이 작품은 NIAID, NIH에서 교내 기금에 의해 지원됩니다.

1. Dictyostelium의 discoideum의 chemotactic 관할 세포의 준비 <ol><li> chemoattractant 캠프 22 떨고 문화 D3 - T 리치 미디어의 성장 수확 전지 ° C.에 chemotactic 아르 D.의 discoideum 세포를 생성하려면 </li><li> (DB 버퍼 5 MM 나 2 HPO 4, 5 MM KH 2 PO 4, 2 MM MgCl 2, 0.1 MM CaCl 2 포함) 이외의 영양 발달 버퍼에 두 번 세포를 씻으십시오. </li><li> 2x10 7 셀 / ML의 밀도에 DB 버퍼의 세포를 다시 일시 중지합니다. </li><li> 한 시간 만이라도 22 100 RPM ° C에서 250 ML 플라스크 10 ML 세포를 흔들어. </li><li> 75 NM 캠프의 최종 농도, 캠프 pulsing 치료로 지정된 프로세스를 달성 6 시간 이상 매 6 분마다 10 ML 세포에 7.5 μm의 캠프 주식 100 μl를 제공합니다. 캠프 pulsing 처리, D.의 5~6시간 후 discoideum 전지 캠프 기울기 방향으로 능력 chemotactic된다. </li><li> 5 분 200g에서 원심 분리하여 세포를 수집하고 DB 버퍼로 resuspend 셀 2.5 MM의 카페인을 포함, 22에서 200 rpm으로 흔들 ° C를 20 분에 대한 chemotactic 상황에 셀 basolate 수 있습니다. </li></ol> 2. 가시와 manipulatable chemoattractant 기울기의 이미징 chemotaxing 세포 <ol><li> 1 μm의 캠프 및 DB 버퍼에 0.1μg/μl에서 알렉사 594의 신선한 30 μl 솔루션 Backfill은 micropipette. </li><li> micropipette 홀더에 Femtotip를 첨부하고 압력 공급 장치로 튜브, Eppendorf FemtoJet 시스템을 연결합니다. </li><li> 안정적인 경사도를 확립하기 위해 지속적으로 압력을 제공하기 위해 micromanipulator를 전동 micromanipulator (Eppendorf TransferMan NK2)로 micropipette 어셈블리를 연결합니다. </li><li> 공촛점 현미경에 기름 40X 렌즈를 통해 DB 버퍼 6 ML 가득한 한 잘 LabTek 챔버를 마운트하고 볼 분야에 밝은 필드 광학, 센터 Femtotip을 사용합니다. </li><li> 압력 공급 장치의 전원을 켜고 캠프 / 알렉사 594 혼합물의 그라디언트를 확립 70 고전력 증폭기에 대한 보상 압력 (PC)를 설정합니다. </li><li> 캠프와 543 nm의 레이저 라인을 사용하여 여기 알렉사 594 형광 원하는 농도의 혼합 모니터링하여 캠프 그라데이션을 시각화. </li><li> 원하는 위치로 micropipette를 놓고 1 순위, 위치 2, 및 세포를 노출하기 위해 경사도를 조작하는 3 순위로 설정하는 micromanipulator의 자동 위치 기능을 사용하십시오. </li></ol> 3. 고정되어 nonpolarized 전지 시스템 야영 기울기 감지에 관여 영상 신호 이벤트를 용이하게 <ol><li> 카페인 치료 후, 3 분 500g에 세포 원심 분리기의 나누어지는를 제거합니다. </li><li> 버퍼를 제거하고 2.5 MM의 카페인을 포함하는 신선한 DB 버퍼와 5x10 5 셀 / ML로 세포를 희석. </li><li> 4 잘 챔버 각각 잘 수있는 단일 잘 챔버 또는 0.4 ML에 세포 현탁액 1 ML을 적용합니다. </li><li> 10 분 동안 신중하게 무소속의 세포를 제거하고 동일한 볼륨 대체 버퍼를 피펫 세포가 준수하도록 허용합니다. </li><li> 현미경 원하는 세포를 찾아 이미지를 시작합니다. </li><li> 꾸준한 그라데이션에 노출 세포의 구성 요소를 신호의 역학을 모니터하기위한 실험은 실험하기 전에 10 분 5.0 μm의 (최종 농도) Latrunculin B와 세포를 치료. </li></ol> 4. 동시 모니터링 heterotrimeric G 단백질 활성화 및 PIP 3 생산 <ol><li> CAMP 세포 공동 표현 GαCFP 및 YFPGβ을 (G 세포)와 세포 PIP 3 표시기 PH - GFP (PH 세포)가 7 표현.을 개발 pulsing </li><li> 1시 1분 비율 및 플레이트들을 한 잘 또는 4 - 음 실에서 함께 세포의 이러한 두 종류를 섞습니다. </li><li> 작성하고 10 나노미터 넓이로 464에서 624 nm의에 대한 스펙트럼 범위 내에서 람다 스택 수집 모드를 사용하여 방출 지문 참조 곡선 CFP, YFP와 GFP를 저장합니다. </li><li> G 세포와 464에서 10 nm의 증가와 544 nm의에 대한 스펙트럼 범위 내에서 동일한 람다 스택 수집 모드를 사용하여 PH 세포에서 PIP 3 제작에 동시에 이미지 G 단백질 활성화. </li><li> G.에서 개별 채널에 CFP와 YFP 강도를 분리 수학적 람다 스택의 각 형광단의 기여를 계산에 저장 CFPand YFP 및 방출 지문을 사용하여 자이스 혈구 510META 소프트웨어의 선형 Unmixing 기능을 적용 </li><li> 같은 전략을 통해 수학적 PH 세포에 GFP 강도를 계산에 저장 GFP 배경 방출 지문을 사용하여 선형 Unmixing 기능을 적용할 수 있습니다. </li></ol> 5. 대표 결과 : <ol><li> D.의 훌륭한 모델 시스템 GPCR 중재 chemotaxis에 대한 discoideum. 사회 아메바, D. discoideum는 수명주기 동안 인상적인 chemotaxis를 전시하고 있습니다. 의 유전자 및 생화 학적 이점으로 인해, D. D는 강력한 s을 (를) 제공ystem는 chemotaxis를 연구하기 위해 서요. </li><li> 가시와 manipulatable chemoattract 필즈 세포의 Chemotaxis. 