Name: Forfatter Spotlight: Afsløring af dynamikken i mekaniske og biokemiske signaler i forskning i dyremorfogenese
Uploaded: 2024-01-19T13:55:00
Description: Watch this Scientific Journal Video about Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research at JoVE.com

Vi vil gerne takke Dr. Jennifer A. Zallen for at levere Sqh-GFP Drosophila-linjen og støtte til den indledende udvikling af denne teknik og Dr. Francois Schweisguth for at levere Notch-GFP Drosophila-linjen . Dette arbejde blev støttet af finansiering fra National Natural Science Foundation of China (32270809) til HH Yu, generøs økonomisk og personalestøtte fra School of Life Sciences, SUSTech og finansiering til Y. Yan fra Shenzhen Science and Technology Innovation Commission / JCYJ20200109140201722.

Forsøgene blev udført i overensstemmelse med retningslinjer og godkendelse fra School of Life Sciences, SUSTech University. Den anvendte organisme er Drosophila melanogaster, og genotyperne er Notch-Knockin-GFP (kromosom X) og Sqh-sqh-GFP (kromosom II), generøst leveret af laboratorierne af henholdsvis Dr. Francois Schweisguth (Institute Pasteur) og Dr. Jennifer Zallen (Sloan Kettering Institute). Mens denne protokol primært fokuserer på aspekter af antistofmærkning og levende billeddannelse, henvises til ...

Forbedring af proteinvisualisering med antistofmedieret Live Imaging

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Denne præsenterede procedure skitserer den specialiserede metode til fluorescensmærkning med brugerdefinerede antistoffer og efterfølgende injektion i tidlige stadier af Drosophila-embryoner . Denne teknik letter visualisering i realtid af proteiner eller posttranslationelle modifikationer, der findes i lave mængder og typisk er vanskelige at observere gennem konventionelle GFP / mCherry-mærkningsmetoder.
Der bør udvises forsigtighed, når denne metode udvides til at omfatte kva...

Immunofluorescens er en hjørnestensteknik i moderne cellebiologi, der oprindeligt blev udviklet af Albert Coons, som muliggør påvisning af molekyler i deres oprindelige cellulære rum og karakterisering af de molekylære sammensætninger af subcellulære organeller eller maskiner1. Sammen med genetiske manipulationer hjælper immunofluorescens med at etablere det nu velaccepterede koncept, at proteinlokalisering er afgørende for dets funktion2. Bortset fra specifikke primære antistoffer og lyse fluorescerende farvestoffer afhænger succesen med denne teknik af en foreløbig proces kaldet fiksering og permeabilisering, som bevarer cellulære morfologier, immobiliserer antigener og øger tilgængeligheden af antistoffer i intracellulære rum. Uundgåeligt ville fikserings- og permeabiliseringsprocessen dræbe celler og afslutte alle biologiske processer3. Derfor giver immunofluorescens kun snapshots af proteiners livsrejse. Imidlertid er mange biologiske processer såsom cellemigration og delinger dynamiske, hvilket kræver undersøgelse af proteinadfærd på en rumlig-tidsmæssigt løst måde 4,5.
For at undersøge proteindynamikken i levende organismer er der udviklet levende billeddannelsesmetoder baseret på genetisk kodede fluorescerende proteiner såsom grønt fluorescerende protein (GFP)6 og højhastighedskonfokale mikroskoper. Kort fortalt kan proteinet af interesse genetisk manipuleres til at blive smeltet sammen med GFP7 og derefter ektopisk udtrykt fra virale eller gærpromotorer såsom cytomegalovirus (CMV)8 eller opstrøms aktiveringssekvens (UAS)9. Fordi GFP er autofluorescerende i naturen, kræves der ingen fluoroforkoblede antistoffer for at afsløre lokaliseringen af målproteiner, hvilket omgår nødvendigheden af indledende fikseringsprocesser eller permeabilisering. I løbet af de sidste to årtier er fluorescerende mærker, der spænder over hele spektret af bølgelængder, blevet udviklet10, hvilket muliggør flerfarvet levende billeddannelse af flere målproteiner på samme tid. Sammenlignet med kemisk manipulerede fluorescerende farvestoffer som AlexaFluor eller ATTO er autofluorescensen af disse genetisk kodede fluorescerende proteiner imidlertid relativt svag og ustabil, når den udtrykkes fra endogene promotorer, især under levende billeddannelse over længere tidsskalaer10. Mens denne mangel kan afbødes ved overekspression af fluorescerende mærkede målproteiner, forstyrrer mange med enzymatiske aktiviteter såsom kinaser og fosfataser alvorligt normale biologiske processer, hvis de ikke udtrykkes på fysiologiske niveauer.
Denne protokol præsenterer en metode, der muliggør fototabel antistofbaseret målbelysning i en live billedopsætning, hvilket i det væsentlige tillader immunofluorescens uden fikserings- eller permeabiliseringsprocessen (figur 1). Gennem en simpel NHS-baseret primær aminreaktion11 kan man konjugere fluorescerende farvestoffer som AlexaFluor 488 eller 594 med stort set ethvert primært antistof eller GFP / HA / Myc nanobody12. Ved at udnytte en udviklingsfunktion, at alle Drosophila embryonale celler deler en fælles cytoplasma under syncytium fase13, kan man opnå antigenbinding og belysning på tværs af hele embryoner efter injektion af farvestofkonjugerede antistoffer. Med ekspanderende biblioteker af endogent mærkede proteiner, der er tilgængelige i Drosophila og andre modelsystemer14, kan denne metode potentielt udvide anvendelser af disse biblioteker ved at afsløre dynamikken i fluorescerende mærkede proteiner med lav overflod og andre ikke-fluorescerende mærkede (HA / Myc-mærkede) proteiner i levende væv.

