The authors of this paper would like to thank Prof. Jiayan Xie for technical assistance and equipment usage. This work was financially supported by the National Natural Science Foundation of China (41406137).

1. 셀 제조 <ol><li> 세포 복구 <ol><li> 37 ° C의 물을 욕조에 액체 질소 탱크로부터 동결 보존 인간 배아 신장 293 (HEK293) 세포를 옮기고 1 연속 진탕 - 2 분. </li><li> 10 cm 세포 배양 접시에 평형화 성장 배지 10 ㎖ (둘 베코 변형 이글 배지 (DMEM), 10 % 소 태아 혈청 (FBS), 1 % 페니실린 - 스트렙토 마이신 용액)을 추가한다. 부드럽게 평형 성장 매체에 해동 세포를 추가합니다. </li><li> 4 시간 - 2 95 %의 공기와 5 % CO2를 함유하는 가습 분위기 37 ° C의 물 재킷 CO 2 인큐베이터에서 세포를 인큐베이션. </li><li> 후 2 - 4 시간 동안 세포가 부착 된 것을 확인한다. 48 시간 - 또 다른 24의 평형 성장 매체와 문화를 교체합니다. 참고 :이 디메틸 설폭 사이드 (DMSO)를 제거하고 세포 손상을 방지하기 때문에 평형 성장 매체를 변경하면 유용합니다. </li></ol></li><li> &lt;강한&gt; 셀 통로 <ol><li> 24 일 후 - 세포가 합류에 가까운 성장을 48 시간, 평형화 성장 배지를 폐기하고 인산 완충 식염수 2 ㎖ (PBS)으로 세포를 세척 하였다. 부드럽게 접시를 소용돌이. </li><li> PBS를 폐기하고 트립신 EDTA 1 ㎖ (식염수, 0.25 % 트립신 및 0.02 % EDTA)를 추가한다. 완전히 세포를 소화하기 위해 접시를 조심스럽게 소용돌이와 트립신 - EDTA 솔루션을 대기음. 3 분 동안 37 ° C의 물 재킷 CO 2 인큐베이터에서 접시를 넣어. 참고 :이 기간 동안, 세 개의 새로운 10 cm 세포 배양 접시를 준비하고 각 평형 성장 매체의 10 mL를 넣어. </li><li> 세포가 접시의 바닥에서 해제 한 경우 광학 현미경을 사용하여 결정합니다. 접시에 평형 성장 매체의 2 ML을 추가합니다. 피펫 팅하여 혼합하고 즉시 새로운 접시의 각 세포 현탁액을 0.6 ㎖의 피펫. 부드럽게 피펫 팅하여 혼합하고, 세포를 37 ℃, 5 % CO 2에서 배양 할 수있다. </li></ol></lI&gt; </ol> 2. 셀 시드 <ol><li> 24 일 후 - 계대 세포가 합류에 가까운 성장을 48 시간, 평형화 성장 배지를 버리고 PBS 2 ml로 세포를 세척한다. 부드럽게 접시를 소용돌이. </li><li> PBS를 폐기하고 트립신 EDTA 1 ㎖를 추가한다. 완전히 세포를 소화하기 위해 접시를 조심스럽게 소용돌이와 트립신 - EDTA를 대기음. 3 분 동안 37 ° C의 물 재킷 CO 2 인큐베이터에서 접시를 넣어. 주 :이 기간 동안, 각각의 웰에 평형화 성장 배지 2ml를 첨가하여 6 웰 플레이트를 준비한다. </li><li> 현미경 관찰 하에서 세포 접시 바닥으로부터 상승하는 한 경우 접시 평형화 성장 배지 1 ㎖를 추가한다. 피펫 팅하여 혼합하고 즉시 제조 6- 웰 플레이트 (평형화 성장 배지 2 mL를 셀리스 6 웰 플레이트)의 각 웰에 세포 현탁액 0.1 ㎖ (양의 세포 성장 밀도에 따라 조정)을 피펫. 부드럽게 세포를 피펫으로 혼합 허용37 ° C에서 배양하고, 5 % CO 2. </li></ol> 3. 세포 형질 <ol><li> 리포좀 제제로 형질 감염 방법 (1) 참고 : 형질 전환 시약 1 재료의 표를 참조하십시오. <ol><li> 95 % 합류 - 다음 날, 세포는 85해야합니다 있는지 확인하십시오. </li><li> 무균 마이크로 원심 튜브에 플라스미드 DNA (500 NG / μL) 및 감소 된 혈청 배지 100 μL 3㎍을 추가하고 부드럽게 혼합한다. 실온에서 5 분 동안 인큐베이션. </li><li> 시약 1 및 다른 마이크로 원심 튜브 환원 혈청 배지 100 μL 9 μL를 준비하고 부드럽게 혼합한다. 실온에서 5 분 동안 인큐베이션. </li><li> 희석 시약 1 (전체 부피 = 215 μL)로 희석하고 혼합물을 플라스미드 수확기. 