Name: ensemble force spectroscopy by shear forces
Uploaded: 2022-07-26T13:00:00
Description: Watch this Scientific Journal Video about Ensemble Force Spectroscopy by Shear Forces at JoVE.com

Detta forskningsarbete stöddes av National Science Foundation [CBET-1904921] och National Institutes of Health [NIH R01CA236350] till H. M.

OBS: Alla buffertar och kemiska reagenser som används i detta protokoll är listade i tabellen Material.
1. Beredning av skjuvkraftmikroskopet
OBS: Skjuvkraftmikroskopet innehåller två delar, en reaktionsenhet (homogenisator) och en detektionsenhet (fluorescensmikroskop). Okularets förstoring är 10x och förstoringen av objektivlinsen (luft) är 4x.
<ol><li>Montera homogenisatorn och mikroskopet på ett monteringsbord. Använ...

<ol>
	<li>Neuman, K. C., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+force+spectroscopy:+Optical+tweezers,+magnetic+tweezers+and+atomic+force+microscopy.">Single-molecule force spectroscopy: Optical tweezers, magnetic tweezers and atomic force microscopy.</a> Nature Methods. 5 (6), 491-505 (2008).</li><li>Woodside, M. T., 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=Nanomechanical+measurements+of+the+sequence-dependent+folding+landscapes+of+single+nucleic+acid+hairpins.">Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (16), 6190-6195 (2006).</li><li>Grandbois, M., Beyer, M., Rief, M., Clausen-Schaumann, H., Gaub, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+strong+is+a+covalent+bond.">How strong is a covalent bond.</a> Science. 283 (5408), 1727-1730 (1999).</li><li>Strick, T. R., Allemand, J. F., Bensimon, D., Croquette, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+supercoiled+DNA.">Behavior of supercoiled DNA.</a> Biophysical Journal. 74 (4), 2016-2028 (1998).</li><li>Yang, D., Ward, A., Halvorsen, K., Wong, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+single-molecule+force+spectroscopy+using+a+centrifuge.">Multiplexed single-molecule force spectroscopy using a centrifuge.</a> Nature Communications. 7, 11026 (2016).</li><li>Su, 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=Light-responsive+polymer+particles+as+force+clamps+for+the+mechanical+unfolding+of+target+molecules.">Light-responsive polymer particles as force clamps for the mechanical unfolding of target molecules.</a> Nano Letters. 18 (4), 2630-2636 (2018).</li><li>Kirkness, M. W. H., Forde, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+assay+for+proteolytic+susceptibility:+Force-induced+collagen+destabilization.">Single-molecule assay for proteolytic susceptibility: Force-induced collagen destabilization.</a> Biophysical Journal. 114 (3), 570-576 (2018).</li><li>Astumian, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamics+and+kinetics+of+molecular+motors.">Thermodynamics and kinetics of molecular motors.</a> Biophysical Journal. 98 (11), 2401-2409 (2010).</li><li>Bekard, I. B., Asimakis, P., Bertolini, J., Dunstan, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+shear+flow+on+protein+structure+and+function.">The effects of shear flow on protein structure and function.</a> Biopolymers. 95 (11), 733-745 (2011).</li><li>Chistiakov, D. A., Orekhov, A. N., Bobryshev, Y. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+shear+stress+on+endothelial+cells:+go+with+the+flow.">Effects of shear stress on endothelial cells: go with the flow.</a> Acta Physiologica. 219 (2), 382-408 (2017).</li><li>Hu, C., Jonchhe, S., Pokhrel, P., Karna, D., Mao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+unfolding+of+ensemble+biomolecular+structures+by+shear+force.">Mechanical unfolding of ensemble biomolecular structures by shear force.</a> Chemical Science. 12 (30), 10159-10164 (2021).</li><li>Sedghi Masoud, 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=Analysis+of+interactions+between+telomeric+i-motif+DNA+and+a+cyclic+tetraoxazole+compound.">Analysis of interactions between telomeric i-motif DNA and a cyclic tetraoxazole compound.</a> ChemBioChem. 19 (21), 2268-2272 (2018).</li><li>Abraham Punnoose, J., 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=Adaptive+and+specific+recognition+of+telomeric+G-quadruplexes+via+polyvalency+induced+unstacking+of+binding+units.">Adaptive and specific recognition of telomeric G-quadruplexes via polyvalency induced unstacking of binding units.</a> Journal of the American Chemical Society. 139 (22), 7476-7484 (2017).</li><li>Dhakal, 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=Coexistence+of+an+ILPR+i-motif+and+a+partially+folded+structure+with+comparable+mechanical+stability+revealed+at+the+single-molecule+level.">Coexistence of an ILPR i-motif and a partially folded structure with comparable mechanical stability revealed at the single-molecule level.</a> Journal of the American Chemical Society. 132 (26), 8991-8997 (2010).</li><li>Hu, C., Tahir, R., Mao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+mechanochemical+sensing.">Single-molecule mechanochemical sensing.</a> Accounts of Chemical Research. 55 (9), 1214-1225 (2022).</li></ol>