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	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sangon Biotech</td>
			<td>&#160;A620014&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Antibody Labeling Kit</td>
			<td>Invitrogen&#160;</td>
			<td>A20185</td>
			<td>Purification column from step 1.6 is included in this kit</td>
		</tr>
		<tr>
			<td>Biological Microscope</td>
			<td>SOPTOP</td>
			<td>EX20</td>
			<td>Eyepiece lens: PL 10X/20. Objective lens: 10x/0.25</td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Clorox&#174;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>TW100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5245</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>FALCON</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccation chamber&#160;</td>
			<td>LOCK&#38;LOCK</td>
			<td>HSM8200</td>
			<td>320ml</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Mshot</td>
			<td>MZ62</td>
			<td>Eyepiece lens: WF10X/22mm.</td>
		</tr>
		<tr>
			<td>Double-sided Tape</td>
			<td>Scotch</td>
			<td>665</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Super Tweezer</td>
			<td>VETUS</td>
			<td>ST-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#8482; Cover Glasses: Rectangles</td>
			<td>Fisherbrand</td>
			<td>12-545F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#8482; Superfrost&#8482; Plus Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>VETUS</td>
			<td>33A-SA</td>
			<td></td>
		</tr>
		<tr>
			<td>Halocarbon oil 27</td>
			<td>Sigma-Aldrich</td>
			<td>H8773-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Halocarbon oil 700</td>
			<td>Sigma-Aldrich</td>
			<td>H8898-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>Sigma-Aldrich</td>
			<td>H2198-1L</td>
			<td>Heptane glue is made of double-sided tape immersed in heptane</td>
		</tr>
		<tr>
			<td>Dehydration reagent&#160;</td>
			<td>TOKAI</td>
			<td>1-7315-01</td>
			<td>Fill to 90% volume of the dessication chamber</td>
		</tr>
		<tr>
			<td>Manual Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>M3301R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>World Precision Instruments</td>
			<td>PUL-1000</td>
			<td>Procedure: step 1, Heat: 290, Force:300, Distance:1.00, Delay:50. 
			Step 2, Heat: 290, Force:300, Distance:2.21, Delay:50&#160;</td>
		</tr>
		<tr>
			<td>Pneumatic picopump</td>
			<td>World Precision Instruments</td>
			<td>PV 830</td>
			<td>Eject: 20 psi;&#160; Range: 100ms; Duration: timed</td>
		</tr>
		<tr>
			<td>PY20</td>
			<td>Santa Cruz</td>
			<td>SC-508</td>
			<td></td>
		</tr>
		<tr>
			<td>Square petri dishes</td>
			<td>Biosharp</td>
			<td>BS-100-SD</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP nanobody</td>
			<td>Chromotek</td>
			<td>gt</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing low-abundance proteins and post-translational modifications in living drosophila embryos via fluorescent antibody injection

