Name: a direct force probe for measuring mechanical integration between the nucleus and the cytoskeleton
Uploaded: 2018-07-29T13:30:00.000+00:00
Duration: 5 min 47 s
Description: Watch this Scientific Journal Video about A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton at JoVE.com

Bu eser NIH R01 EB014869 tarafından desteklenmiştir.

1. hücre görüntüleme için hazırlanması
Not: Doğrudan gücü probun (DFP) herhangi bir yapisan hücre türü için kullanılabilir. Burada, NIH 3T3 fare fibroblastlar modeli hücre satır olarak bu iletişim kuralı için kullanılır.
<ol><li>Kültür NIH 3T3 fibroblast hücreleri içinde Dulbecco'nın modifiye kartal Orta (% 10 donör Sığır serum ve % 1 ile DMEM) penisilin-streptomisin 35-mm cam alt çanağı kadar istenen confluency. 37 ° C ve % 5 CO2hücreleri korumak.<ol>
<li>Tüm 35-mm cam alt yemekler 5 µg/mL fibronektin (veya benzer ECM protein) ile NIH 3T3 hücreleri görüntüleme için tohum önce kat emin olun. Not: Tam hücreleri yaymak gerekir ve yemek için deneme üzerinde yapışık. DFP yöntemin işe yaraması confluency açısından herhangi bir kısıtlama yoktur.</li>
	</ol>
</li>
	<li>Deney hemen önce hücreleri iki kez tam büyüme orta ile tek bir yıkama ardından PBS ile yıkayın.</li>
	<li>Tam büyüme orta 3 mL Cam alt yemek için ekleyin.</li>
</ol>2. mikroskobu ve resim alma
Not: Bir ters floresans mikroskobu (veya eşdeğeri) ile üreticinin önerilerini göre yan kol için yüklü micromanipulator. Mikroskop da 37 ° C sıcaklıkta ve % 5 CO2 düzeyinde korumak için çevre bir odası ile outfitted. Bir micromanipulator ve microinjector için mikroskop bağlı gereklidir de. Bir petrol daldırma 40 x / 1.3 NA veya 60 x / 1,49 NA (ya da eşdeğer hedefleri) deneyler için tavsiye edilir. Mikroskop bir titreşim yalıtım masaya monte edilmelidir.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58038/58038fig1.jpg"> Resim 1 . Nükleer deformasyon ve mikroskop odaklanarak A. en büyük nükleer deformasyon ve nükleer deformasyon gevşeme. En büyük nükleer deformasyon hesaplamadan önce nükleer şekillerin arka kenarları ilk deforme çekirdeği çeviri için düzeltmek için çakıştı. Micropipette ipucu dekolmanı Şu anda çekirdeğin şekli ilk nükleer şeklin üzerine çekerek önce overlaid. Alan iki şekil arasındaki farkı Δ ölçülmüştürbir1. En büyük nükleer deformasyon ΔA1 orijinal nükleer alanına göre bölünmüş olarak tanımlanmıştır. Benzer şekilde, ikinci bir parametre, ΔA2, son kararlı duruma nükleer şekli özgün nükleer şeklindeki micropipette dekolmanı sonra overlaying tarafından tanımlı. B. hücre uçak A'da odaklanmak ve odak düzlemi micropipette ipucu bulmak için uçak B kadar hareket ettirin. Görüntüleme sırasında micropipette sağa (turuncu ok yönünü) çevrildi. Bu rakam Neelam ve arkdeğiştirildi. 15. <a href="https://www.jove.com/files/ftp_upload/58038/58038fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<ol><li>Üreticinin iletişim kuralı başına microinjector açın.</li>
	<li>Bir daldırma yağ damlalık kullanmak, Daldırma yağ objektif lens üzerinde tek bir damla uygulanır.</li>
	<li>Çanak sıkıca çanak tutucusuna klamp ve çanak tutucu sahne alanı'na yük. Not: Hücreleri 37 ° C ve % 5 CO2 deneme boyunca muhafaza edilmelidir.</li>
	<li>Hücreleri (Uçak A, Şekil 1B) odak haline getirmek için hedefi yüksekliğini ayarlayın.</li>
	<li>Mikroskop sahne alanı ilgi bir hücreyi bulmak için hareket ederler.</li>
	<li>Oyun çubuğu damlalıklı tutucu üst konuma taşımak için micromanipulator üzerinde döndürün. 0.5 µm çapı uç micropipette damlalıklı tutucu üzerine yükleyin.<ol>
