이 연구는 보건 서비스 교부금에 의해 지원되었다 AI - 34431 알레르기 및 전염병 국립 연구소에서와 VA 메디컬 연구 기금 (DAH로)로 (DAH)을.

1. 유리 슬라이드에 Leptospira의 고정 <ol><li> Leptospira interrogans는 클래스 BSL2 병원체이다. 라이브 전지 작업은 같은 장갑을 끼고, 실험실 - 코팅 및 살균 - 모자 pipetting 단계로 적절한 처리가 필요합니다. </li><li> 30 ° C 그들이 도달할 때까지 중순에 약 6 일 늦은 로그 단계 (5 X 10 8 셀 / ML 5 × 10 7 밀도). 1 %의 토끼 혈청과 보충, EMJH medium11에서 Leptospira 그로 </li><li> 상온에서 7 분 ~ 2000 XG에서 원심 분리하여 문화를 수확. </li><li> 흡인에 의해 표면에 뜨는를 제거하고 부드럽게 인산염의 펠렛을 resuspend 5 X 10 8 셀 / ML의 최종 농도 호수 (PBS) -5 MM에게 MgCl 2, pH7.2를 버퍼. </li><li> 두 잘 챔버 유리 슬라이드의 각 웰에 세포 현탁액 1 ML을 추가하고 30 품어 ° C를 80 분에 대한 세포를 준수하도록합니다. </li><li> 조심스럽게 흡인하여 언바운드 세포가 들어있는 액체를 제거합니다. </li><li> / 잘 PBS - 5 MM MgCl 2 2 % paraformaldehyde 1 ML 추가하여 유리 슬라이드에 남아있는 그대로 박테리아를 수정. 30 40 분 품어 ° C. 이러한 슬라이드 표면 단백질의 노출 평가를위한 것입니다. </li><li> 하위 표면 단백질을 평가 제어 슬라이드에, 얼음처럼 차가운 메탄올 1 ML / 물론 100 %로 고정하여 외부 막을 permeabilize. 20 분 -20 ° C에서 알을 품다. 메탄올은 여러 가지 방법으로 행동 : 그것은 외부 막을 permeabilizes 단백질 denaturates하고, 유리 슬라이드에 수정 세포. </li><li> 열망의 요원을 고정 제거합니다. </li></ol> 2. 특정 항체와 라벨링 <ol><li> / 잘 차단 버퍼 (Difco Leptospira 농축 EMJH) 1 ML 추가하여 불특정 바인딩을 차단합니다. 90 분 30 ° C에서 알을 품다. </li><li> 특정 항체 (면역 토끼 세라 드나 마우스 단클론 항체, OmpL54 10 특정 여기 polyclonal 토끼 세라, FlaA1 12, 및 OmpL1 13)과 아무런 항체를 포함하지 미리 면역 토끼 세라이나 마우스 고행 유체 (부정적인 컨트롤로 활용시)에 희석 버퍼를 차단. 각 항체에 대한 Dilutions는 경험적으로 세포에 항원 - 항체 반응과 단백질의 풍부한, 항체 titer에 따라 결정해야합니다. 일반적인 범위는 1:600​​으로 1시 50분입니다. </li><li> 흡인에 의해 차단 버퍼를 제거하고 1 ML / 음의 희석 차 항체를 추가합니다. </li><li> 1 시간 30 ° C에서 알을 품다. </li><li> 흡인하여 액체를 제거하고 PBS (1 ML / 음)와 우물을 세 번 씻으십시오. </li></ol> 3. leptospires의 시각화 <ol><li> 1 ML / 음의 알렉사 희석 플루어 488 - 라벨 보조 항체 (염소 안티 - 토끼 IgG 또는 염소 방지 마우스 IgG)를 1:2000 및 형광 핵산 얼룩, 4&#39;6 - diamidino - 2 - 페닐 - 인돌 dihydrochloride을 (추가 DAPI)는 0.25 μg / 버퍼를 차단에 ML의 최종 농도로 희석. 이 단계는 항체 바인딩 및 바인딩, 각각 항체의 독립 모든 spirochetes의 존재의 확인을 보장합니다. </li><li> ° C 45 분 30에 슬라이드를 품어. </li><li> 흡인하여 액체를 제거하고 증류수 (1 ML / 음)로 두 번 PBS와 한 우물을 씻으십시오. </li><li> 실과 유리 슬라이드와 ~ 10 분 건조 공기의 접착 스트립을 제거합니다. </li><li> 골드 방지 퇴색 설치 매체 (슬라이드 당 20 μl 2 X)과 24 X 50mm 커버 슬립을 연장 추가합니다. </li><li> 어둠 속에서 실온에서 하룻밤 품어. 이 단계는 설치 매체 (강화) 치료하도록하는 것이 필요합니다. </li><li> 매니큐어로 커버 슬립을 봉쇄. </li><li> DAPI에 대한 시안 / 블루 감지 필터와 알렉사 플루어 488에 녹색 검색 필터를 사용하여 형광 현미경으로 얼룩을 시각화. 샘플의 정확한 표현이 만들어되었는지 확인하려면 각 샘플에 대한 전체 챔버 영역을 평가할 수 있는지 확인하십시오. </li><li> 이미징 각 샘플에 대한 대표적인 필드로 데이터를 기록합니다. 이미지 알렉사 플루어 488 형광이 모든 샘플에 대해 같은 노출 시간을 사용하면. 일관된 노출 시간을 사용하면 테스트 항원 대 컨트롤과 함께 결과를보다 정확한 비교를하실 수 있습니다. </li></ol> 4. 대표 결과 : 표면 및 하위 표면 단백질에 대한 항체를 사용하여 Leptospira interrogans 세포와 표면 IFA 결과의 예는 그림 2에 표시됩니다. 