부드럽게 혼합하고, 실온에서 20 분 동안 배양한다. </li><li> 대기음 세포 배양 접시 오프 매체, 및 FBS없이를 1.5 ml의 신선한 DMEM을 추가한다. </li><li> DNA-TRANSF의 215 μL를 추가ection 시약을 각 웰에 적하 혼합하고, 플레이트를 선회하여 부드럽게 혼합한다. </li><li> 8 시간 - 4 CO 2, 37 ℃에서 인큐베이션하고, 5 %. 10 % FBS와 신선한 DMEM로 교체하십시오. </li><li> 모든 실험을 실행하기 전에 48 시간 - 24 다른 CO 2, 37 ℃에서 세포를 인큐베이션하고, 5 %. </li></ol></li><li> 리포좀 제제로 형질 감염 방법 2 참고 : 형질 전환 시약 2 재료의 표를 참조하십시오. <ol><li> 무균 마이크로 원심 튜브에 플라스미드 DNA 2 μg의 (500 NG / μL), 시약 2 4 μL, 100 μL의 혈청 감소 배지를 추가한다. 부드럽게 혼합하고, 실온에서 15 분 동안 배양한다. </li><li> 적가 방식으로 세포에 형질 전환 혼합물을 추가한다. </li><li> 모든 실험을 실행하기 전에, 37 ℃에서 48 시간 및 5 % CO 2 - 형질 감염 후 24 세포를 배양한다. </li></ol></li></ol> 커버 전표 4. 세포 배양 <ol><li> 레배양 6 웰 플레이트에서 성장 배지를 이동. </li><li> 칼슘 및 마그네슘없이 PBS 1 ㎖로 세포 단층을 세척한다. </li><li> 0.5 mL의 트립신 EDTA를 추가한다. 신중하게 접시를 소용돌이 및 트립신 - EDTA를 대기음. 37 ℃에서 3 분, 5 % CO 2의 배양 플레이트를 인큐베이션. 실제의 배양 시간은 사용되는 세포주에 따라 달라진다. 주 :이 기간 동안 새로운 12 웰 플레이트를 준비하여 멸균 커버 슬립 각 웰에 직경 15mm의 추가. 리신, 라미닌, 콜라겐으로 코팅 된 커버 슬립은 쉽게 분리 HEK293 세포에 대한 세포 부착을 향상시킬 수있다. </li><li> 세포가 분리 될 때, 각 웰에 1 개 ㎖의 사전 - 가온 완전 성장 배지를 추가한다. 피펫 팅하여 혼합하고 바로 슬립 커버로 12- 웰 플레이트에 세포를 필요한 수를 옮긴다. 한 ML에 총 볼륨을 조정하기 위해 각 웰에 신선한 전체 성장 매체를 추가 </li><li> 37 ° C에서 24 시간 및 5 % CO 2 - 16 세포를 인큐베이션. 참고 : 30 후 -60 분은, 세포의 대부분은 멸균 커버 슬립에 부착 한 것이다. </li></ol> 5. 공 초점 현미경 감지 <ol><li> 셀룰러 현지화 <ol><li> 16 일 후 - 세포를 12 웰 플레이트에서 멸균 커버 슬립에 가까운 합류로 성장 24 시간, 5 μM의 최종 농도를 제공하는, 각 웰에, 멤브레인 프로브 DII를 추가한다. 세포는 37 ° C에서 30 분, 5 % CO 2 동안 배양 할 수있다. </li><li> 30 분 후, 평형화 성장 배지를 제거하고 차가운 PBS로 두 번 세포를 씻어 (2 - 11 °의 C). </li><li> 각 웰 (PBS)에서 4 % 파라 포름 알데히드 용액 1 ㎖를 첨가하여 세포를 고정한다. 조심스럽게 15 분 동안 교반한다. 주의 : 파라 포름 알데히드는 피부 접촉이나 흡입 중간 독성이며, 가능성이 발암 물질로 지정. 화학은 파라 포름 알데히드의 무게 및 취급 중에 표기 후드 배기 밸런스 격납 등의 보호 대책을 연기. &lt;/ 리&gt; <li> PBS 두 번 고정 된 세포를 씻으십시오. μL DAPI (4 &#39;, 6-diamidino-2-페닐 -) 용액 (0.5 ㎍ / mL)을 각 웰 200-300 추가하고, 실온에서 10 분 동안 배양한다. </li><li> PBS로 두 번 세포를 씻으십시오. 조심스럽게 판에서 커버 전표를 제거하고 여분의 PBS를 심지. 장착 매체 (50 % V / V 글리세롤 / 물) 한 방울 로케이션 현미경 슬라이드 상 전환. 슬라이드에서 초과 장착 매체를 제거합니다. </li></ol></li><li> 휴대 내면화 <ol><li> 16 일 후 - 세포를 12 웰 플레이트에서 멸균 커버 슬립에 가까운 합류로 성장 24 시간, 평형화 성장 배지를 제거하고 신선한 추가 DMEM 배지 (37 ° C)를 가온. 37 ° C에서 1 시간, 5 % CO 2 세포를 인큐베이션. </li><li> 30 분 동안 (0, 10-12, 10-9 및 10-6 M) 서로 다른 농도로 리간드를 세포를 처리하고, 다른 시간 10 -6 M의 리간드 (0 그들을 취급10, 30 및 60 분). </li><li> (- 8 °의 C 2) 차가운 PBS로 두 번 세포를 씻으십시오. 