Visualisering af proteiner i levende celler ved hjælp af GFP (Green Fluorescent Protein) og andre fluorescerende tags har i høj grad forbedret forståelsen af proteinlokalisering, dynamik og funktion. Sammenlignet med immunfluorescens afspejler levende billeddannelse mere præcist proteinlokalisering uden potentielle artefakter som følge af vævsfiksering. Det er vigtigt, at levende billeddannelse muliggør kvantitativ og tidsmæssig karakterisering af proteinniveauer og lokalisering, hvilket er afgørende for at forstå dynamiske biologiske processer såsom cellebevægelse eller deling. En væsentlig begrænsning ved fluorescerende mærkningsmetoder er imidlertid behovet for tilstrækkeligt høje proteinekspressionsniveauer for at opnå vellykket visualisering. Derfor kan mange endogent mærkede fluorescerende proteiner med relativt lave ekspressionsniveauer ikke detekteres. På den anden side kan ektopisk ekspression ved hjælp af virale promotorer undertiden føre til proteinfejllokalisering eller funktionelle ændringer i fysiologiske sammenhænge. For at løse disse begrænsninger præsenteres en tilgang, der anvender meget følsom antistofmedieret proteindetektion i levende embryoner, der i det væsentlige udfører immunfluorescens uden behov for vævsfiksering. Som bevis på princippet kan endogent GFP-mærket Notch-receptor, der næppe kan påvises i levende embryoner, visualiseres med succes efter antistofinjektion. Desuden blev denne tilgang tilpasset til at visualisere posttranslationelle modifikationer (PTM'er) i levende embryoner, hvilket muliggør påvisning af tidsmæssige ændringer i tyrosinphosphoryleringsmønstre under tidlig embryogenese og afslører en ny subpopulation af phosphotyrosin (p-Tyr) under apikale membraner. Denne fremgangsmåde kan modificeres til at rumme andre proteinspecifikke, mærkespecifikke eller PTM-specifikke antistoffer og bør være kompatibel med andre injektionsmodtagelige modelorganismer eller cellelinjer. Denne protokol åbner nye muligheder for levende billeddannelse af proteiner eller PTM'er med lav forekomst, der tidligere var udfordrende at detektere ved hjælp af traditionelle fluorescerende mærkningsmetoder.

Immunofluorescence is a cornerstone technique of modern cell biology originally developed by Albert Coons, which enables the detection of molecules at their native cellular compartments and characterization of the molecular compositions of subcellular organelles or machineries1. Coupled with genetic manipulations, immunofluorescence helps establish the now well-accepted concept that protein localization is essential for its function2. Aside from specific primary antibodies and bright fluorescent dyes, the success of this technique relies on a preliminary process named fixation and permeabilization, which preserves cellular morphologies, immobilizes antigens, and increases the accessibility of antibodies into intracellular compartments. Inevitably, the fixation and permeabilization process would kill cells and terminate all biological processes3. Therefore, immunofluorescence only provides snapshots of the life journey of proteins. However, many biological processes such as cell migration and divisions are dynamic in nature, requiring investigation of protein behaviors in a spatial-temporally resolved manner4,5.
To examine protein dynamics in living organisms, live imaging methods based on genetically encoded fluorescent proteins such as green fluorescent protein (GFP)6 and high-speed confocal microscopes have been developed. Briefly, the protein of interest can be genetically manipulated to be fused with GFP7, and then ectopically expressed from viral or yeast promoters such as cytomegalovirus (CMV)8 or upstream activation sequence (UAS)9. Because GFP is autofluorescent in nature, no fluorophore-coupled antibodies are required to reveal the localization of target proteins, which bypasses the necessity of preliminary processes of fixation or permeabilization. Over the last two decades, fluorescent tags spanning the whole spectrum of wavelength have been developed10, enabling multi-color live imaging of several target proteins at the same time. However, compared to chemically engineered fluorescent dyes such as AlexaFluor or ATTO, the autofluorescence of these genetically encoded fluorescent proteins is relatively weak and unstable when expressed from endogenous promoters, especially during live imaging over longer time scales10. While this shortfall can be mitigated by over-expressing fluorescently tagged target proteins, many with enzymatic activities such as kinases and phosphatases severely disrupt normal biological processes if not expressed at physiological levels.
This protocol presents a method that enables photostable antibody-based target illumination in a live image setup, essentially allowing immunofluorescence without the process of fixation or permeabilization (Figure 1). Through a simple NHS-based primary amine reaction11, one can conjugate fluorescent dyes such as AlexaFluor 488 or 594 with essentially any primary antibody or GFP/HA/Myc nanobody12. Taking advantage of a developmental feature that all Drosophila embryonic cells share a common cytoplasm during the syncytium stage13, one can achieve antigen binding and illumination across entire embryos after the injection of dye-conjugated antibodies. With expanding libraries of endogenously tagged proteins available in Drosophila and other model systems14, this method can potentially broaden applications of these libraries by revealing dynamics of low-abundance fluorescently tagged proteins and other non-fluorescently tagged (HA/Myc-tagged) proteins in living tissues.