<li>Micropipette için hücre adezyon önlemek için oda sıcaklığında 0.3 mg/mL PLL-g-PEG çözüm 1 h için micropipette ucuyla önceden tedavi. Yapışma için emme herhangi bir baskı olmadan çekirdeği için micropipette dokunmadan da çekirdek uzak micropipette çeviri sınayın. Yapışma yokluğu nükleer deformasyon ve çeviri tam bir eksikliği ayırt edilebilir. Not: Lütfen paketi açmak için üretim önerileri uygulayın.</li>
	</ol>
</li>
	<li>Objektif odak düzlemi uçak A ve uçak b hücrenin üstüne üzerinde daha iyi denetim ayarlayarak yükseltmek (Şekil 1B, bkz: adım 2.4).</li>
	<li>Micromanipulator kaba denetimine ayarlayın. Tamamen odak haline gelene kadar micropipette micropipette, siluet için izleyerek micropipette uçak B aşağı yavaşça.</li>
	<li>Micropipette ipucu odak olduğunda, micromanipulator iyi denetimine ayarlayın.</li>
	<li>(Uçak A, Şekil 1B) hücrenin Ekvator uçak amaca daha düşük ve micropipette yaklaşık 15 µm (Şekil 1B, kesik micropipette) Uçak A yukarıda için daha düşük.</li>
	<li>Tazminat basınç (Pc) istenilen basınç için microinjector olarak ayarlayın; Basıncı dengelemek için birkaç saniye bekleyin. Not: Optimum basınç ayar noktası hücre türüne ve denemenin belirli hedeflere göre değişir. Çoğu durumda, 300 hPa iyi bir başlangıç noktası olur.</li>
	<li>Micropipette micromanipulator panelde temiz ayarı kullanarak ve hava kabarcıkları micropipette ucundan ortaya emin olmak için kontrol tıkalı değil emin olun.</li>
	<li>Ucu ucunu hafifçe nükleer yüzey dokunuyor kadar yavaş yavaş micropipette düşürerek hücreye yerleştirin. Not: ne zaman micropipette düşürücü, micropipette ucunu silüeti odak haline gelir olarak açık olacak. Micropipette çekirdek dokunamaz önce objektif odak yükseltmek ve micropipette çekirdeği (aynı x-y koordinatı, daha yüksek z-uçak) ile hizalayın. Odak (uçak A, Şekil 1B) çekirdeği Ekvator uçağa geri dönmek ve yavaş yavaş micropipette ucunu alt.</li>
	<li>Bir mühür micropipette uç ve nükleer membran arasında böylece atmosfer micropipette Tüp sonuna açılış mikroenjeksiyon sisteminden basınç kaynağı tüp keserek oluşturun. Bu adım bir negatif basınç nükleer yüzeyde eşit Pc oluşturur.</li>
	<li>Mikroskoplar resim koleksiyonu yazılımı ile görüntüleri elde etmek. AVI-edinme (video) veya nd-edinme (görüntüleri) görüntü collection yazılım ayarlayın. Not: yazılım edinme herhangi bir görüntüleme için gerçek zamanlı video görüntüleme veya Hızlandırılmış görüntü satın almalar ile bir kısa süre aralığı ayarlayın.</li>
	<li>Kanal görüntüleme karşılık gelen floresan için geçiş (Yani, GFP, RFP, vb) ve görüntüleme başlar.</li>
	<li>Micropipette İpucu (sağa doğru Şekil 1B) hücre gövdesinden çekin çevirmek kadar çekirdek micropipette ayırır. Not: ucu pozitif x-yön boyunca (görüş alanı sağa) çekin. Programlanmış ve bilgisayar tarafından kontrol çekme oranı veya joystick el ile hareket edebilir. Biz çekerek arasında herhangi bir korelasyon bulunmadı oranı ve nükleer deformasyon15 zorlamak için öncelikle elastik bir yanıt düşündüren.</li>
</ol>3. veri analizi
<ol><li>Herhangi bir temel görüntü işleme yazılımı ile görüntü analizi gerçekleştirin. Nükleer deformasyon ölçüde uzunluğu zorlanma (Ɛ) ya da (Şekil 1A) alan zorlanma sayılabilir. Denklem 1, kullanarak uzunluğu zorlanma ölçmek nerede L ve M0 temsil eden en fazla deformasyon ve ilk konum, çekirdeği uzunlukları sırasıyla. <img alt="Equation 1" src="/files/ftp_upload/58038/58038eq1.jpg">(Denklem 1)</li>
</ol>