여기, 우리는 처음 10 μg / ML 혼합 2 μm의 캠프의 희석 시리즈 캠프 농도와 형광 염료 알렉사 594의 강도 사이의 선형 관계를 얻을 수있는 간단한 방법을 보여 알렉사 594 (그림 2A). 다음, 우리는 알렉사 594 (그림 그림. 2B)의 강도에 의해 그라디언트의 캠프 농도의 양적 측정을 수립, 또한, 그라디언트를 시각화하는 쉬운 방법을 제공합니다. </li><li> 세포 운동성가. 전지 분극 및 방향 감지와 uncoupled 굴지의 중합 억제제는 기존의 형태학의 극성을 제거하고 방향 감지의 세포 &#39;능력 (그림 3A)를 유지하면서 또한 세포의 움직임을 방지합니다. 표시 및 manipulatable 캠프 자극의 고용 예를 균일하게 적용 stimulations하거나 그라데이션을 위해 입력을 보장합니다. 이 방법은 그라디언트 감지 기기의 주요 신호 구성 요소 CAMP 유발 재배포의 정량 분석​​이 가능합니다. 이러한 신호 구성 요소의 측정 spatiotemporal 역학은 우리가 그라디언트 (그림 3B)에 편광 생화 학적 반응을 생성하는 동안 신호 네트워크가 균일한 stimulations에 적응을 실현 방법을 이해하실 수 있습니다. </li><li> 안정된 구배에 노출시 신호 네트워크를 chemosensing의 속도론의 체계적 측정. 방향 - 감지은 세포가 처음 그라디언트가 발생할 때 각 구성 요소는 세포 분극의 설립에 기여하는 방법 구성 요소를 이해하기 위해 신호의 역학 / 속도론을 측정하는 중요한 그것입니다. 높은 템포 - 공간 해상도 라이브 셀 이미징의 응용, 우리는 먼저 꾸준한 캠프 기울기 (그림 4A - C)에 노출된 세포의 biphasic PIP3 생산을 보여줍니다. 라이브 셀 이미징을 적용, 우리는 체계적으로의 역학을 측정해야 방향 감지 PIP3 제작 (그림 4D, E)에 캠프 자극에서 특정 신호를 네트워크를. </li><li> 균일하게 적용 캠프 자극에 동시 모니터링 heterotrimeric G 단백질 활성화 및 PIP 세 생산. 포스터 공명 에너지 전송 (약식은 무서워) 캠프 자극시 heterotrimeric G 단백질 활성화 (분리)를 모니터링하는 효율적인 방법을 제공합니다. 여기, 우리는 (그림 5 변화와 PIP의 멤브레인 translocation 각각 3 프로브, G 및 PH 세포에서 PH - GFP를 걱정 모니터링하여 heterotrimeric G 단백질 활성화 및 PIP 세 생산의 동시 측정을위한 편리한 쉽게 채택할 시스템을 설명 ). 어떤이 일시적 PIP 세 생산을 실행하는 동안 균일하게 적용 캠프 자극이 지속 G 단백질 활성화를 트리거합니다. </li></ol><img src="/files/ftp_upload/3128/3128fig1.jpg" alt="그림 1"/> 그림 1 : D.의 훌륭한 모델 시스템 GPCR의 중재를 chemotaxis에 대한 discoideum. A. 제도 방향 감지의 간단한 신호 경로를 보여줍니다. B. 캠프 그라디언트가 D.의 빠른 chemotaxis를 유도 discoideum 세포. 세포는 PIP 3 프로브, PH - GFP (녹색)을 표현한다. 그라데이션 (레드) 594 알렉사에 의해 시각입니다. 스케일 바 = 50μm. <img src="/files/ftp_upload/3128/3128fig2.jpg" alt="그림 2"/> 그림 2 : 가시와 manipulatable chemoattract 필즈 세포의 Chemotaxis. A. 그래프는 10 μg / ML 알렉사 594 혼합 2 μm의 캠프의 희석 시리즈 캠프 농도와 형광 염료 알렉사 594의 강도 사이의 선형 관계를 보여줍니다. 선형 관계에 의해 그라디언트의 캠프 농도 B. 양적 측정 A에서 형광 염료 알렉사 594의 캠프 농도와 강도의 <img src="/files/ftp_upload/3128/3128fig3.jpg" alt="그림 3"/> 그림 3 : 세포 운동성가 세포 분극 및 방향 감지와 uncoupled 있습니다. A. 이미지 굴지 중합 억제제 Latrunculin B의 치료에 의해 ​​고정되어 세포가 방향 감지의 기능을 유지할 것으로 나타났습니다. 세포는 PIP 3 프로브, PH - GFP (녹색)을 표현한다. 그라데이션 (레드) 594 알렉사에 의해 시각입니다. B. Manipulatable 캠프 자극과 고정되어 전지 시스템은 방향 감지의 핵심 질문을 해결하실 수 있습니다. 스케일 바 = 10μm. <img src="/files/ftp_upload/3128/3128fig4.jpg" alt="그림 4"/> 그림 4 : 지속적인 그라디언트에 노출시 신호 네트워크를 chemosensing의 속도론의 체계적 측정. A. 몽타쥬는 꾸준한 캠프 기울기 (적색)에 노출된 세포의 biphasic PIP 3 제작 (녹색). B. 이미지 C. C로 표시 PIP 세 생산의 속도론의 측정에 대한 관심 영역 (ROIs)를 보여줍니다 보여줍니다 . 동력학 O꾸준한 그라데이션에 노출 세포의 F PIP 세 생산. D. 제도 PIP 3 제작 캠프 자극의 방향 감지의 신호 네트워크를 보여줍니다. 안정된 구배에 노출시 그들의 운동은 E에서 동일한 색상 고체 라인에 표시됩니다. <img src="/files/ftp_upload/3128/3128fig5.jpg" alt="그림 5"/> 그림 5 : GPCR 신호 네트워크의 동시 모니터링 멀티 이벤트. A. 제도 변화와 각각 PIP3 프로브, G 및 PH 세포에서 PH - GFP의 멤브레인 translocation을 걱정 모니터링하여 heterotrimeric G 단백질 활성화 및 PIP 세 생산의 동시 측정을 보여줍니다. G와 PH 세포의 무지개 이미지 B. 장면 보여줍니다 어떤이 과도 PIP3 생산을 실행하는 동안 균일하게 적용 캠프 자극은 주변 세포에 영구 G 단백질 활성화를 트리거 것을. 시간 점 4.9s, 10.2s 및 20.4s에 대한 전 (개로) 및 자극 후 수 있습니다. G 단백질 활성화하고 균일하게 적용 캠프 자극시 PIP3 생산 C. 속도론가.