Protokollet som beskrivs i detta manuskript möjliggör realtidsundersökning av utvecklingen av en ensembleuppsättning biomolekylära strukturer genom skjuvkraft. Resultaten som presenteras här understryker att DNA-i-motivstrukturer kan utvecklas med skjuvkraft. Utvecklingen av det ligandbundna i-motivet och skjuvkraftsaktiverade klickreaktionerna var proof-of-concept-applikationer för denna ensemblekraftspektroskopimetod.
Figur 1 visar instrumentets inställn...

I enmolekylär kraftspektroskopi1 (SMFS) har de mekaniska egenskaperna hos enskilda molekylära strukturer studerats med sofistikerade instrument såsom atomkraftmikroskopet, optisk pincett och magnetisk pincett 2,3,4. Begränsad av samma riktningskrav för molekylerna i de kraftgenererande/detekterande inställningarna eller det lilla synfältet i magnetisk pincett och miniatyrcentrifugkraftmikroskopet (MCF)5,6,7,8, kan endast ett begränsat antal molekyler undersökas samtidigt med hjälp av SMFS. Den låga genomströmningen av SMFS förhindrar dess breda tillämpning inom molekylärigenkänningsfältet, vilket kräver involvering av en stor uppsättning molekyler.
Skjuvflöde ger en potentiell lösning för att applicera krafter på en massiv uppsättning molekyler9. I ett vätskeflöde inuti en kanal, ju närmare kanalytan, desto långsammare är flödeshastigheten10. En sådan flödeshastighetsgradient orsakar skjuvspänning som är parallell med gränsytan. När en molekyl placeras i detta skjuvflöde omorienterar molekylen sig själv så att dess långa axel ligger i linje med flödesriktningen, eftersom skjuvkraften appliceras på den långa axeln11. Som ett resultat av denna omorientering förväntas alla molekyler av samma typ (storlek och längd på handtag) att rikta sig i samma riktning medan de upplever samma skjuvkraft.
Detta arbete beskriver ett protokoll för att använda ett sådant skjuvflöde för att utöva skjuvkraft på en massiv uppsättning molekylära strukturer, vilket exemplifieras av DNA-i-motivet. I detta protokoll genereras ett skjuvflöde mellan rotorn och statorn i en homogenisatorspets. Den aktuella studien fann att den vikta DNA-i-motivstrukturen kunde utvecklas med skjuvhastigheter på 9724-97245 s−1. Dessutom hittades en dissociationskonstant på 36 μM mellan L2H2-4OTD-liganden och i-motivet. Detta värde överensstämmer med värdet på 31 μM mätt med gelskiftanalysen12. Vidare används den nuvarande tekniken för att veckla ut i-motivet, vilket kan exponera den kelaterade kopparen (I) för att katalysera en klickreaktion. Detta protokoll gör det alltså möjligt för en att utveckla en stor uppsättning i-motivstrukturer med billiga instrument på rimlig tid (kortare än 30 min). Med tanke på att skjuvkraftstekniken drastiskt ökar genomströmningen av kraftspektroskopin kallar vi denna teknik ensemblekraftspektroskopi (EFS). Detta protokoll syftar till att tillhandahålla experimentella riktlinjer för att underlätta tillämpningen av denna skjuvkraftbaserade EFS.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3K MWCO Amicon</td>
			<td>Millipore Sigma</td>
			<td>ufc900324</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>VWR</td>
			<td>VWRC0143-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Calfluor 488 azide</td>
			<td>Click Chemistry Tools</td>
			<td>1369-1</td>
			<td></td>
		</tr>
		<tr>
			<td>CuCl</td>
			<td>Thermo&#160;</td>
			<td>ACRO270525000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispersion tip</td>
			<td>Switzerland</td>
			<td>PT-DA07/2EC-B101</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA oligos</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dye</td>
			<td>IDT</td>
			<td>/5Cy5/</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Janpan</td>
			<td>Nikon TE2000-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>Switzerland</td>
			<td>PT 3100D</td>
			<td></td>
		</tr>
		<tr>
			<td>HPG</td>
			<td>Santa Cruz Biotechnology</td>
			<td>cs-295271</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>VWR</td>
			<td>VWRC26760.295</td>
			<td></td>
		</tr>
		<tr>
			<td>MES</td>
			<td>VWR</td>
			<td>VWRCE169-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Quencher</td>
			<td>IDT</td>
			<td>/3IAbRQSp/</td>
			<td></td>
		</tr>
		<tr>
			<td>TBTA</td>
			<td>Tokyo Chemical Industry</td>
			<td>T2993</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>VWR</td>
			<td>VWRCE133-100G</td>
			<td></td>
		</tr>
	</tbody>
</table>

