injecting fluorescently labeled antibodies in live drosophila embryos and imaging of low abundance proteins

Verbetering van eiwitvisualisatie met antilichaam-gemedieerde live beeldvorming

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.

Auteur in de schijnwerpers: onthulling van de dynamiek van mechanische en biochemische signalen in onderzoek naar morfogenese bij dieren 

Visualisatie van eiwitten met een lage abundantie en posttranslationele modificaties in levende Drosophila-embryo's via fluorescerende antilichaaminjectie

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.

Watch this Scientific Journal Video about Injecting Fluorescently Labeled Antibodies in Live Drosophila  Embryos and Imaging of Low Abundance Proteins at JoVE.com

Injecting Fluorescently Labeled Antibodies in Live Drosophila  Embryos and Imaging of Low Abundance Proteins  

Injecteren van fluorescerend gelabelde antilichamen in levende Drosophila-embryo's en beeldvorming van eiwitten met een lage abundantie

Vul om te beginnen elke naald met vijf microliter Alexa Fluor-gelabelde GFP-nanobody-oplossing. Plaats een dekglaasje van 25 bij 25 millimeter op een glasplaatje. Pipetteer 40 microliter Halocarbon-olie op het glaasje en verdeel het langs de omtrek van het dekglaasje.Plaats de punt van de naald onder de injectiemicroscoop tegen de rand van het dekglaasje ondergedompeld in olie. Pas de injectieluchtdruk van de PicoPump dienovereenkomstig aan. Vervang het lege glasplaatje door het voorbereide Drosophila embryoglaasje.Zet de punt van de injectienaald loodrecht op de voorste-achterste as van het embryo. Gebruik de XY-stadiummanipulator om embryo's naar de naald te leiden totdat de punt het midden van de dooier bereikt en pomp twee tot drie keer om antilichamen in de dooier te brengen. Met de XY-stadiummanipulator stuurt u embryo's na injectie weg van de naald en gaat u naar het volgende embryo.Laat de embryo's bij 25 graden Celsius incuberen tot ze het gewenste ontwikkelingsstadium hebben bereikt. Identificeer na incubatie de embryo's onder een lens met laag vermogen en heldere veldverlichting en schakel over naar een 40X- of 63X-lens voor fluorescerende live-beeldvorming met hoge resolutie. Na injectie verbetert de signaal-ruisverhouding van de notch-receptor aanzienlijk, vergelijkbaar met de signaalkwaliteit van immunofluorescentie.Bovendien maakte antilichaaminjectie de temporele karakterisering van de lokalisatie van de inkeping mogelijk met tussenpozen van 45 seconden zonder een duidelijk verlies van signaalintensiteit gedurende een beeldvormingsvenster van vijf minuten. Live beeldvorming van embryonaal fosfotyrosine toonde aan dat tyrosinefosforylering sterk verrijkt is bij de driecellige overgangen. Bovendien werd een nieuwe tweede populatie van fosfotyrosinesignalen waargenomen onder het midden van het apicale membraan.Tweekleurige beeldvorming onthulde dat deze populatie van fosfotyrosinesignalen zich in de buurt van mediaal myosine bevindt. Bovendien vertoonde de mediale populatie van fosfotyrosine vergelijkbare pulsatiele coalescentie- en dissipatiepatronen, zoals aangetoond voor mediaal myosine.

Research

JoVE Journal

Developmental Biology

Injecting Fluorescently Labeled Antibodies in Live Drosophila Embryos and Imaging of Low Abundance Proteins

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 ...

Fluorescent Labeling of Antibodies for Imaging Low Abundance Proteins in Drosophila

Fluorescent Labeling of Antibodies for Imaging Low Abundance Proteins in  Drosophila   

Fluorescerende etikettering van antilichamen voor het afbeelden van eiwitten met een lage abundantie in Drosophila

Preparation and Alignment of Drosophila Embryos for Injecting Fluorescently Labeled Antibodies

Preparation and Alignment of Drosophila  Embryos for Injecting Fluorescently Labeled Antibodies  

Voorbereiding en uitlijning van Drosophila-embryo's voor het injecteren van fluorescerend gelabelde antilichamen