실질적인 형광이 표면 (그림 2A) 노출 관심 단백질에 대한 손상 leptospires과 샘플에 감지되면 시험은 긍정적인 것으로 간주됩니다. 보통 긍정적인 검사 (여기 OmpL54을 표면 노출)에, 약 같은 형광 강도는 그것은 표면 하의에 대한 항체를 사용하여 바깥쪽 - 멤브레인 무결성에 대한 부정적인 컨트롤을 포함하는 것이 필수적입니다 (그림 2A) 온전하고 permeabilized 모두 세포에서 관찰됩니다 선호 periplasmic 단백질 (예 : leptospiral FlaA1, 그림. 2B). 데이터는 외부 세포막이 손상 듀리 유지했음을 표시하는 경우에만NG 실험, 하위 표면 단백질에 대한 즉, 항체가 그대로 세포 (그림에서와 같이 permeabilized 샘플에 비해 형광 또는 훨씬 약한 형광 안돼. 2B)에 반응하지 그것은 관심의 단백질 (S)가 있다고 결론을 내렸다 수 있습니다 표면은 노출. 추가 부정적인 제어로 사전에 면역 세라 (polyclonal 항체의 경우)와 실험의 포함은 모호하지 않은 데이터를 위해 필수적이며, 그대로 또는 permeabilized 세포 (그림 2A, 2C)에 상당한 형광를 얻을 수 없습니다. 얼룩 DAPI은 부정적인 결과가 (NO 알렉사 플루어 488 형광) 관찰 때 leptospires을 시각화하는 것이 필요합니다. propidium 요오드화물의와 Counterstaining 것은 DAPI에 수용 대안입니다. 이 방법은 같은 풍요의 다른 항원에 비해 하나의 항원에 있지만 적은 표면에 노출된 epitopes로 인정되고 antigenic epitopes를 노출의 여러 표면으로 인해 발생했을 수 있습니다 질적보다는 밝은 형광으로 양적됩니다. 따라서 형광 강도는 등 서로 다른 단백질 간의 손상 대 permeabilized 세포 형광하지 농도와 같은 항체의 반응을 비교하는 데 사용할 수 있습니다. 이 구별은 훨씬 적은 형광이 permeabilized 세포 (그림 2C)에 비해 그대로 세포에서 관찰 어디에 모호한 결과를 얻을 때 중요하다. 이러한 결과 중 단백질 (그대로 세포의 일부가 아닌 구체적인 배경 얼룩 포함) 하위 표면 위치에 있음을 나타냅니다, 항체는 여러 수도 우선적으로 메탄올의 permeabilization 또는 단백질 후 노출이 아닌 기본 epitopes에 구속력이 지역화 사이트 같은 다른 spirochete, Borrelia burgdorferi 14 ERP 및 ELP 단백질에 대해 설명했다. 각각의 경우에, 이와 같은 모호한 IFA 결과가 관심의 단백질 표면 노출 여부 10 여부를 확인하는 등 표면 biotinylation 또는 표면 proteolysis로 대체 접근이 필요합니다. <img src="/files/ftp_upload/2805/2805fig1.jpg" alt="그림 1"/> Leptospira interrogans spirochetes의 단백질 형광 분석 표면에 대해 그림 1. 플로우 차트. 첫째, 세포 현탁액 1 ML은 두 잘 챔버 슬라이드의 각 잘 추가 (각 실험에 대해 적어도 두 개의 슬라이드)와 준수 leptospires위한 incubated입니다. 다음 leptospires 그대로 외부 멤브레인 (OM)와 permeabilized OM과 샘플 1 ML / 물론 100 % 차가운 메탄올과 샘플 / 음 2 % paraformaldehyde (PFA) 1 ML 추가하여 챔버 슬라이드에 고정됩니다. 다음 leptospires 30에서 표면 하의 단백질 (주 AB의) · 90 분 C에 대한 제어 항체와 함께 표면 노출 단백질을 인식 항체와 incubated 있습니다. 그런 다음, 기본 항체는 / 잘 알렉사 플루어 488 복합 이차 항체 1 ML과 부화에 의해 감지됩니다. 마지막으로, 세포는 형광 현미경에 의해 시각입니다. <img src="/files/ftp_upload/2805/2805fig2.jpg" alt="그림 2"/> leptospiral 단백질의 그림 2. immunofluorescence 표면 분석. Leptospira interrogans spirochetes는 면역 및 Pre - 면역 세라와 읽혔어요하고, 부정적인 결과는 spirochetes의 존재를 입증하는 DAPI counterstain와 결합했다. 녹색 형광 필터는 알렉사 플루어 세포 DNA에 바인딩 DAPI를 검색에 노출 단백질 및 파랑 / 시안 형광 필터를 표면에 바인딩 488 레이블 차 항체를 감지하는 데 사용되었다. A) Leptospires는 표면에 노출된 단백질 OmpL54 10 인식 항체와 탐지. B) 세포는 periplasmic 표면 하의 단백질, FlaA1 (대조군)에 대한 항체와 탐지. C) 세포 leptospiral porin, OmpL1에 대한 항체와 탐지. leptospires에 토끼 세라의 바인딩은 알렉사 플루어 488 복합 염소 안티 - 토끼 IgG의 파편 발견했다. 모든 이미지는 4 초 동안 노출 후 촬영됩니다.