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+elastic+properties+of+the+nuclear+envelope.">Characterization of the elastic properties of the nuclear envelope.</a> Journal of the Royal Society Interface. 2 (2), 63-69 (2005).</li><li>Pagliara, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxetic+nuclei+in+embryonic+stem+cells+exiting+pluripotency.">Auxetic nuclei in embryonic stem cells exiting pluripotency.</a> Nature Materials. 13 (6), 638-644 (2014).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+mechanical+characterization+of+the+cell+nucleus+by+atomic+force+microscopy.">In situ mechanical characterization of the cell nucleus by atomic force microscopy.</a> ACS Nanotechnology. 8 (4), 3821-3828 (2014).</li><li>Krause, M., Te Riet, J., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+compressibility+of+tumor+cell+nuclei+by+combined+atomic+force-confocal+microscopy.">Probing the compressibility of tumor cell nuclei by combined atomic force-confocal microscopy.</a> Physical Biology. 10 (6), 065002 (2013).</li><li>Neelam, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+force+probe+reveals+the+mechanics+of+nuclear+homeostasis+in+the+mammalian+cell.">Direct force probe reveals the mechanics of nuclear homeostasis in the mammalian cell.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (18), 5720-5725 (2015).</li><li>Pajerowski, J. 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K., Lele, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actomyosin+tension+exerted+on+the+nucleus+through+nesprin-1+connections+influences+endothelial+cell+adhesion,+migration,+and+cyclic+strain-induced+reorientation.">Actomyosin tension exerted on the nucleus through nesprin-1 connections influences endothelial cell adhesion, migration, and cyclic strain-induced reorientation.</a> Biophysical Journal. 99 (1), 115-123 (2010).</li><li>Neelam, S., Dickinson, R. B., Lele, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+for+understanding+the+nuclear+force+balance+in+living,+adherent+cells.">New approaches for understanding the nuclear force balance in living, adherent cells.</a> Methods. 94, 27-32 (2016).</li></ol>

Yazarlar ifşa gerek yok.

(Çekirdek nerede sitoiskeleti bilgisini iletmiyor) her iki izole çekirdeği gerektirdiğinden sitoiskeleti çekirdekle mekanik entegrasyonu ölçme micropipette aspirasyon16gibi en yeni yöntemleri için bir meydan okuma olduğunu veya çekirdekleri askıya alınmış hücrelerindeki (hücre dışı güçleri, çekiş güçleri gibi eksik durumda). Güç çekirdeğine hücre yapışık bir membran17,18biaxial baskı uygulayarak uygulanm?...

Patolojiler kanser gibi nükleer şekli ve yapısı1,2, genellikle bir 'yumuşama' çekirdek3,4tarafından yayımlanır değişiklikleri içerir. Nükleer mekanik deformasyon direnci genellikle izole çekirdeği5' e bir kuvvet uygulayarak karakterize.
Hücre çekirdeğinde tatlı sitoiskeleti için bağlayıcı Nucleoskeleton ve sitoiskeleti (LINC) karmaşık6,7,8,9tarafından bağlanır. Sonuç olarak, çekirdek mekanik sitoiskeleti ile ve hücre-terkedilemeyen yapışıklıklar, hücre dışı matriks ile entegre edilmiştir. Mekanik yapışık hücreleri içinde çekirdek sondalama mekanik Bu entegrasyon içgörü sağlayabilir. Micropipette aspirasyon10,11ve atomik kuvvet mikroskobu12,13,14çekirdeği canlı hücreler içinde işlemek için yöntemler içerir. Biz son zamanlarda bir yaşam yapisan hücre15çekirdeğinde doğrudan mekanik Kuvvetleri uygulandığı bir doğrudan kuvvet sonda (DFP) nitelendirdi.
Burada, mikroskobu tesislerinde doğrudan yapisan bir hücre çekirdeğinde için bilinen, nano ölçekli mekanik kuvvet uygulamak için yaygın olarak kullanılabilir bir mikroenjeksiyon sistemi kullanan yordamı anahat. Bir femtotip (0.5 µm çapı micropipette tıp) monte ve bir tüp tarafından mikroenjeksiyon sisteme bağlı. Kültür çanak yüzeyine göre 45 ° açıyla konumlandırılmış uç, bitişik kadar nükleer yüzeye indirilir. Tüp sonra bağlantısı kesilen ve nükleer yüzeyinde negatif bir emme basınç oluşturur ve nükleer yüzeye karşı micropipette ipucu mühürler atmosfere açtı. Çeviri micropipette uç çekirdeği deforme ve sonunda (büyüklüğü yük bağlı olarak), micropipette ilişkisi kesildi. Bu dekolmanı çekirdek ve hücre, tarafından sarf geri yükleme (dirençli) kuvvetleri micropipette tarafından uygulanan emiş gücü eşit oluşur. Çözümleme yapılır çekirdeği, deplasman ölçerek uzunluğu zorlanma (denklem 1) veya alan zorlanma (Şekil 1A).

