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 forskningsarbeidet ble støttet av National Science Foundation [CBET-1904921] og National Institutes of Health [NIH R01CA236350] til HM.

MERK: Alle buffere og kjemiske reagenser som brukes i denne protokollen er oppført i tabellen Materialer.
1. Forberedelse av skjærkraftmikroskopet
MERK: Skjærkraftmikroskopet inneholder to deler, en reaksjonsenhet (homogenisator) og en deteksjonsenhet (fluorescensmikroskop). Forstørrelsen av okularet er 10x, og forstørrelsen av objektivlinsen (luft) er 4x.
<ol><li>Monter homogenisatoren og mikroskopet på et monteringsbord. B...

<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 manuskriptet tillater sanntidsundersøkelse av utfoldelsen av et ensemblesett av biomolekylære strukturer med skjærkraft. Resultatene som presenteres her understreker at DNA-i-motivstrukturer kan utfoldes med skjærkraft. Utfoldelsen av det ligandbundne i-motivet og skjærkraftaktiverte klikkreaksjoner var proof-of-concept-applikasjoner for denne ensemblekraftspektroskopimetoden.
Figur 1 viser instrumentoppsettet. Homogenisatorgener...

I enkeltmolekylkraftspektroskopi 1 (SMFS) har de mekaniske egenskapene til individuelle molekylære strukturer blitt studert av sofistikerte instrumenter som atomkraftmikroskopet, optisk pinsett og magnetisk pinsett 2,3,4. Begrenset av det samme direksjonalitetskravet til molekylene i kraftgenererende / detekterende oppsett eller det lille synsfeltet i magnetisk pinsett og miniatyrsentrifugekraftmikroskopet (MCF) 5,6,7,8, kan bare et begrenset antall molekyler undersøkes samtidig ved hjelp av SMFS. Den lave gjennomstrømningen av SMFS forhindrer dens brede anvendelse i molekylær anerkjennelsesfelt, noe som krever involvering av et stort sett med molekyler.
Skjærstrøm gir en potensiell løsning for å påføre krefter på et massivt sett med molekyler9. I en væskestrøm inne i en kanal, jo nærmere kanaloverflaten, jo langsommere strømningshastigheten10. En slik strømningshastighetsgradient forårsaker skjærspenning som er parallell med grenseflaten. Når et molekyl er plassert i denne skjærstrømmen, reorienterer molekylet seg slik at den lange aksen justerer seg med strømningsretningen, ettersom skjærkraften påføres den lange aksen11. Som et resultat av denne nyorienteringen forventes alle molekylene av samme type (størrelse og lengde på håndtakene) å justere seg i samme retning mens de opplever samme skjærkraft.
Dette arbeidet beskriver en protokoll for å bruke en slik skjærstrøm for å utøve skjærkraft på et massivt sett med molekylære strukturer, som eksemplifisert av DNA i-motivet. I denne protokollen genereres en skjærstrøm mellom rotoren og statoren i en homogenisatorspiss. Den foreliggende studien fant at den brettede DNA i-motivstrukturen kunne utfoldes med skjærhastigheter på 9724-97245 s−1. Dessuten ble det funnet en dissosiasjonskonstant på 36 μM mellom L2H2-4OTD-liganden og i-motivet. Denne verdien er i samsvar med verdien på 31 μM målt ved gelskiftanalysen12. Videre brukes den nåværende teknikken til å utfolde i-motivet, som kan eksponere chelatert kobber (I) for å katalysere en klikkreaksjon. Denne protokollen gjør det dermed mulig å utfolde et stort sett med i-motivstrukturer med rimelige instrumenter innen rimelig tid (kortere enn 30 min). Gitt at skjærkraftteknikken drastisk øker gjennomstrømningen av kraftspektroskopien, kaller vi denne teknikken ensemble force spectroscopy (EFS). Denne protokollen tar sikte på å gi eksperimentelle retningslinjer for å lette anvendelsen av denne skjærkraftbaserte 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