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+Ras+signaling+at+the+leading+edge+regulates+PI3K,+cell+polarity,+and+directional+cell+movement.">Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement.</a> J. Cell Biol. 167, 505-505 (2004).</li><li>Meili, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoattractant-mediated+transient+activation+and+membrane+localization+of+Akt/PKB+is+required+for+efficient+chemotaxis+to+cAMP+in+Dictyostelium.">Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium.</a> EMBO J. 18, 2092-2092 .</li><li>Parent, C. A., Blacklock, B. J., Froehlich, W. M., Murphy, D. B., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein+signaling+events+are+activated+at+the+leading+edge+of+chemotactic+cells.">G protein signaling events are activated at the leading edge of chemotactic cells.</a> Cell. 95, 81-81 (1998).</li><li>Funamoto, S., Meili, R., Lee, S., Parry, L., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+regulation+of+3-phosphoinositides+by+PI+3-kinase+and+PTEN+mediates+chemotaxis.">Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis.</a> Cell. 109, 611-611 (2002).</li><li>Iijima, M., Devreotes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+suppressor+PTEN+mediates+sensing+of+chemoattractant+gradients.">Tumor suppressor PTEN mediates sensing of chemoattractant gradients.</a> Cell. 109, 599-599 (2002).</li><li>Haastert, P. J. V. a. n., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis:+signalling+the+way+forward.">Chemotaxis: signalling the way forward.</a> Nat Rev Mol Cell Biol. 5, 626-626 (2004).</li></ol>