<table><tbody><tr><td>FluoroDish</td><td>WPI</td><td>FD35</td><td></td></tr><tr><td>SYTO 59</td><td>ThermoFisher Scientific</td><td>S11341</td><td></td></tr><tr><td>Femtotips&amp;nbsp;</td><td>Eppendorf</td><td>930000043</td><td></td></tr><tr><td>InjectMan NI2</td><td>Eppendorf</td><td>NA</td><td>discontinued, current equivalent model: InjectMan 4</td></tr><tr><td>FemtoJet</td><td>Eppendorf</td><td>NA</td><td>Current model FemtoJet 4i</td></tr><tr><td>Plan Fluor oil immersion 40x</td><td>Nikon</td><td>NA</td><td></td></tr><tr><td>Apo TIRF oil immersion 60x</td><td>Nikon</td><td>NA</td><td></td></tr><tr><td>Donor Bovine Serum (DBS)</td><td>ThermoFisher Scientific</td><td>16030074</td><td>NIH 3T3 serum</td></tr><tr><td>Dulbecco&#039;s Modification of Eagle&#039;s (DMEM)</td><td>Mediatech cellgro</td><td>MT10013CVRF</td><td>NIH 3T3 medium</td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Mediatech</td><td>MT30004CIRF</td><td>NIH 3T3 medium supplement</td></tr><tr><td>Immersion Oil Type LDF Non-Fluorescing</td><td>Nikon</td><td>77007</td><td>Immersion oil for objective lens&amp;nbsp;</td></tr></tbody></table>

a direct force probe for measuring mechanical integration between the nucleus and the cytoskeleton

Çekirdeği mekanik özellikleri mekanik Kuvvetleri hücrelerinde üretilen onun yanıt belirler. Çekirdek ile sitoiskeleti tatlı sürekli olduğundan, yöntemleri mekanik davranışını yapisan hücrelerdeki soruşturma için ihtiyaç vardır. Burada, doğrudan gücü probun (DFP) doğrudan bir yaşam yapisan Hücre çekirdeğinde kuvvet uygulamak için bir araç olarak tartışmak. Emme nükleer yüzeye dar bir micropipette ekleyin. Micropipette deforme ve çevirmek çekirdek neden olan çekirdek uzak çevrilir. Geri yükleme kuvvetleri emiş gücüne eşit olduğunda, çekirdek ayırır ve Melosun rahatlatır. Emiş basıncı kesin olarak bilindiğinden, güç nükleer yüzeyi bilinir. Bu yöntem nano ölçekli güçleri deforme ve yapışık Hücre çekirdeğinde çevirmek için yeterli ve kuvvetler direnmeye çekirdek etkinleştirmek hücre iskeleti elemanlar ortaya koymuştur. DFP hücresel ve nükleer bileşenleri nükleer mekanik özellikleri canlı hücreler'katkılarıyla incelemek için kullanılabilir.

Şekil 2A bir NIH 3T3 fare fibroblast çekirdeği zorlayarak gösterir. Micropipette ucunu sağa çevrilir, çekirdeği deforms ve sonunda micropipette ucundan ayırır. Çekirdek uzunluğu suşu emiş gücü (Şekil 2B) ile artacaktır görülmektedir. Çekirdeği (kenar çekerek micropipette) ön kenarındaki nükleer bir çıkıntı oluşturan ve trailing edge özgün konumundan yerinden olduğunu. Çıkıntı uzunluğu çekir...