ensemble force spectroscopy by shear forces

Enmolekylära tekniker baserade på fluorescens och mekanokemiska principer ger överlägsen känslighet vid biologisk avkänning. På grund av bristen på hög genomströmningskapacitet är dock tillämpningen av dessa tekniker begränsad inom biofysik. Ensemblekraftspektroskopi (EFS) har visat hög genomströmning i undersökningen av en massiv uppsättning molekylära strukturer genom att omvandla mekanokemiska studier av enskilda molekyler till molekylära ensembler. I detta protokoll utvecklades DNA-sekundära strukturer (i-motiv) i skjuvflödet mellan rotorn och statorn på en homogenisatorspets vid skjuvhastigheter upp till 77796/s. Effekterna av flödeshastigheter och molekylstorlekar på skjuvkrafterna som i-motivet upplevde demonstrerades. EFS-tekniken avslöjade också bindningsaffiniteten mellan DNA-i-motiv och ligander. Dessutom har vi demonstrerat en klickkemisk reaktion som kan aktiveras av skjuvkraft (dvs mekano-klickkemi). Dessa resultat fastställer effektiviteten av att använda skjuvkraft för att kontrollera konformationen av molekylära strukturer.

Figur 1 beskriver den mekaniska utfällningen och realtidsavkänningen av ensemblemolekyler i EFS. I figur 1B observerades fluorescensintensiteten hos i-motiv-DNA öka med skjuvhastigheten från 9 724 s−1 till 97 245 s−1 i en pH 5,5 MES-buffert. Som kontroll ökades inte fluorescensintensiteten när samma i-motiv-DNA skjuvades med en hastighet av 63 209 s−1 i en pH 7,4 MES-buffert. Detta beror på att i-mot...

Watch this Scientific Journal Video about Ensemble Force Spectroscopy by Shear Forces at JoVE.com

Ensemble Force Spectroscopy by Shear Forces

Ensemblekraftspektroskopi (EFS) är en robust teknik för mekanisk utveckling och realtidsavkänning av en ensembleuppsättning biomolekylära strukturer i biofysiska och biosensingfält.

Ensemble Force Spektroskopi av Shear Forces

Single-molecule techniques based on fluorescence and mechanochemical principles provide superior sensitivity in biological sensing. However, due to the ...