각 웰 (PBS)에서 4 % 파라 포름 알데히드 용액 1 ㎖를 첨가하여 세포를 고정한다. 조심스럽게 15 분 동안 교반한다. </li><li> PBS로 두 번 세포를 씻으십시오. 조심스럽게 판에서 커버 전표를 제거하고 여분의 PBS를 심지. 장착 매체 (50 % V / V 글리세롤 / 물) 한 방울 로케이션 현미경 슬라이드 상 전환. 슬라이드에서 초과 장착 매체를 제거합니다. </li></ol></li><li> 공 초점 현미경 <ol><li> 수은 램프 전력, PC 현미경, 스캐너, 레이저를 포함, 공 초점 현미경의 하드웨어를 켭니다. 소프트웨어를 실행, 운영 체제 계정에 로그인 구성을 확인하고 레이저를 선택합니다. 지침에 따라 순서대로 공 초점 현미경 시스템의 다른 단위를 켭니다. </li><li> 현미경 단계에 준비된 슬라이드를 놓습니다. 스테이지 제어기를 사용하여 대물 렌즈를 통해 샘플을 놓고. 삼나무 기름 한 방울에 추가오일 침지 렌즈가 선택 될 때 커버 슬립한다. 렌즈에 직면 슬라이드에 커버 전표를 놓습니다. </li><li> 형광 현미경 아래에서 관심의 세포를 찾아보십시오. </li><li> 스캔 모드로 전환합니다. 레이저 파워 및 형광 발광의 스펙트럼 범위를 선택. 튜닝 레이저 강도 및 기타 매개 변수에 의해 높은 품질 공 촛점 이미지를 캡처합니다. </li></ol></li></ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Expression+Profile+of+the+Gonadotropin-Releasing+Hormone+Receptor+in+Common+Chinese+Cuttlefish,+Sepiella+japonica.">Identification and Expression Profile of the Gonadotropin-Releasing Hormone Receptor in Common Chinese Cuttlefish, Sepiella japonica.</a> J Exp Zool A Ecol Genet Physiol. , (2016).</li><li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+activation+of+the+G+protein-coupled+receptor+BNGR-A21+by+the+neuropeptide+corazonin+from+the+silkworm,+Bombyx+mori,+dually+couples+to+the+G(q)+and+G(s)+signaling+cascades.">Specific activation of the G protein-coupled receptor BNGR-A21 by the neuropeptide corazonin from the silkworm, Bombyx mori, dually couples to the G(q) and G(s) signaling cascades.</a> J Biol Chem. 288, 11662-11675 (2013).</li><li>Lu, Z. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning,+Characterization,+and+Expression+Profile+of+Estrogen+Receptor+in+Common+Chinese+Cuttlefish,+Sepiella+japonica.">Cloning, Characterization, and Expression Profile of Estrogen Receptor in Common Chinese Cuttlefish, Sepiella japonica.</a> J Exp Zool A Ecol Genet Physiol. 325, 181-193 (2016).</li></ol>

The authors declare no conflicts of interest.

여기에 제시된 프로토콜은 HEK293 세포에서 세포 내 발현 및 위치 파악 GPCR 융합 단백질의 내재화를 검출하는 효율적이고 쉽게 해석 방법을 제공한다. 이 기술은 쉽게 HEK293 세포 BMN 및 봄 빅스 모리 (16)로부터 corazonin 수용체의 세포 내재화 제이션을위한 많은 다른 유전자와 세포 유형에 대해 적응 될 수있다. 이 강력한 분석의 성공적인 응용 프로그램은...

