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

Dette forskningsarbejde blev støttet af National Science Foundation [CBET-1904921] og National Institutes of Health [NIH R01CA236350] til H. M.

BEMÆRK: Alle buffere og kemiske reagenser, der anvendes i denne protokol, er angivet i tabelmaterialerne.
1. Fremstilling af forskydningskraftmikroskopet
BEMÆRK: Forskydningskraftmikroskopet indeholder to dele, en reaktionsenhed (homogenisator) og en detektionsenhed (fluorescensmikroskop). Forstørrelsen af okularet er 10x, og forstørrelsen af objektivlinsen (luft) er 4x.
<ol><li>Saml homogenisatoren og mikroskopet på et monte...

<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>

Protokollen beskrevet i dette manuskript tillader realtidsundersøgelse af udfoldelsen af et ensemblesæt af biomolekylære strukturer ved forskydningskraft. De resultater, der præsenteres her, understreger, at DNA-i-motivstrukturer kan udfoldes ved forskydningskraft. Udfoldelsen af det ligandbundne i-motiv og de forskydningskraftaktiverede klikreaktioner var proof-of-concept-applikationer til denne ensemblekraftspektroskopimetode.
Figur 1 viser instrumentopsætn...

I enkeltmolekylekraftspektroskopi1 (SMFS) er de mekaniske egenskaber af individuelle molekylære strukturer blevet undersøgt af sofistikerede instrumenter såsom atomkraftmikroskopet, optisk pincet og magnetisk pincet 2,3,4. Begrænset af det samme retningskrav til molekylerne i de kraftgenererende/detekterende opsætninger eller det lille synsfelt i magnetiske pincetter og miniaturecentrifugekraftmikroskopet (MCF)5,6,7,8, kan kun et begrænset antal molekyler undersøges samtidigt ved hjælp af SMFS. Den lave gennemstrømning af SMFS forhindrer dens brede anvendelse i molekylærgenkendelsesfeltet, hvilket kræver involvering af et stort sæt molekyler.
Forskydningsstrøm giver en potentiel løsning til at anvende kræfter på et massivt sæt molekyler9. I en væskestrøm inde i en kanal, jo tættere på kanaloverfladen, jo langsommere er strømningshastigheden10. En sådan strømningshastighedsgradient forårsager forskydningsspænding, der er parallel med grænseoverfladen. Når et molekyle placeres i denne forskydningsstrømning, omorienterer molekylet sig selv, så dets lange akse stemmer overens med strømningsretningen, da forskydningskraften påføres den lange akse11. Som et resultat af denne omorientering forventes alle molekyler af samme type (størrelse og længde af håndtag) at justere i samme retning, mens de oplever den samme forskydningskraft.
Dette arbejde beskriver en protokol til at bruge en sådan forskydningsstrøm til at udøve forskydningskraft på et massivt sæt molekylære strukturer, som eksemplificeret ved DNA i-motivet. I denne protokol genereres en forskydningsstrøm mellem rotoren og statoren i en homogenisatorspids. Den nuværende undersøgelse viste, at den foldede DNA i-motivstruktur kunne udfoldes ved forskydningshastigheder på 9724-97245 s−1. Desuden blev der fundet en dissociationskonstant på 36 μM mellem L2H2-4OTD-liganden og i-motivet. Denne værdi er i overensstemmelse med værdien 31 μM målt ved gelforskydningsassay12. Endvidere bruges den nuværende teknik til at udfolde i-motivet, som kan udsætte det chelaterede kobber (I) for at katalysere en klikreaktion. Denne protokol giver således mulighed for at udfolde et stort sæt i-motivstrukturer med billige instrumenter inden for en rimelig tid (kortere end 30 min). I betragtning af at forskydningskraftteknikken drastisk øger gennemstrømningen af kraftspektroskopien, kalder vi denne teknik ensemble force spectroscopy (EFS). Denne protokol har til formål at give eksperimentelle retningslinjer for at lette anvendelsen af denne forskydningskraftbaserede 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