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관심 없음 충돌 선언하지 않습니다.

우리의 표면 IFA 기술은 다양한 borrelial 15, 16 leptospiral 12, 17 단백질의 표면 노출을 증명하기 위해 사용하는 것과 비슷합니다. 표면 IFA 방법의 내용은 유리 슬라이드에 충실하기 위해 Leptospira 세포의 경향을 활용하면서 외부 멤브레인 무결성을 유지하기 위해 세포의 조작을 최소​​화하기 위해 설계되었습니다 여기에 설명되어 있습니다. 이 메서드는 이전에 다음 ID로 출판 프로토?...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> 두 잘 챔버 유리 슬라이드 </td><td> 랩 - 테크 </td><td> 177380 </td><td></td></tr><tr><td> 토끼 혈청 </td><td> 로클랜드 Immunochemicals </td><td> D209 - 00-0100 </td><td></td></tr><tr><td> Leptospira 농축 EMJH </td><td> BD Difco </td><td> 279510 </td><td></td></tr><tr><td> 알렉사 플루어 488 염소 안티 - 토끼 IgG (H + L) </td><td> Invitrogen / 분자 프로브 </td><td> A - 11034 </td><td></td></tr><tr><td> 알렉사 플루어 488 염소 안티 - 마우스 IgG (H + L) </td><td> Invitrogen / 분자 프로브 </td><td> A - 11029 </td><td></td></tr><tr><td> 4&#39;6 - diamidino - 2 - 페닐 - 인돌 dihydrochloride (DAPI) </td><td> Invitrogen / 분자 프로브 </td><td> D1306 </td><td></td></tr><tr><td> 골드 안티 페이드 설치 매체를 연장 </td><td> Invitrogen / 분자 프로브 </td><td> P36930 </td><td> 사용하기 전에 실온에 평형 </td></tr><tr><td> Fisherfinest 프리미엄 커버 유리 </td><td> 어부 </td><td> 12-544-14 </td><td></td></tr><tr><td> 형광 현미경 </td><td> 자이스 혈구 </td><td> Axioskop 40 </td><td></td></tr></tbody></table>