세포를 수송하는화물 피킹 기계를 가지고 세포 외 물질 - 리간드 등, 미생물 영양분, 및 막 관통 단백질 -로 정보, 에너지 및 다른 목적 1,2 셀있다. 그것은 세포 항상성, 조직의 기능, 및 전체 세포 생존에 중요하다. 형광 융합 단백질의 세포 - 기반 표현은 신호 경로 (3)의 위치 파악 및 G 단백질 결합 수용체 (GPCR)와 같은 막 횡단 수용체의 내재화를 연구 할 수있는 강력한 방법이다. 직접 형광 검출 기술과 결합 된 해파리 빅토리아 (Aequorea victoria)로부터의 녹색 형광 단백질 (GFP)로 태그 된 융합 단백질의 발현은, 넓은 수용을 얻은 단백질 타겟팅 연구 4, 5, 6. GFP 기반 detecti에는 고정 아티팩트 (7)을 피하면서 살아있는 세포의 실시간 영상을 허용하는 장점이있다. 다목적 클로닝 벡터 일련 9는 다양한 세포 8에 GFP 단백질 융합체의 발현을 용이하도록 구성되어있다. 및 pEGFP-N1 벡터는 포유류 세포에서보다 밝은 형광 발현 야생형 GFP에서 최적화 된 개선 된 녹색 형광 단백질 (EGFP)를 코딩하는 널리 상용화 된 플라스미드 벡터이다. EGFP의 형광 신호가 포유 동물 세포에서 발현 관찰, 형광 현미경, 바람직하게는 공 촛점 현미경으로 488 nm에서 여기 및 507 nm에서의 방출과 함께 이루어져야한다. 세포 내 위치 파악 및 수용체의 리간드 - 매개 된 내재화를 모니터링 된 GPCR (10)의 신호 전달과 기능을 조사하기 위해 일반적인 기법 </s11&gt;까지. 대부분의 경우에는 내재화 된 GPCR의 탈감작 (작용제의 존재에도 불구하고, 자극 반응의 감쇠) (12, 13)로 이끈다. 내재화 수용체 리소좀에 통합 저하하거나 수용체는 상기 실험에서 사용 된 세포주의 특성에 따라 다시 세포 표면에 재활용 될 수 수있다. 고나 도트로 핀 방출 호르몬 수용체 (GnRHRs)는 GPCR 패밀리에 속하고, 세포 외 아미노 말단 세포 내 -COOH 단자 전형적인 GPCR 단백질 구조를 가지고 있고, 일곱 막 횡단 (TM)는 14 도메인들. (Sepiella 자포니카에서 GnRHR 유전자) 재조합 플라스미드 Sj에 GnRHR-EGFP의 형질 감염 및 (HEK293 세포에서 발현) EGFP 태깅 에스 GnRHR 공 초점 현미경의 검출은 기존에보고 된교활한 및 GFP 형광은 막 횡단 단백질 제이션 분석법 15 기자로 설치되었다. 이제, S. 자포니카 성선 자극 호르몬 방출 호르몬 (GnRH에 Sj에)에 의해 활성화 Sj에 GnRHR의 내재화는 공 촛점 현미경으로 시간 및 용량 - 의존적 방식으로 입증되었다.

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			<td>High glucose 4.0 mM L-glutamine</td>
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			<td height="21" style="height:21px;width:300px;">Foetal Bovine Serum (FBS)</td>
			<td style="width:236px;">PuFei</td>
			<td style="width:119px;">1101-100</td>
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			<td height="21" style="height:21px;width:300px;">Phosphate Buffered Saline (PBS)</td>
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			<td>reduced serum media</td>
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			<td style="width:236px;">Roche</td>
			<td style="width:119px;">6366236001</td>
			<td>Reagent 2</td>
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			<td style="width:236px;">Beyotime</td>