Enkeltmolekyleteknikker baseret på fluorescens og mekanokemiske principper giver overlegen følsomhed i biologisk sensing. På grund af manglen på høje gennemstrømningskapaciteter er anvendelsen af disse teknikker imidlertid begrænset i biofysik. Ensemble force spektroskopi (EFS) har vist høj gennemstrømning i undersøgelsen af et massivt sæt molekylære strukturer ved at konvertere mekanokemiske undersøgelser af individuelle molekyler til molekylære ensembler. I denne protokol blev DNA's sekundære strukturer (i-motiver) udfoldet i forskydningsstrømmen mellem rotoren og statoren på en homogenisatorspids ved forskydningshastigheder op til 77796/s. Virkningerne af strømningshastigheder og molekylære størrelser på forskydningskræfterne oplevet af i-motivet blev demonstreret. EFS-teknikken afslørede også bindingsaffiniteten mellem DNA i-motiver og ligander. Desuden har vi demonstreret en klikkemireaktion, der kan aktiveres af forskydningskraft (dvs. mekano-klikkemi). Disse resultater fastslår effektiviteten af at anvende forskydningskraft til at kontrollere konformationen af molekylære strukturer.

Figur 1 skitserer den mekaniske udfoldelse og realtidsregistrering af ensemblemolekyler i EFS. I figur 1B blev fluorescensintensiteten af i-motiv-DNA observeret at stige med forskydningshastigheden fra 9.724 s-1 til 97.245 s-1 i en pH 5,5 MES-buffer. Som kontrol blev fluorescensintensiteten ikke øget, når det samme i-motiv-DNA blev skåret med en hastighed på 63.209 s-1 i en pH-buffer på 7,4 MES. Dette skylde...

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

Ensemble Force Spectroscopy by Shear Forces

Ensemble force spectroscopy (EFS) er en robust teknik til mekanisk udfoldelse og realtidsføling af et ensemblesæt af biomolekylære strukturer i biofysiske og biosensing felter.