Watch this Scientific Journal Video about A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton at JoVE.com

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

Çekirdek ve sitoiskeleti mekanik bütünleşmesi ölçmek için bir doğrudan kuvvet sonda

Bu protokol için doğrudan bir yaşam Hücre çekirdeğinde kontrollü bir kuvvet uygulamak için micropipette yöntemi açıklanmaktadır. Bu tahlil sorgulama yaşayan, yapisan hücredeki nükleer mekanik özellikleri sağlar.

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

a-direct-force-probe-for-measuring-mechanical-integration-between

Research

JoVE Journal

Biochemistry

15.2K Görüntüleme.  University of Florida. Bu protokol için doğrudan bir yaşam Hücre çekirdeğinde kontrollü bir kuvvet uygulamak için micropipette yöntemi açıklanmaktadır. Bu tahlil sorgulama yaşayan, yapisan hücredeki nükleer mekanik özellikleri sağlar.

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

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Yöntem HCN1 ve TRIP8b arasında protein-protein etkileşimi küçük molekül önleyicilerinin tespit edilmesi için

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Biyokimya

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

Förster Rezonans Enerji Transferi (FRET) -force biyosensörlerin kullanılması için Protokol Nükleer LINC Kompleksi karşısında Mekanik Kuvvetleri Ölçmek için

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

Biyomühendislik

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 ...

Gelişim Biyolojisi

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Kuvvet duyarlı Protein Dynamics floresan teknikleri bir arada kullanarak canlı hücrelerdeki ölçümü

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

Fazlararası Hücre Çekirdek Mekanik Özellikleri Probe Biyofiziksel Tahliller: Yüzey Gerilme Uygulama ve Microneedle Manipülasyon

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Biyoloji

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be "locked" and prevented from refolding to enable "storing" of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently "erased" by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions.

This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces &gt;4.7pN in real-time and can integrate forces over time.

Bağışıklık Hücreleri Tarafından Geçici Piconewton Reseptör Kuvvetlerini Haritalamak için DNA Gerilim Probları

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical ...

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.

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 ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

İskelet Kas Biyopsilerinden Miyofibrillerin İzlenmesi ve Nano-Newton Çözünürlük Transdüseri ile Kontraktil Fonksiyonun Belirlenmesi

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.

Here, we present a protocol for reconstituting microtubule bundles in vitro and directly quantifying the forces exerted within them using simultaneous optical trapping and total internal reflection fluorescence microscopy. This assay allows for nanoscale-level measurement of the forces and displacements generated by protein ensembles within active microtubule networks.

Yeniden yapılandırılmış aktif mikrotübül demetleri içindeki kuvvetleri doğrudan ölçme

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as ...

Combining 3D Magnetic Force Actuator and Multi-Functional Fluorescence Imaging to Study Nucleus Mechanobiology

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The cellular mechano-sensation of extracellular mechanical stimuli is believed to be through the membrane receptors, the associated protein complex, and the cytoskeleton. Recent advances in mechanobiology demonstrate that the cell nucleus in cytoplasm itself can independently sense mechanical stimuli simultaneously. However, a mechanistic understanding of how the cell nucleus senses, transduces, and responds to mechanical stimuli is lacking, mainly because of the technical challenges in accessing and quantifying the nucleus mechanics by conventional tools. This paper describes the design, fabrication, and implementation of a new magnetic force actuator that applies precise and non-invasive 3D mechanical stimuli to directly deform the cell nucleus. Using CRISPR/Cas9-engineered cells, this study demonstrates that this tool, combined with high-resolution confocal fluorescent imaging, enables the revelation of the real-time dynamics of a mechano-sensitive yes-associated protein (YAP) in single cells as a function of nucleus deformation. This simple method has the potential to bridge the current technology gap in the mechanobiology community and provide answers to the knowledge gap that exists in the relation between nucleus mechanotransduction and cell function.

This study presents a new protocol to directly apply mechanical force on the cell nucleus through magnetic microbeads delivered into the cytoplasm and to conduct simultaneous live-cell fluorescent imaging.

Çekirdek Mekanobiyolojisini Incelemek için 3D Manyetik Kuvvet Aktüatörü ve Çok Fonksiyonlu Floresan Görüntülemeyi Birleştirmek

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The ...