Enkeltmolekylteknikker basert på fluorescens og mekanokjemiske prinsipper gir overlegen følsomhet i biologisk sensing. På grunn av mangelen på høy gjennomstrømningskapasitet er anvendelsen av disse teknikkene imidlertid begrenset i biofysikk. Ensemblekraftspektroskopi (EFS) har vist høy gjennomstrømning i undersøkelsen av et massivt sett med molekylære strukturer ved å konvertere mekanokjemiske studier av individuelle molekyler til molekylære ensembler. I denne protokollen ble DNA-sekundære strukturer (i-motiver) brettet ut i skjærstrømmen mellom rotoren og statoren til en homogenisatorspiss ved skjærhastigheter opp til 77796/s. Effektene av strømningshastigheter og molekylstørrelser på skjærkreftene som oppleves av i-motivet ble demonstrert. EFS-teknikken avslørte også bindingsaffiniteten mellom DNA i-motiver og ligander. Videre har vi demonstrert en klikkkjemireaksjon som kan aktiveres av skjærkraft (dvs. mekano-klikkkjemi). Disse resultatene fastslår effektiviteten av å bruke skjærkraft for å kontrollere konformasjonen av molekylære strukturer.

Figur 1 skisserer den mekaniske utfoldelsen og sanntidsfølingen av ensemblemolekyler i EFS. I figur 1B ble fluorescensintensiteten til i-motiv-DNA observert å øke med skjærhastigheten fra 9,724 s−1 til 97,245 s−1 i en pH 5,5 MES-buffer. Som en kontroll ble fluorescensintensiteten ikke økt når det samme i-motiv-DNA ble skåret med en hastighet på 63 209 s−1 i en pH 7,4 MES-buffer. Dette skyldes at i-m...

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 teknikk for mekanisk utfoldelse og sanntidsregistrering av et ensemblesett av biomolekylære strukturer i biofysiske og biosenserende felt.

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 ...