Detta protokoll ger en hög genomströmningsteknik för att studera den intermolekylära bindningen mellan DNA och ligander. Denna teknik har visat hög genomströmning i undersökningen av massiva uppsättningar biomolekylära strukturer. Det gör det genom att omvandla de mekaniska kemiska egenskaperna hos enskilda molekyler till molekylära ensembler.Jag tycker att det är ett väldigt steg framåt experiment men det finns få kritiska steg. Till exempel inriktningen av mikroskop, homogenisator och reaktionsröret. Dessa tre saker bör vara perfekt anpassade för att exakt spåra förändringen i fluorescens.Börja med att montera homogenisatorn och mikroskopet på ett monteringsbord. Efter att ha använt skyddsglasögon, slå på fluorescensmikroskopet och justera sedan homogenisatorn för att se till att excitationsljusstrålen passerar genom mitten av homogenisatorns dispergeringsspets. Förbered sedan en plattbottnad reaktionskammare som är fem centimeter hög och 1,5 x 1,5 kvadratcentimeter i tvärsnitt, så att det valda kammarmaterialet inte fluorescerar för att minska bakgrunden.Kläm sedan fast reaktionskammaren på provsteget i fluorescensmikroskopet och justera sedan kammaren så att den håller homogenisatorns dispergeringsspets, så att spetsen ligger något ovanför reaktionskammarens bottenyta. Justera sedan homogenisatorns och kammarens vertikala läge tillsammans för att säkerställa att mikroskopets fokus ligger på ytan av dispersionsspetsen. Justera sedan spridningsspetsens horisontella läge för att säkerställa att detekteringsområdet är inställt mellan rotorn och delstaten.Slå på de fluorescerande kanalerna, enligt det fluorescerande färgämnet som används i experimentet. Före höghastighetsskjuvningsexperimentet användes avjoniserat vatten för att testa skjuvningen med låg skjuvningshastighet. Förbered först ett humant telomeriskt i-motiv-DNA märkt med ett färgämne och en släckare i dess två ändar i avjoniserat vatten som beskrivs i manuskriptet.Späd sedan DNA till fem mikromolar i 30 millimolar MES-buffert vid pH 5,5 eller pH 7,4. Till DNA-lösningen, tillsätt ligand L2H24OTD"i ett koncentrationsområde från noll till 60 mikromolar och blanda lösningen försiktigt i 10 minuter för att vika i-motivstrukturerna utan ljus. Kontrollera och minimera bakgrundsfluorescensintensiteten hos reaktionskammaren som är fylld med avjoniserat vatten med hjälp av det fluorescerande mikroskopet utan skjuvning.Ställ sedan in parametrarna för CCD-kameran med hjälp av programvaran med exponeringstid som 0,5 sekunder, CCD-känslighet på 1 600 och inspelningstid lika med 20 minuter. Använd en lång pipett, tillsätt DNA-lösningen i den tomma och rena reaktionskammaren och täck reaktionskammaren med en svart låda. Starta sedan homogenisatorn för att utföra skjuvning med en vald skjuvningshastighet som sträcker sig från 9 724 per sekund till 97 245 per sekund i 20 minuter, med CCD-kameran påslagen för att spela in data.Efter experimentet, ta bort kammaren och tvätta den med avjoniserat vatten. Förbered i-motiv-DNA och avjoniserat vatten. Inkubera sedan 10 mikromolärt i-motiv-DNA och 300 mikroliter 30 millimolar tris-buffert kompletterat med 150 mikromolar kopparklorid och 300 mikromolar askorbinsyra i 10 minuter för att vika i-motivstrukturerna.Därefter ultrafiltrerar lösningen med en ultrafiltreringsanordning vid en centrifugalkraft på 14 300 gånger G.Fyll på lösningen till cirka 500 mikroliter med 30 millimolära trisbuffertar kompletterade med 300 mikromolar askorbinsyra efter varje filtrering. Efter uppsamling av restlösningen, gör en slutlig volym på 300 mikroliter genom att tillsätta 30 millimolar tris kompletterad med 300 mikromolar askorbinsyra tillsammans med 20 mikromolar CalFluor 488 azid, 20 mikromolar HPG och 10 mikromolar TBTA. När reagenserna har tillsatts, flytta lösningen till det mörka rummet.Kontrollera och minimera sedan bakgrundsfluorescensintensiteten hos reaktionskammaren fylld med avjoniserat vatten med hjälp av mikroskopet före skjuvningsexperimenten. Tillsätt sedan DNA-lösningen i den tomma reaktionskammaren med en lång pipett och starta sedan homogenisatorns skjuvning med en skjuvhastighet på 63 209 per sekund i 20 minuter med CCD-kameran påslagen. Efter experimentet, ta bort kammaren och tvätta den med avjoniserat vatten.Fluorescensintensiteten hos i-motiv-DNA observerades öka med den rena hastigheten från 9 724 per sekund till 97 245 per sekund i en pH 5,5 MES-buffert. Fluorescensintensiteten ökade emellertid inte när samma i-motiv-DNA skjuvades med en hastighet av 63 209 per sekund, eftersom det inte viks vid pH 7,4 i MES-buffert. Ligandbindningen till DNA-i-motivmolekylerna som utsattes för det rena flödet utvärderades vilket visade att ökningen av fluorescensintensiteten blev mindre uppenbar med ökande ligandkoncentration.Dissociationskonstanten bestämdes för interaktionen mellan liganden och i-motivet och befanns vara 34 mikromol. Det kopparkelaterade i-motivet vecklades ut med skjuvhastigheten 63, 209 per sekund, och den frigjorda kopparn utlöste den fluorgena klickreaktionen, vilket resulterade i ökad fluorescensintensitet över tiden. För det första, se till att detekteringsområdet är inställt mellan rotorn och staten.För det andra, kontrollera och minimera fluorescensbakgrunden. Och slutligen, se till att du filtrerar reaktionen innan du utför klickreaktion. Denna metod öppnar en port för att utforska mekanisk hållfasthet, vikning och utfällning av alla typer av polymerer.Det kan vara andra DNA-strukturer, proteiner eller andra polymerer och deras interaktion med andra typer av molekyler. Som sagt kan flera andra makromolekyler studeras med hjälp av detta tillvägagångssätt.

