.</li>
	<li>Formvar kaplı coverslip Gözlem odası yerleştirin ve PBS 500 µL ile kapağı.</li>
	<li>Formvar sayfası AFM iletişim modu ve 0.5 nN kuvvet ayar noktası olarak kullanarak delik kenarlığını inceden inceye gözden geçirmek.</li>
	<li>Cam coverslip yüzey boy ölçümleri için başvurmak ve Formvar kalınlık kesit yüksekliğini olarak değerlendirmek (Formvar toplu iş başına en az 10 ölçümleri gerçekleştirmek).</li>
</ol>3. tohum hücreleri üzerinde ızgaralar
<ol><li>Steril bir başlık altında bir şerit (12 mm x 3 mm) çift taraflı yapışkan bant bir coverslip koy.</li>
	<li>Formvar kaplı ızgara cımbızla ve (sadece ızgara kenarında teybe bağlı olması gerekir) yapıştırıcı şerit üzerinde ters koydu. Formvar film coverslip karşısında gereken.</li>
	<li>Bu cihaz bir kültürde de koy.</li>
	<li>Yer RPMI 104 makrofajlar içeren FCS olmadan bir 10 µL damlacık insan monosit16' dan farklı. Not: Formvar kaplama zarar önlemek için pipet ucu kılavuzla dokunmak emin olun.</li>
	<li>30 dk (37 ° C, % 5 CO2) uygun hücreleri izin vermek için kuluçkaya.</li>
	<li>Kadar iyi 2 mL % 10 FCS içeren RPMI ile doldurun.</li>
	<li>Aygıt hücreleri AFM gözlem önce uygun izin için 37 ° C ve % 5 CO2 2 h için kuluçkaya.</li>
</ol>4. Podosome kaynaklı deformasyonlar topografya ölçümleri
<ol><li>İki katına taraflı yapışkan bant iki şeritler bir cam alt Petri kabına sadık. Not: İki şeritler arasındaki mesafe kılavuz çapı daha küçük olmalıdır.</li>
	<li>Dikkatle makrofajlar yapışkan bandı üzerinden ile kaplama Kılavuzu bağlantısını kesin.</li>
	<li>Izgarayı cam dış yüzey hücreleri için ters çevirmek ve Izgarayı iki yapışkan şeritleri arasında sopa.</li>
	<li>Izgarayı iki ile yeni iki katına taraflı yapışkan şeritler Düzeltme: kılavuz böylece merkezi alan kılavuz AFM ipucu için erişilebilir bırakarak iki yapışkan şeritleri arasında gözükeceksin. Not: ızgara tamir ve değil bükülmüş emin olun; Aksi takdirde AFM prensibine göre Formvar yüzey yaklaşmak mümkün olmayabilir.</li>
	<li>Petri kabına 10 mM HEPES (pH 7,4) ile desteklenmiş önceden ısıtılmış 37 ° C Kültür ortamının (RPMI % 10 FCS ile) 2 mL ile doldurun.</li>
	<li>Kültür tabak bir 37 ° C yemek ısıtıcı yerleştirin.</li>
	<li>Yemek ısıtıcı AFM sahne alanı'nda yükleyin.</li>
	<li>37 ° C'de stabilize sistem izin</li>
	<li>Kişi modu bir 0.5 nN kuvvetle podosomes bulmak için bir ilk küresel görüntü olun ve 3 Hz değerinde Formvar topografya ile yaklaşık 20 nm büyük pixel inceden inceye gözden geçirmek. Not: ızgara kenarlarına tarama kaçının.</li>
</ol>

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Hiçbir çıkar çatışmaları ilan etti.

Malzeme özellikleri
Formvar, bizim durumumuzda deforme membran için malzeme seçimi birkaç şartları yerine getirmek gerekiyor. Malzeme için görünür ışık şeffaf olması ve gözlemler parlak alan ve floresan mikroskopi izin vermek için sınırlı otomatik floresans sergilemek gerekir. İnce film pürüzlülüğü de 10 olmalıdır nm açık hücre kaynaklı çıkıntılar gözlenmesi AFM görüntüleme tarafından izin veren ve hücre adezyon topografik herhangi bi...