Denne protokollen gir en høy gjennomstrømningsteknikk for å studere den intermolekylære bindingen mellom DNA og ligander. Denne teknikken har vist høy gjennomstrømning i undersøkelsen av massive sett med biomolekylære strukturer. Det gjør det ved å konvertere de mekaniske kjemiske egenskapene til individuelle molekyler til molekylære ensembler.Jeg tror det er et veldig skritt fremover eksperiment, men det er få kritiske skritt. For eksempel justering av mikroskop, homogenisator og reaksjonsrøret. Disse tre tingene bør være perfekt justert for å nøyaktig spore endringen i fluorescens.Begynn med å montere homogenisatoren og mikroskopet på et monteringsbord. Etter å ha brukt vernebriller, slå på fluorescensmikroskopet og juster deretter homogenisatoren for å sikre at eksitasjonslysstrålen passerer gjennom midten av dispergeringsspissen til homogenisatoren. Lag deretter et flatt bunnet reaksjonskammer som er fem centimeter i høyden og 1,5 x 1,5 kvadratcentimeter i tverrsnitt, slik at det valgte kammermaterialet ikke fluorescerer for å redusere bakgrunnen.Etterpå klemmer du reaksjonskammeret på prøvetrinnet i fluorescensmikroskopet og justerer deretter kammeret for å holde dispergeringsspissen til homogenisatoren, slik at spissen er litt over den nederste overflaten av reaksjonskammeret. Deretter justerer du den vertikale posisjonen til homogenisatoren og kammeret sammen for å sikre at mikroskopets fokus er på overflaten av dispergeringsspissen. Juster deretter den horisontale posisjonen til dispergeringsspissen for å sikre at deteksjonsområdet er satt mellom rotoren og statene.Slå på fluorescerende kanaler, i henhold til fluorescerende fargestoff som ble brukt i forsøket. Før høyhastighetsskjæringseksperimentet brukte avionisert vann for å teste skjæringen med lav skjærehastighet. Først må du forberede et humant telomert i-motiv DNA merket med henholdsvis et fargestoff og en slukker i de to endene i avionisert vann som beskrevet i manuskriptet.Fortynn deretter DNA til fem mikromolar i 30 millimolar MES-buffer ved pH 5,5 eller pH 7,4. Til DNA-løsningen, tilsett ligand L2H24OTD"i et konsentrasjonsområde på null til 60 mikromolar og bland løsningen forsiktig i 10 minutter for å brette i-motivstrukturene uten lys. Kontroller og minimer bakgrunnsfluorescensintensiteten til reaksjonskammeret som er fylt med avionisert vann ved hjelp av fluorescerende mikroskop uten skjæring.Still deretter parametrene til CCD-kameraet ved hjelp av programvaren med eksponeringstid som 0.5 sekunder, CCD-følsomhet på 1.600 og opptakstid lik 20 minutter. Bruk en lang pipette, tilsett DNA-løsningen i det tomme og rene reaksjonskammeret og dekk reaksjonskammeret med en svart boks. Start deretter homogenisatoren for å utføre skjæring med en valgt skjærhastighet fra 9.724 per sekund til 97, 245 per sekund i 20 minutter, med CCD-kameraet slått på for å registrere dataene.Etter forsøket, fjern kammeret og vask det med avionisert vann. Forbered i-motiv DNA og avionisert vann. Deretter inkuberer du 10 mikromolar i-motiv DNA og 300 mikroliter 30 millimolar tris buffer supplert med 150 mikromolar cupric chloride og 300 mikromolar askorbinsyre i 10 minutter for å brette i-motivstrukturene.Deretter ultrafiltrerer løsningen med en ultrafiltreringsanordning ved en sentrifugalkraft på 14, 300 ganger G.Fyll løsningen til ca. 500 mikroliter med 30 millimolar tris buffere supplert med 300 mikromolar askorbinsyre etter hver filtrering. Etter å ha samlet restløsningen, gjør du et endelig volum på 300 mikroliter ved å tilsette 30 millimolar tris supplert med 300 mikromolar askorbinsyre sammen med 20 mikromolar CalFluor 488 Azide, 20 mikromolar HPG og 10 mikromolar TBTA. Når reagensene er tilsatt, flytt løsningen til det mørke rommet.Kontroller deretter og minimer bakgrunnsfluorescensintensiteten til reaksjonskammeret fylt med avionisert vann ved hjelp av mikroskopet før skjæreeksperimentene. Deretter legger du DNA-løsningen i det tomme reaksjonskammeret med en lang pipette, og start deretter homogenisatorskjæringen med en skjærhastighet på 63.209 per sekund i 20 minutter med CCD-kameraet slått på. Etter forsøket, fjern kammeret og vask det med avionisert vann.Fluorescensintensiteten til i-motiv-DNA ble observert å øke med den rene hastigheten fra 9.724 per sekund til 97.245 per sekund i en pH 5.5 MES-buffer. Fluorescensintensiteten ble imidlertid ikke økt når det samme i-motiv-DNA ble kuttet med en hastighet på 63.209 per sekund, da det ikke brettes ved pH 7,4 i MES-buffer. Ligandbindingen til DNA i-motivmolekylene utsatt for ren strømning ble evaluert som viste at økningen av fluorescensintensiteten ble mindre tydelig med økende ligandkonsentrasjon.Dissosiasjonskonstanten ble bestemt for samspillet mellom liganden og i-motivet og ble funnet å være 34 mikromol. Det kobberchelaterte i-motivet ble brettet ut med skjærhastigheten 63.209 per sekund, og det frigjorte kobberet utløste den fluorgene klikkreaksjonen, noe som resulterte i økt fluorescensintensitet over tid. Først må du sørge for at deteksjonsområdet er satt mellom rotoren og statene.For det andre, sjekk og minimer fluorescensbakgrunnen. Og til slutt, sørg for at du filtrerer reaksjonen før du utfører klikkreaksjon. Denne metoden åpner en inngangsport for å utforske mekanisk styrke, folding og utfolding av alle slags polymerer.Det kan være andre DNA-strukturer, proteiner eller andre polymerer, og deres interaksjon med andre typer molekyler. Som sagt kan flere andre makromolekyler studeres ved hjelp av denne tilnærmingen.

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.

Gransker Receptor-ligandsysternene med Cellulosome med AFM-baserte Single-molekyl kraft 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 skjæradhesjon Kart Bruke 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.

Produksjon av Konsentrert, Lipid-baserte Oxygen mikrobobleutskillere Emulsjoner av High Shear Homogenisering og Serial Konsentrasjon

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 av maksimal isometrisk Force generert av permeabilized skjelettmuskulatur Fibers

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 and Force spektroskopi av Supported lipidbilag

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 protokoll for å bruke Förster Resonance Energy Transfer (FRET) -force Biosensorer måle mekaniske krefter over hele 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.

Deteksjon av Endotoxin i Nano-formuleringer bruker Limulus Amoebocyte 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 ved vekslende 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 ...