ensemble-force-spectroscopy-by-shear-forces

Research

JoVE Journal

Bioengineering

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

Termodynamik för membranprotein Folding Mätt fluorescensspektroskopi

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

Bakteriell Immobilisering for Imaging genom atomkraftsmikroskopi

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Undersöka Receptor-ligand System av Cellulosome med AFM-baserad Single-molekyl Force spektroskopi

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 &#181;m biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

Generering av Shear Adhesion Karta Använda SynVivo Syntetisk Mikrovaskulära Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using sonication, mechanical agitation, microfluidic devices, or homogenization. Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver oxygen intravenously during medical emergencies, reversing life-threatening hypoxemia, and preventing subsequent organ injury, cardiac arrest, and death. We present methods for scaled-up production of highly oxygenated microbubbles using a closed-loop high-shear homogenizer. The process can produce 2 L of concentrated LOMs (90% by volume) in 90 min. Resulting bubbles have a mean diameter of ~2 &#956;m, and a rheologic profile consistent with that of blood when diluted to 60 volume %. This technique produces LOMs in high capacity and with high oxygen purity, suggesting that this technique may be useful for translational research labs.

We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

Tillverkning av Koncentrerad, Lipid-baserade Oxygen mikrobubblor Emulsioner av hög Shear Homogenisering och Serial Koncentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Mätning av Högsta Isometrisk kraft som genereras av permeabiliserade Skelett muskelfibrer

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Atomic Force Microscopy Imaging och Force-spektra enligt stöds lipiddubbelskikt

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

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

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.

Ett protokoll för användning av Förster resonansenergiöverföring (FRET) -force Biosensorer för att mäta mekaniska krafter över 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. ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Påvisande av Endotoxin i Nano-formuleringar använder Limulus immobiliserat Lysate (LAL) analyser

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

3D-utskrift av In Vitro Hydrogel Microcarriers genom alternerande viskös-tröghetskraft jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...