Forfatter Spotlight: Afsløring af dynamikken i mekaniske og biokemiske signaler i forskning i dyremorfogenese 

Visualisering af proteiner med lav overflod og posttranslationelle modifikationer i levende Drosophila-embryoner via fluorescerende antistofinjektion

We aim to understand how mechanical and biochemical signals correlate animal morphogenesis, including the detection and the response to forces by cells and molecules. More molecules have been discovered to be mechanosensitive, functioning differently under varying levels of tension. However, the physiological context and the function of these force-sensitive events remain largely unknown.Due to the dynamic nature of force-sensitive events, our field largely relies on high-speed fluorescence imaging and time-lapse recording to capture molecular and cellular behaviors. Many fluorescently-tagged proteins are not bright enough to be imaged, and it requires spatial-temporal resolution without significant bleach. On the other hand, traditional immunofluorescence methods can only capture a snapshot of molecular and cellular fixture after fixation.This method paves the way to dynamically track the localization of many low-abundance protein receptors of major signaling pathways, such as Notch, Wnt, and the BMP pathways. Eventually, in addition to the traditional linear signaling cascades, we can pinpoint and where the signaling events occur at the subcellular scales.

For at demonstrere fordelene ved antistofinjektionsmetoden i forhold til fluorescerende tag-baseret levende billeddannelse eller immunofluorescens tilvejebringes to casestudier, der karakteriserer den dynamiske lokalisering af en transmembranreceptor med lav forekomst, Notch, og en type posttranslationel modifikation kaldet tyrosinphosphorylering i levende embryoner.
Notch signalaktivitet spiller en vigtig rolle i bestemmelse af celleskæbne under embryogenese og voksenorganhomeostase<sup clas...

Watch this Scientific Journal Video about Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research at JoVE.com

Denne protokol beskriver den tilpassede antistofbaserede fluorescensmærkning og injektion i tidlige Drosophila-embryoner for at muliggøre levende billeddannelse af proteiner med lav overflod eller posttranslationelle modifikationer, der er udfordrende at detektere ved hjælp af traditionelle GFP / mCherry-tag-tilgange.

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

Vi sigter mod at forstå, hvordan mekaniske og biokemiske signaler korrelerer dyrs morfogenese, herunder detektion og respons på kræfter fra celler og molekyler. Flere molekyler har vist sig at være mekanofølsomme og fungerer forskelligt under forskellige spændingsniveauer. Imidlertid forbliver den fysiologiske kontekst og funktionen af disse kraftfølsomme begivenheder stort set ukendt.På grund af den dynamiske karakter af kraftfølsomme begivenheder er vores felt stort set afhængig af højhastighedsfluorescensbilleddannelse og time-lapse-optagelse for at fange molekylær og cellulær adfærd. Mange fluorescerende mærkede proteiner er ikke lyse nok til at blive afbildet, og det kræver rumlig-tidsmæssig opløsning uden signifikant blegemiddel. På den anden side kan traditionelle immunfluorescensmetoder kun fange et øjebliksbillede af molekylær og cellulær fiksering efter fiksering.Denne metode baner vejen for dynamisk at spore lokaliseringen af mange proteinreceptorer med lav overflod af større signalveje, såsom Notch, Wnt og BMP-veje. Til sidst kan vi ud over de traditionelle lineære signalkaskader lokalisere, og hvor signalhændelserne forekommer på de subcellulære skalaer.

visualizing-low-abundance-proteins-post-translational-modifications

Research

JoVE Journal

Developmental Biology

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.