Ensemble Force Spektroskopi af Shear Forces

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

Denne protokol giver en høj gennemstrømningsteknik til at studere den intermolekylære binding mellem DNA og ligander. Denne teknik har vist høj gennemstrømning i undersøgelsen af massivt sæt biomolekylære strukturer. Det gør det ved at omdanne de enkelte molekylers mekaniske kemiske egenskaber til molekylære ensembler.Jeg mener, at det er et meget vanskeligt eksperiment, men der er få kritiske skridt. For eksempel justeringen af mikroskop, homogenisator og reaktionsrøret. Disse tre ting skal være perfekt justeret for nøjagtigt at spore ændringen i fluorescens.Begynd med at samle homogenisatoren og mikroskopet på et monteringsbord. Efter brug af beskyttelsesbriller skal du tænde fluorescensmikroskopet og derefter justere homogenisatoren for at sikre, at excitationslysstrålen passerer gennem midten af homogenisatorens dispergeringsspids. Forbered derefter et fladbundet reaktionskammer, der er fem centimeter i højden og 1,5 x 1,5 kvadratcentimeter i tværsnit, hvilket sikrer, at det valgte kammermateriale ikke fluorescerer for at reducere baggrunden.Derefter klemmes reaktionskammeret på fluorescensmikroskopets prøvetrin, og kammeret justeres derefter for at holde homogenisatorens dispergeringsspids, hvilket sikrer, at spidsen er lidt over reaktionskammerets bundflade. Juster derefter homogenisatorens og kammerets lodrette position sammen for at sikre, at mikroskopets fokus er på overfladen af dispergeringsspidsen. Juster derefter dispergeringsspidsens vandrette position for at sikre, at detektionsområdet er indstillet mellem rotoren og staten.Tænd for de fluorescerende kanaler i henhold til det fluorescerende farvestof, der blev brugt i eksperimentet. Før højhastighedsforskydningseksperimentet brugte deioniseret vand til at teste forskydningen med en lav forskydningshastighed. Først skal du forberede et humant telomerisk i-motiv-DNA mærket med henholdsvis et farvestof og en slukker i dets to ender i deioniseret vand som beskrevet i manuskriptet.Derefter fortyndes DNA'et til fem mikromolære i 30 millimolar MES-buffer ved pH 5,5 eller pH 7,4. Til DNA-opløsningen tilsættes ligand L2H24OTD"i et koncentrationsområde på nul til 60 mikromolar og bland opløsningen forsigtigt i 10 minutter for at folde i-motivstrukturerne uden lys. Kontroller og minimer baggrundsfluorescensintensiteten af reaktionskammeret, der er fyldt med deioniseret vand ved hjælp af det fluorescerende mikroskop uden klipning.Indstil derefter parametrene for CCD-kameraet ved hjælp af softwaren med eksponeringstid som 0,5 sekunder, CCD-følsomhed på 1.600 og optagetid svarende til 20 minutter. Brug en lang pipette til at tilsætte DNA-opløsningen i det tomme og rene reaktionskammer og dække reaktionskammeret med en sort boks. Start derefter homogenisatoren for at udføre klipning med en valgt forskydningshastighed fra 9.724 pr. sekund til 97, 245 pr. sekund i 20 minutter, med CCD-kameraet tændt for at optage dataene.Efter eksperimentet skal du fjerne kammeret og vaske det med deioniseret vand. Forbered i-motiv DNA og deioniseret vand. Derefter inkuberes 10 mikromolære i-motiv-DNA og 300 mikroliter 30 millimolar tris-buffer suppleret med 150 mikromolær kobberchlorid og 300 mikromolær ascorbinsyre i 10 minutter for at folde i-motivstrukturerne.Dernæst ultrafiltrere opløsningen med en ultrafiltreringsanordning ved en centrifugalkraft på 14.300 gange G.Genopfyld opløsningen til ca. 500 mikroliter med 30 millimolar trisbuffere suppleret med 300 mikromolær ascorbinsyre efter hver filtrering. Efter opsamling af restopløsningen fremstilles et slutvolumen på 300 mikroliter ved at tilsætte 30 millimolar tris suppleret med 300 mikromolær ascorbinsyre sammen med 20 mikromolære CalFluor 488 Azide, 20 mikromolære HPG og 10 mikromolære TBTA. Når reagenserne er tilsat, skal du flytte opløsningen til det mørke rum.Kontroller og minimer derefter baggrundsfluorescensintensiteten af reaktionskammeret fyldt med deioniseret vand ved hjælp af mikroskopet før skæreforsøgene. Tilsæt derefter DNA-opløsningen i det tomme reaktionskammer med en lang pipette, og start derefter homogenisatorforskydningen med en forskydningshastighed på 63.209 pr. Sekund i 20 minutter med CCD-kameraet tændt. Efter eksperimentet skal du fjerne kammeret og vaske det med deioniseret vand.Fluorescensintensiteten af i-motiv DNA blev observeret at stige med den rene hastighed fra 9.724 per sekund til 97, 245 per sekund i en pH 5,5 MES buffer. Imidlertid blev fluorescensintensiteten ikke øget, når det samme i-motiv-DNA blev klippet med en hastighed på 63.209 pr. Sekund, da det ikke foldes ved pH 7,4 i MES-buffer. Limandbindingen til DNA i-motivmolekylerne udsat for den rene strøm blev evalueret, hvilket viste, at stigningen i fluorescensintensiteten blev mindre tydelig med stigende ligandkoncentration.Dissociationskonstanten blev bestemt for interaktionen mellem liganden og i-motivet og viste sig at være 34 mikromol. Det kobber en chelaterede i-motiv blev udfoldet med 63, 209 per sekund forskydningshastighed, og den frigivne kobber udløste den fluorgene klikreaktion, hvilket resulterede i øget fluorescensintensitet over tid. For det første skal du sørge for, at detektionsområdet er indstillet mellem rotoren og tilstanden.For det andet skal du kontrollere og minimere fluorescensbaggrunden. Og til sidst skal du sørge for at filtrere reaktionen, før du udfører klikreaktion. Denne metode åbner en gateway til at udforske mekanisk styrke, folde og udfolde alle slags polymerer.Det kan være andre DNA-strukturer, proteiner eller andre polymerer og deres interaktion med andre slags molekyler. Som sagt kunne flere andre makromolekyler studeres ved hjælp af denne tilgang.

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.

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).

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.

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 af Shear Vedhæftning kort Brug SynVivo Syntetisk Mikrovaskulære 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.

Fremstilling af koncentreret, Lipid-baserede Oxygen Microbubble Emulsioner af High Shear Homogenisering og 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.

Måling af Maximum Isometrisk Kraft Dannet med permeabiliseres Skeletal muskelfibre

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 mikroskopi Imaging og Kraft spektroskopi af understøttede lipiddobbeltlag

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.

En protokol til brug af Förster Resonance Energy Transfer (FRET) -Force Biosensorer at måle mekaniske kræfter på tværs af 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.

Påvisning af Endotoxin i Nano-formuleringer bruger Limulus Amoebocyte Lysate (LAL) Assays

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-udskrivning af in vitro hydrogelmikrobærere ved vekslende viskøs inertial kraftstråle

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 ...