Hayvan hücrelerinin fiziksel olarak matrix ve onların çevre1oluşturan diğer hücreleri ile etkileşim. Bu onlar için geçirme, cesetleri içselleştirmesi, dış bilgi edinme veya ayırt etmek gereklidir. Bu tür işlemlerde, mekanik Kuvvetleri hücre oluşturmak için gereken ve çok sayıda çalışma son yıllarda gösterdiği gibi kuvvetler oluşturmak ve çevresine yoklama için hücre yeteneği Örneğin nükleer silahların yayılmasına karşı yönlendiren biyolojik davranışını etkiler veya farklılaşma2,3. Buna karşılık, hücresel kuvvetler ölçümü kuvvet oluşturma Yönetmeliği çalışma ve onun dolaylı hücre davranış ve doku kader4,5anlamak için bir büyük yardımcısıdır.
Son yıllarda bir hücre üzerinde onun çevre6uygulamayın kuvvetleri ölçmek için yöntemlerin geliştirilmesi sayesinde çok sayıda tanık olduk. Bunların çoğunluğu onlar mobil probları veya deforme substrat çekmek gibi hücreleri sarfetmek çekiş güçleri açığa vesile olmuştur. Ancak, mekanik Kuvvetleri çıkıntı hücre dışı ortama dahil ölçüm teknikleri bir eksikliği muzdarip ve iyi değil karakterize bugüne kadar.
Bu sınırlamayı aşmak için uygun belgili tanımlık substrate sarf kuvvetleri ölçmek için bir yöntem mevcut. Bu canlı hücreler hücreleri tarafından yüzey deformasyon ölçmek ve katılan kuvvetleri anlamak mümkün hale dik yönde deforme ince bir elastik levha üzerinde kaplama oluşur. Substrat topografya atomik kuvvet mikroskobu kullanarak nano çözünürlüğü ile ölçülür ve deformasyon kuvvetlerden değerlendirilmesi protrusive hücresel yapılar7,8, geometri bilgisine dayanıyor 9.
Burada, kurulum ve uygulama podosomes, protrusive yapışma yapıları için üç boyutlu ortamlarda10,11mezenkimal onların göç makrofajlar tarafından kurulan tarafından oluşturulan kuvvetleri ölçmek için açıklamak, 12,13,14,15,16,17. Bu teknik kuvvet oluşturma ve çıkıntı içeren birçok hücresel süreçler, Yönetmelikte anlayışı peşin olacak inanıyorum.

<table><tbody><tr><td>200 mesh nickel grids</td><td>Electron Microscopy Sciences</td><td>G200-Ni</td><td></td></tr><tr><td>Filter paper</td><td>Sigma-Aldrich</td><td>1001-055</td><td></td></tr><tr><td>Microscope slides</td><td>Fisher Scientific</td><td>10235612</td><td></td></tr><tr><td>White stickers 26 x 70 mm</td><td>Avery</td><td>DP033-100</td><td></td></tr><tr><td>Film casting device with valve in its outlet</td><td>Electron Microscopy Sciences</td><td>71305-01</td><td></td></tr><tr><td>Razorblades</td><td>Electron Microscopy Sciences</td><td>72000</td><td></td></tr><tr><td>Ethanol</td><td>VWR</td><td>1.08543.0250</td><td></td></tr><tr><td>Acetone</td><td>VWR</td><td>20066.321</td><td></td></tr><tr><td>Formvar 0.5% solution in ethylene dichloride</td><td>Electron Microscopy Sciences</td><td>15820</td><td></td></tr><tr><td>12 mm coverslips</td><td>VWR</td><td>631-0666</td><td></td></tr><tr><td>Inverted microscope</td><td>Carl Zeiss</td><td>Axiovert 200</td><td></td></tr><tr><td>Atomic Force Microscope</td><td>JPK Instruments</td><td>NanoWizard III</td><td></td></tr><tr><td>Temperature-controlled sample holder&amp;nbsp;</td><td>JPK Instruments</td><td>BioCell</td><td></td></tr><tr><td>Silicon nitride cantilever with a nominal spring constant of 0.01 N/m</td><td>Veeco Instruments</td><td>MLCT-AUHW</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Double-sided adhesive tape</td><td>APLI AGIPA</td><td>118100</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>31870-025</td><td></td></tr><tr><td>FCS</td><td>Sigma-Aldrich</td><td>F7524</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H0887</td><td></td></tr><tr><td>35 mm glass-bottom Petri dishes</td><td>WPI</td><td>FD35-100</td><td></td></tr></tbody></table>

protrusion force microscopy: a method to quantify forces developed by cell protrusions

Çok sayıda biyolojik bağlamlarda hayvan hücrelerinin fiziksel olarak mekanik Kuvvetleri geliştirerek onların çevre ile etkileşim gerekir. Bunlar arasında çekiş güçleri iyi karakterize olmuştur, ama teknikleri onların substrat uygun hücrelere tarafından sarf çıkıntı kuvvetler ölçümü izin veren bir eksikliği olduğunu. Biz onların yüzey üzerinde yapışık hücreleri tarafından sarf çıkıntı kuvvetleri ölçmek için deneysel bir kurulum tasarlanmıştır. Bu yüzey hücreleri uyumlu Formvar kağıt üzerinde kaplama deforme ve elde edilen topografya nanometre ölçeğinde atomik kuvvet mikroskobu (AFM) tarafından eşleştirilir. Kuvvet değerleri daha sonra protrusive hücresel yapılar geometri üzerinde dayalı deformasyon profil bir analiz ayıklanır. Bu nedenle, bir yaşam hücre bireysel çıkıntılı birimleri tarafından sarf kuvvetleri zamanla ölçülebilir. Bu teknik, kuvvet oluşturma ve çıkıntı içeren birçok hücresel süreçler, Yönetmelikte sağlayacaktır. Burada, insan makrofajlar tarafından kurulan podosomes tarafından oluşturulan protrusive kuvvetleri ölçmek için uygulama açıklar.