관심 없음 충돌 선언하지 않습니다.

세포의 chemotactic 관할 단계에 도달하는 과정 야생 유형 D. discoideum 세포, 그건 소요 약 5 세포가 잘 편광 세포 형태학 및 신속한 세포 마이 그 레이션 (그림 1)을 표시하는 동안 잘 chemotactic 관할 무대로 그들을 유도하기 위해 상온에서 개발을 pulsing ~ 6 시간. 이러한 pulsing, 온도 및 다른 유전 배경에 대한 캠프 농도 등 여러 가지 요인은, chemotactic 유능한 단계에 도달하는 과정...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> D3 - T 성장 미디어 </td><td> KD 의료 </td><td></td><td></td></tr><tr><td> 카페인 </td><td> 시그마 </td><td></td><td></td></tr><tr><td> Latrunculin B </td><td> 분자 프로브 </td><td></td><td></td></tr><tr><td> 알렉사 594 </td><td> 분자 프로브 </td><td></td><td></td></tr><tr><td> CAMP </td><td> 시그마 </td><td></td><td></td></tr><tr><td> ChronTrol XT 프로그램 타이머 </td><td> ChronTrol 법인 </td><td></td><td></td></tr><tr><td> Miniplus 세 연동 펌프 </td><td> Gilbson </td><td></td><td></td></tr><tr><td> 플랫폼 로터리 흔드는 </td><td></td><td></td><td></td></tr><tr><td> FemtoJet microcapillary 압력 공급 장치 </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> 단일, 4 잘 연구실 - 테크 II coverglass 챔버 </td><td> Nalge Nunc 인터내셔널 </td><td></td><td></td></tr><tr><td> LSM 510 META 또는 동급 형광 현미경 </td><td> 자이스 혈구 </td><td></td><td> 40X 1.3 NA 또는 60X 1.4 NA 오일 DIC 계획 - Neofluar 목표 렌즈 </td></tr><tr><td> 올림푸스 X81 또는 동급 </td><td> 하늘 </td><td></td><td> 100X 1.47 NA TIRF 객관적인 렌즈를 필요 </td></tr></tbody></table>

