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

단백질 국소화 분석을 위한 금 나노입자의 직접 합성

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.

저자 스포트라이트: Direct synthesis of EM-Visible gold nanoparticles in cells for Protein Localization Analysis with Well-Preserved Ultrastructure(잘 보존된 초세구조를 이용한 단백질 국소화 분석을 위한 세포 내 EM-Visible 금 나노입자의 직접 합성)

잘 보존된 미세 구조를 가진 단백질 국소화 분석을 위한 세포에서 EM-Visible Gold Nanoparticles의 직접 합성

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  

사파이어 디스크를 사용한 포유류 세포를위한 금 나노 입자 합성

안정한 HeLa 세포를 함유하는 사전 준비된 HPF 및 FSF 처리된 사파이어 디스크를 취하여 PBS-A 완충액으로 5분 동안 세척하고 이 과정을 3회 반복합니다. 사파이어 디스크를 실온에서 1밀리리터의 PBS-A 버퍼가 들어 있는 35mm 배양 접시로 옮깁니다. 배양 접시 내의 PBS-A 완충액을 1밀리리터의 환원액으로 교체하고 실온에서 1시간 동안 인큐베이션한다.환원액 1 밀리리터에 금 전구체를 준비하고 즉시 와동 처리하여 혼합하십시오. 이중 증류수에 80 밀리리터의 500 밀리몰 D- 페니실라 민을 금 전구체 용액에 첨가한다. 그리고 즉시 소용돌이.배양 접시의 용액을 1 밀리리터의 금 전구체 용액으로 교체하고 섭씨 4도에서 2 시간 동안 배양합니다. 새로 제조된 수소화붕소나트륨 용액 20 내지 100 마이크로리터를 금 전구체 용액에 첨가하고, 즉시 잘 섞이도록 흔들어주고, 실온에서 5분 동안 배양한다. 용액을 2밀리리터의 PBS-A 완충액으로 교체하여 반응을 중지시킨다.그런 다음 제조업체의 지침에 따라 에폭시 수지 혼합물을 준비하고 사파이어 디스크를 평평한 바닥 매립 캡슐로 옮기고 실온에서 최소 30분 동안 수지에 침투시킵니다. 시편을 섭씨 60도의 오븐에서 최소 18시간 동안 중합합니다. 사파이어 디스크를 노출시키기 위해 면도기를 사용하여 시편 블록을 조심스럽게 다듬어 중합된 시편의 초박형 부분을 준비합니다.사파이어 디스크로 블록 팁을 액체 질소에 몇 초 동안 담그고 실온을 해동합니다. 동결-해동 작업을 여러 번 반복하여 블록에서 디스크를 분리합니다. EGFP-MTn-KDEL 단백질은 말초 ER 루멘과 핵 외피의 핵 주위 공간에 독점적으로 분포 된 2-5 나노 미터 크기의 금 나노 입자로 나타났다.잘 보존된 울트라 구조는 태그가 부착된 단백질의 단일 분자 식별을 가능하게 하고 ER 미토콘드리아 상호 작용과 같은 세포 기관 상호 작용의 분석을 용이하게 했습니다. Ost4-EGFP-MTn 단백질의 나노 입자는 핵 외피의 외막에서 ER 막을 묘사했다. 마찬가지로, Mito-acGFP-MTn 발현 세포는 미토콘드리아 기질에서 특이적 표지를 나타내었다.

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