Name: Gold Nanoparticle Synthesis for Mammalian Cells Using Sapphire Discs
Uploaded: 2023-04-28T12:20:00
Description: Watch this Scientific Journal Video about Gold Nanoparticle Synthesis for Mammalian Cells Using Sapphire Discs at JoVE.com

The video outlines a detailed protocol for preparing sapphire discs with HeLa cells for imaging and analysis of protein distribution. It involves several steps, including washing, incubation with reducing and gold precursor solutions, polymerization in epoxy resin, and freeze-thaw techniques, leading to the visualization of proteins in cellular structures.

gold nanoparticle synthesis for mammalian cells using sapphire discs

Síntesis directa de nanopartículas de oro para el análisis de localización de proteínas

In the postgenomic era, the development of green fluorescent protein (GFP) as a single-molecule reporter for light microscopy has revolutionized the field of modern life science research1,2. For decades, electron microscopy (EM) has been a powerful tool for intuitively observing the cellular ultrastructure with nanoscale resolution3; however, the precise identification and localization of protein molecules remain challenging.
The most commonly used EM labeling technique is the immunoelectron microscopy (IEM) labeling technique, which is based on the antigen-antibody reaction. However, although many techniques have been developed in the field of IEM labeling, including pre-embedding IEM and post-embedding IEM (on resin sections or hydrated cryosections), it still suffers from low labeling efficiency (&#60;10%)4,5, which is related to the sample preparation and antibody quality. To overcome these limitations, developing genetically encoded tags has great application potential.
Two main types of EM tags have been thoroughly explored in recent years. One type is the DAB staining method, which utilizes tags such as APEX2 to oxidize 3,3&#39;-diaminobenzidine (DAB) to osmiophilic polymers for EM visualization6,7,8,9,10,11,12. It enables the labeling of high-abundance proteins in subcellular regions but is not suitable for single-molecule counting. The other type utilizes metal-binding proteins, such as ferritin13 and metallothionein (MT)14,15,16,17,18,19,20,21,22,23,24,25,26, to generate electron-dense metal deposits in situ for EM visualization. Only the latter has real potential for single-molecule visualization and counting. The molecular size of ferritin is too large (~450 kD) for it to be used as a promising tag, whereas the small size (~5 kD) of MT and its ability to bind various ions through its 20 cysteines have attracted great attention. Several labs have tried to label purified MT-fusion proteins or MT-expressing cells by incubating directly with Au+. These attempts have initially proved that MT tags can bind gold ions to form high-contrast signals, but none have really achieved the effective identification of individual proteins in cells, and they are not widely applicable14,15,16,17,18,19,20,21,22,23.
The ANSM-based AuNP synthesis technique, which involves synthesizing 2-6 nm-sized AuNPs directly on cysteine-rich tags (e.g., MT, the MT variants MTn and MT&#945;, AFP) as electron-dense labels for EM visualization, is the first reliable and applicable approach for protein labeling and single molecule detection in cells24,25,26. It allows for the specific synthesis of AuNPs on isolated tag fusion proteins and has achieved an unprecedented labeling efficiency in nonfixed or chemically fixed prokaryotic (E. coli) and eukaryotic (S. pombe) cells. However, implementing the same protocol in more advanced systems such as mammalian cells or even tissues involves additional challenges, such as the more complex intracellular redox homeostasis and more fragile cellular structure.
This study presents a clonable EM labeling technology, which combines the novel ANSM-based AuNP synthesis technique for labeling genetically encoded cysteine-rich tags (MT) with the HPF/FSF-rehydration-HPF/FSF sample preparation method, enables the unambiguous single-molecule identification of tagged proteins in the ER membrane, ER lumen, and mitochondrial matrix in HeLa cells. The current method combines the characteristics of high labeling efficiency, a high signal-to-noise ratio, single-molecule labeling, and strong universality, and this method has broad application prospects in life science research.