immuno-fluorescence assay of leptospiral surface-exposed proteins

박테리아 표면 단백질은 호스트 세포와 직접 접촉과 환경 1 영양분의 이해에 관여하고 있습니다. 이러한 이유로, 세포 지방화는 박테리아 단백질의 기능 역할에 대한 통찰력을 제공할 수 있습니다. 세균성 단백질의 표면 지방화는 pathogenicity의 메커니즘에 관련된 독성 요소의 식별 향한 중요한 단계입니다. leptospiral 세포막의 2-5 fractionating위한 방법은 lipoproteins 대 transmembrane의 OMPs으로 바깥쪽 - 막 단백질 (OMPs)의 특정 클래스에 대한 선택 수 있으며, 따라서 misclassification 발생할 수 있습니다. 이 가능성은 구조적 차이가 어떻게 그들이 바깥 막으로 연결되어 있기 때문입니다. Lipoproteins은 N - 터미널 지질 잔기 (세명 지방산)과 지질 이중층의 phospholipids 6, 7 사이의 소수성 상호 작용을 통해 세포막과 연결되어 있습니다. 반면, transmembrane의 OMPs 일반적으로 배럴과 같은 구조 8, 9 배열 β - 시트 amphipathic에 의해 지질 이중층에 통합되어 있습니다. 또한, 바깥쪽 - 막의 단백질의 존재는 반드시 단백질 또는 도메인이 표면에 노출되는 것을 보장하지 않습니다. Spirochetal 외부 점막이 하위 표면 단백질의 세포와 포함의 부드러운 조작과 관련된 할거 그러므로 연약과 방법 것으로 알려져하는 것은 바깥 막의 무결성을 평가하기 위해 제어합니다. 여기, 우리는 직접적으로 손상 leptospires에 단백질의 표면 노출을 평가하기 위해 immunofluorescence 분석 (IFA) 메소드를 제시한다. 이 방법은 항원에 특정한 항체에 의해 leptospiral 표면 단백질의 인식을 기반으로합니다. 여기에, OmpL54 10 특정 항체는 기본, 표면 노출 epitopes에 aftero 바인딩 detetcted 있습니다. 손상 대 permeabilized 세포에 항체 반응의 비교는 단백질 leptospiral 표면에 선택적으로 존재 여부 세포 배포 평가를 가능하게합니다. 외부 막의 무결성이 선호 periplasm에있는 하나 이상의 표면 하의 단백질에 항체를 사용하여 평가해야합니다. 표면 IFA 방법은 특정 항체가 사용할 수있는 어떤 leptospiral 단백질의 표면 노출을 분석하는 데 사용할 수 있습니다. 방법의 유용 성과 한계는 모두 사용된 항체가 기본 epitopes에 바인딩할 수 있는지 여부에 따라 달라집니다. 항체가 자주 재조합 단백질, 기본, 표면 노출 단백질의 epitopes에 대한 사육되기 때문에 인정되지 않을 수 있습니다. 그럼에도 불구하고, 표면 IFA 방법은 그대로 세균 표면의 구성 요소를 공부를위한 유용한 도구입니다. 이 방법은 leptospires뿐만 아니라 다른 spirochetes 및 그람 음성 세균에 대해뿐만 아니라 적용할 수 있습니다. OMPs의 표면 노출에 대한 강한 결론에 대한 여러 셀 로컬 라이제이션 방법을 포함한 종합적인 접근 방식은 10 권장합니다.

Watch this Scientific Journal Video about Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins at JoVE.com

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

leptospiral 단백질의 표면 노출을 평가하기위한 효율적인 방법이 설명되어 있습니다. 방법은 특별히 leptospiral 세포의 연약한 바깥 막의 중단을 방지하도록 설계되었습니다. 이 기술은 몇 가지 부정적인 컨트롤의 고용은 바깥 막과 항체 반응의 특이성의 무결성을 평가해야합니다.

Leptospiral 표면 노출 단백질의 단백질 형광 분석

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

immuno-fluorescence-assay-of-leptospiral-surface-exposed-proteins

Research

JoVE Journal

Immunology and Infection

15.7K 조회수.  University of California, Los Angeles. leptospiral 단백질의 표면 노출을 평가하기위한 효율적인 방법이 설명되어 있습니다. 방법은 특별히 leptospiral 세포의 연약한 바깥 막의 중단을 방지하도록 설계되었습니다. 이 기술은 몇 가지 부정적인 컨트롤의 고용은 바깥 막과 항체 반응의 특이성의 무결성을 평가해야합니다.

비디오: Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

실시간, TCR의 동시 이미징 및 관련 신호 전달 단백질에 대한 TIRF 현미경 기법

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

연구

면역학 및 감염병학

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

손쥐 노로 바이러스에 대한 상패 분석

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

형광 태그 바시니아 바이러스 단백질의 효율적인 생성을위한 방법론

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

형광 단백질을 사용하면 아프리카 Trypanosome에서 Glycosome 역학을 모니터링하려면

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

파지 Phenomics : 생리 학적 접근 방법은 소설 바이러스 성 단백질의 특성을

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Arenavirus 셀 첨부 파일의 측정을위한 고감도 분석

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

정량적 마이크로 중화 분석의 최적화

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Plasmodesmal 지역화 시퀀스 Planta에 단백질의 식별

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

재조합 형 단백질의 생체 외에서 사이트별 Glycosylation에 대 한 대상 시스테인 Thiols

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.