Yukarıdaki Protokolü makrofaj podosomes Formvar substrat üzerine tarafından uygulanan çıkıntı kuvvetleri ölçmek için deneysel kurulum hazırlamak açıklar. Bu AFM kullanarak elde edilir ve Şekil 1' de gösterilmiştir.
JPK veri işleme yazılımı kullanarak podosomes altında çıkıntı topografik bir görüntüsünü analiz ederken, uygun bir üçüncü derece polinom her tarama sat?...

Watch this Scientific Journal Video about Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions at JoVE.com

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

Burada, podosomes uyumlu bir filmde filmin topografik görüntülerin otomatik Analize hazırlanması uygulamak çıkıntı kuvvetleri değerlendirmek için kullanılan Deneysel teknikler ayrıntılı.

Çıkıntı kuvvet mikroskobu: Hücre çıkıntılar tarafından geliştirilen kuvvetleri ölçmek için bir yöntem

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

protrusion-force-microscopy-method-to-quantify-forces-developed-cell

Research

JoVE Journal

Biology

5.5K Görüntüleme.  IPBS, Université de Toulouse, CNRS, UPS. Burada, podosomes uyumlu bir filmde filmin topografik görüntülerin otomatik Analize hazırlanması uygulamak çıkıntı kuvvetleri değerlendirmek için kullanılan Deneysel teknikler ayrıntılı.

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

A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous with the cytoskeleton, methods are needed to probe its mechanical behavior in adherent cells. Here, we discuss the direct force probe (DFP) as a tool to apply force directly to the nucleus in a living adherent cell. We attach a narrow micropipette to the nuclear surface with suction. The micropipette is translated away from the nucleus, which causes the nucleus to deform and translate. When the restoring force is equal to the suction force, the nucleus detaches and elastically relaxes. Because the suction pressure is precisely known, the force on the nuclear surface is known. This method has revealed that nano-scale forces are sufficient to deform and translate the nucleus in adherent cells, and identified cytoskeletal elements that enable the nucleus to resist forces. The DFP can be used to dissect the contributions of cellular and nuclear components to nuclear mechanical properties in living cells.

In this protocol, we describe a micropipette method to directly apply a controlled force to the nucleus in a living cell. This assay allows interrogation of nuclear mechanical properties in the living, adherent cell.

Çekirdek ve sitoiskeleti mekanik bütünleşmesi ölçmek için bir doğrudan kuvvet sonda

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous ...

Biyokimya

Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular stress and traction generation is currently unknown. Therefore, we present here a mechanics-based protocol to probe the influence of gap junction connexin 43 (Cx43) has on endothelial biomechanics by exposing confluent endothelial monolayers to a known Cx43 inhibitor 2,5-dihydroxychalcone (chalcone) and measuring the impact this inhibitor has on tractions and intercellular stresses. We present representative results, which show a decrease in both tractions and intercellular stresses under a high chalcone dosage (2 &#181;g/mL) when compared to control. This protocol can be applied to not just Cx43, but also other gap junctions as well, assuming the appropriate inhibitor is used. We believe this protocol will be useful in the fields of cardiovascular and mechanobiology research.

Here, we present a mechanics-based protocol to disrupt the gap junction connexin 43 and measure the subsequent impact this has on endothelial biomechanics via observation of tractions and intercellular stresses.

Connexin 43 Yapısal Bozulma ile Perturbing Endotel Biyomekanik

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular ...

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

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

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Floresan biyomembran Kuvvet Probe: Tek Hücre üzerinde Reseptör-ligand Kinetiklerinin Eşzamanlı kantitasyonu ve Bağlama kaynaklı Hücre içi Sinyal

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Biyomühendislik

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts&#39; rheology.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.

Hücre mekanik ile boncuk-Alerjik Optik cımbız Drosophila embriyo sondalama

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

Gelişim Biyolojisi

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

Optik Cımbız Kullanarak Hapsetmede Hücre Altı Mekaniğinin Doğrudan Kuvvet Ölçümleri

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

GFP-Aktin ifade Canlı Endotel Hücreleri Aktin Sitoiskelet Çalışması

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

Biyoloji

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

Referanssız Çekiş Kuvveti Mikroskopi Platformunun İmalatı ve Uygulanması

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Traction Force Microscopy to Study B Lymphocyte Activation

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor. Gel rigidity, bead density, and protein functionalization must be optimized for the study of relatively small cells (~ 6 &#181;m) that interact with, and respond specifically to ligands for cell surface receptors.

Here we present a protocol used to perform traction force microscopy experiments on B cells. We describe the preparation of soft polyacrylamide gels and their functionalization, as well as data acquisition at the microscope and a summary of data analysis.

B Lenfosit Aktivasyonunu Incelemek Için Çekiş Kuvveti Mikroskobu

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Mikrodesen Traksiyon Mikroskobu için Desen Oluşturma

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

Büyüme Konisi İlerlemesi için Actin Dynamics, Debriyaj Kavrama ve Çekiş Kuvveti Analizleri

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...