imaging g-protein coupled receptor (gpcr)-mediated signaling events that control chemotaxis of dictyostelium discoideum

대부분의 진핵세포 세포는 자신의 환경에서 화학적 신호의 기울기를 감지하고 이에 따라 1 마이그레이션할 수 있습니다. 이 가이드 셀 마이 그 레이션은 이러한 세포의 연결 2, 3의 면역 세포와 patterning의 인신 매매는 그들의 기능을 수행하는 다양한 세포에 필수적인 것입니다 chemotaxis로라고합니다. G - 단백질 결합 수용체의 큰 가족 (GPCRs)는 생체내 4 셀 마이 그 레이션을 직접하기 위해 chemokines로 알려진 변수 작은 펩티드를 감지합니다. chemotaxis 연구의 최종 목표는 GPCR 기계 감각의 케모카인 그라디언트와 컨트롤 chemotaxis에 선도적인 이벤트를 신호 방법을 이해하는 것입니다. 이를 위해, 우리는 실시간으로, chemoattractant의 그라데이션에 chemoattractants, 세포 운동의 spatiotemporal 농도를 모니터링하는 이미징 기술을 사용, GPCR은 heterotrimeric G - 단백질의 활성화를 중재하고, 5-8 진핵 세포의 chemotaxis에 관련된 세포내 신호 이벤트 . 간단한 진핵 생물, Dictyostelium의 discoideum은 leukocytes의 것과 유사 chemotaxic 동작을 표시하고, D. discoideum는 진핵세포 chemotaxis를 공부에 대한 핵심적인 모델 시스템입니다. 자유 생활 amoebae, D.으로 discoideum 세포가 풍부한 매체 나눕니다. 기아되면 세포들은 multicullular 구조를 형성 CAMP - 중재 chemotaxis를 통해 집계되는 발달 프로그램을 입력합니다. 캠프 chemotaxis에 관련된 대부분의 구성 요소 D.으로 확인되었습니다 discoideum. GPCR에 캠프의 바인딩 (cAR1)는 Gγ 및 Gβγ의 subunits 7, 9, 10로 heterotrimeric의 분리 G - 단백질을 유도. Gβγ subunits는 회전 활성화의 PI3K에서, 세포막 11-13에 PIP 3에 PIP 2 변환 RAS를 활성화해야합니다. PIP 3 따라서 멤브레인 14, 15 이러한 단백질을 모집, pleckstrin의 상동 (PH) 도메인과 단백질에 대한 구속력이 사이트의 역할을합니다. cAR1 수용체의 활성화는 또한 PIP 2 16, 17 PIP 3 dephosphorylates PTEN의 멤브레인 연결을 제어합니다. 분자 메커니즘은 evolutionarily 같은 neutrophils 18 인간 세포의 케모카인 GPCR - 중재 chemotaxis에 보존되어있다. 우리는 D.의 chemotaxis를 공부에 대한 다음과 같은 방법을 제시 discoideum 세포. 1. chemotactic 컴포넌트 세포의 준비. 2. 캠프 기울기에 세포의 영상이 chemotaxis. 3. 한 살 세포에서 G - 단백질 heterotrimeric의 GPCR 유도된 활성화를 모니터링. 4. 실시간으로 한 라이브 세포 이미징 chemoattractant - 트리거 다이나믹 PIP 3 응답. 우리 개발한 이미징 방법은 인간 leukocytes의 chemotaxis을 연구에 적용할 수 있습니다.

