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

Mejora de la visualización de proteínas con imágenes en vivo mediadas por anticuerpos

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.

Autor destacado: Desvelando la dinámica de las señales mecánicas y bioquímicas en la investigación de la morfogénesis animal 

Visualización de proteínas de baja abundancia y modificaciones postraduccionales en embriones vivos de Drosophila mediante inyección de anticuerpos fluorescentes

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  

Inyección de anticuerpos marcados con fluorescencia en embriones vivos de Drosophila y obtención de imágenes de proteínas de baja abundancia

Para comenzar, llene cada aguja con cinco microlitros de solución de nanocuerpo GFP marcada con Alexa Fluor. Coloque un cubreobjetos de 25 por 25 milímetros sobre una superficie deslizante de vidrio. Pipetear 40 microlitros de aceite de Halocarbon en el portaobjetos y extenderlo a lo largo del perímetro del cubreobjetos.Bajo el microscopio de inyección, coloque la punta de la aguja contra el borde del cubreobjetos sumergido en aceite. Ajuste la presión del aire de inyección de la PicoPump en consecuencia. Reemplace el portaobjetos de vidrio vacío con el portaobjetos de embriones de Drosophila preparado.Coloque la punta de la aguja de inyección perpendicular al eje anteroposterior del embrión. Use el manipulador de la etapa XY para guiar a los embriones hacia la aguja hasta que la punta llegue al centro de la yema, y bombee dos o tres veces para infundir anticuerpos en la yema. Con el manipulador de la etapa XY, aleje los embriones de la aguja después de la inyección y pase al siguiente embrión.Dejar que los embriones se incuben a 25 grados centígrados hasta alcanzar la etapa de desarrollo deseada. Después de la incubación, identifique los embriones bajo una lente de baja potencia con iluminación de campo brillante y cambie a una lente de 40X o 63X para obtener imágenes fluorescentes en vivo de alta resolución. Después de la inyección, la relación señal-ruido del receptor notch mejora significativamente en comparación con la calidad de la señal de la inmunofluorescencia.Además, la inyección de anticuerpos permitió la caracterización temporal de la localización de la muesca a intervalos de 45 segundos sin una pérdida aparente de la intensidad de la señal durante una ventana de imágenes de cinco minutos. Las imágenes en vivo de la fosfotirosina embrionaria mostraron que la fosforilación de la tirosina está altamente enriquecida en las uniones tricelulares. Además, se observó una nueva segunda población de señal de fosfotirosina debajo del centro de la membrana apical.Las imágenes de doble color revelaron que esta población de señal de fosfotirosina está cerca de la miosina medial. Además, la población medial de fosfotirosina exhibió patrones de coalescencia y disipación pulsátil similares, como se muestra para la miosina medial.

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 ...

Investigación

Biología del Desarrollo

Fluorescent Labeling of Antibodies for Imaging Low Abundance Proteins in Drosophila

Fluorescent Labeling of Antibodies for Imaging Low Abundance Proteins in  Drosophila   

Marcaje fluorescente de anticuerpos para obtener imágenes de proteínas de baja abundancia en Drosophila

Preparation and Alignment of Drosophila Embryos for Injecting Fluorescently Labeled Antibodies

Preparation and Alignment of Drosophila  Embryos for Injecting Fluorescently Labeled Antibodies  

Preparación y alineación de embriones de Drosophila para la inyección de anticuerpos marcados con fluorescencia