Autor destacado: Síntesis directa de nanopartículas de oro EM-visible en células para el análisis de localización de proteínas con ultraestructura bien conservada  

Síntesis directa de nanopartículas de oro EM-visibles en células para análisis de localización de proteínas con ultraestructura bien conservada

Protein detection and localization are essential in biological research. Our protocol describes a novel EM-labeling technology using genetically-encoded tags which allows single-molecule visualization in situ with well-preserved ultrastructure. It is crucial to avoid over-fixation.The labeling method is based on the activity of cysteines in the tags, which are highly sensitive to aldehyde cross-linking. Our protocol avoids use of aldehyde fixatives and preserves the tag activity and ultrastructure. at the same time.EM labeling has always been challenging, suffering from poor morphology or inefficient labeling. Our protocols synthesizes in situ nanoparticles directly on individual proteins to provide clear and precise localization of the target proteins, with a high signal-to-noise ratio, high efficiency, and specificity. We have so far achieved excellent labeling in isolated proteins, E.coli cells, yeast cells, and HeLa cells.We will explore and optimize the method for application to tissue samples. Combining the prevailing cryo-FIB and cryo-EM technologies will allow us to address important biological questions.

Watch this Scientific Journal Video about Gold Nanoparticle Synthesis for Mammalian Cells Using Sapphire Discs at JoVE.com

Gold Nanoparticle Synthesis for Mammalian Cells Using Sapphire Discs  

Síntesis de nanopartículas de oro para células de mamíferos usando discos de zafiro

Tome un disco de zafiro tratado con HPF y FSF previamente preparado que contenga células HeLa estables, lávelo con tampón PBS-A durante cinco minutos y repita este proceso tres veces. Transfiera los discos de zafiro a una placa de cultivo de 35 milímetros que contenga un mililitro de tampón PBS-A a temperatura ambiente. Reemplace el tampón PBS-A en la placa de cultivo con un mililitro de solución reductora e incube durante una hora a temperatura ambiente.Prepare el precursor de oro en un mililitro de solución reductora e inmediatamente mezcle por vórtice. Agregue 80 microlitros de D-penicilamina de 500 milimolares en agua destilada doble a la solución precursora de oro. E inmediatamente vórtice.Reemplace la solución en el plato cultivado con un mililitro de solución precursora de oro e incube durante dos horas a cuatro grados centígrados. Agregue de 20 a 100 microlitros de la solución de borohidruro de sodio recién preparada a la solución precursora de oro, agite inmediatamente para mezclar bien e incube durante cinco minutos a temperatura ambiente. Reemplace la solución con dos mililitros de tampón PBS-A para detener la reacción.Luego prepare la mezcla de resina epoxi de acuerdo con las instrucciones del fabricante, transfiera los discos de zafiro a cápsulas de incrustación de fondo plano e infiltre con resina durante al menos 30 minutos a temperatura ambiente. Polimerizar la muestra a 60 grados centígrados en un horno durante al menos 18 horas. Prepare secciones ultrafinas de la muestra polimerizada recortando los bloques de la muestra cuidadosamente con una navaja de afeitar para exponer los discos de zafiro.Sumerja las puntas de los bloques con discos de zafiro en nitrógeno líquido durante varios segundos y descongele la temperatura ambiente. Repita la acción de congelación-descongelación varias veces para separar los discos de los bloques. La proteína EGFP-MTn-KDEL apareció como nanopartículas de oro de dos a cinco nanómetros de tamaño distribuidas exclusivamente en el lumen periférico del RE y en el espacio perinuclear de la envoltura nuclear.La ultraestructura bien conservada permitió la identificación de moléculas individuales de proteínas marcadas y facilitó el análisis de las interacciones de orgánulos, como las interacciones de las mitocondrias ER. Las nanopartículas de la proteína Ost4-EGFP-MTn delinearon la membrana ER en la membrana externa de la envoltura nuclear. Del mismo modo, las células que expresan Mito-acGFP-MTn exhibieron un etiquetado específico en la matriz mitocondrial.

Research