Watch this Scientific Journal Video about Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum at JoVE.com

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

여기, 우리는 chemotaxis를 조사에 대한 자세한 라이브 셀 이미징 방법을 설명합니다. 우리는 마이 그 레이션 세포에서 이벤트를 신호의 spatiotemporal 역학을 모니터 형광 현미경 방법을 제시한다. 신호 이벤트의 측정은 더욱 GPCR - 신호 네트워크 chemoattractants 및 제어 진핵 세포의 방향 이주 감지 기울기를 달성 방법을 이해하기 위해 우리를 허용합니다.

이미징 G - 단백질 결합 수용체 (GPCR) - 중재 신호 행사 그 제어 Chemotaxis Dictyostelium Discoideum</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

imaging-g-protein-coupled-receptor-gpcr-mediated-signaling-events

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Biology

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

17.9K 조회수.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. 여기, 우리는 chemotaxis를 조사에 대한 자세한 라이브 셀 이미징 방법을 설명합니다. 우리는 마이 그 레이션 세포에서 이벤트를 신호의 spatiotemporal 역학을 모니터 형광 현미경 방법을 제시한다. 신호 이벤트의 측정은 더욱 GPCR - 신호 네트워크 chemoattractants 및 제어 진핵 세포의 방향 이주 감지 기울기를 달성 방법을 이해하기 위해 우리를 허용합니다.

비디오: Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

Spectrally 해결된 두 광자 현미경을 사용하여 G 단백질 결합 수용체 상호 작용의 생체내 부량에서

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

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생물학

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

G 단백질 결합 수용체에 작용제 활동 Quantifying

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

Unimolecular 바이오 센서를 사용하여 라이브 세포에서 이벤트를 신호의 실시간 모니터링을위한 현미경을 안달

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

초기 자식 작용 이벤트의 셀 이미징 라이브 : Omegasomes을 넘어

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

유전자 인코딩 분자 프로브는 G 단백질 결합 수용체를 연구하는

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

형광 기반의 칼슘 동원 분석에 의해 G 단백질 결합 수용체의 특성

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

TIRF 현미경을 통해 단일 ​​이벤트 해상도에서 G 단백질 결합 수용체의 Clathrin 매개 엔도 시토 시스를 시각화

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular development program, during which single cells chemotax towards aggregation centers1-4. This process is dependent on the release of cyclic adenosine monophosphate (cAMP)5. cAMP is produced in waves through the concerted action of adenylate cyclase and phosphodiesterases, and binds to G protein-coupled cAMP receptors6,7. A widely used assay to analyze the mechanisms involved in the developmental cycle of the lower eukaryote Dictyostelium discoideum is based on the observation of cell aggregation in submerged conditions8,9. This protocol describes the analysis of the role of coronin A in the developmental cycle by starvation in tissue-culture plates submerged in balanced salt solution (BSS)10. Coronin A is a member of the widely conserved protein family of coronins that have been implicated in a wide variety of activities11,12. Dictyostelium cells lacking coronin A are unable to form multicellular aggregates, and this defect can be rescued by supplying pulses of cAMP, suggesting that coronin A acts upstream of the cAMP cascade10. The techniques described in these studies provide robust tools to investigate functions of proteins during the initial stages of the developmental cycle of Dictyostelium discoideum upstream of the cAMP cascade. Therefore, utilizing this aggregation assay may allow the further study of coronin A function and advance our understanding of coronin biology.

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

의 조기 기아 응답에 Coronin (A)의 기능 조사<em&gt; Dictyostelium의 discoideum</em&gt; 집계 분석 실험에 의해

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

이미징 G의 호중구와 같은 HL60 세포에서 화성과 시그널링 이벤트 수용체가 매개 단백질 결합

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

단백질의 품질 관리 시스템을 공부<em&gt; D. discoideum</em&gt; 온도 제어 라이브 셀 이미징을 사용하여

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...