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			<td height="21" style="height:21px;width:300px;">DiI</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">C1036</td>
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			<td height="21" style="height:21px;width:300px;">Antifade Mounting Medium</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">P0126</td>
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			<td height="42" style="height:42px;width:300px;">Paraformaldehyde</td>
			<td style="width:236px;">Sinopharm Chemical Reagent Co.(SCRC)</td>
			<td style="width:119px;">80096618</td>
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			<td height="21" style="height:21px;width:300px;">100 mm cell culture</td>
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			<td style="width:119px;">CS016-0092</td>
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			<td height="21" style="height:21px;width:300px;">12-well plate&#160;</td>
			<td style="width:236px;">ExCell Bio</td>
			<td style="width:119px;">CS016-0093</td>
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			<td height="21" style="height:21px;width:300px;">Cover Glass</td>
			<td style="width:236px;">NEST</td>
			<td style="width:119px;">801007</td>
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			<td height="21" style="height:21px;width:300px;">Microscope Slides</td>
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			<td style="width:119px;">10127105P-G</td>
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			<td height="21" style="height:21px;width:300px;">Thermo Scientific Forma CO2 Incubator</td>
			<td style="width:236px;">Thermo</td>
			<td style="width:119px;">3111</td>
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			<td height="21" style="height:21px;width:300px;">TCS SP5II laser scanning confocal microscope</td>
			<td style="width:236px;">Leica&#160;</td>
			<td style="width:119px;">5100001311</td>
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detection of ligand-activated g protein-coupled receptor internalization by confocal microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

도 1은 공 초점 레이저 주사 현미경 시스템의 일례를 나타낸다. 그림 2는 HEK293 세포 및 pEGFP-N1의 발현을 제공한다. GFP 신호는 세포질 및 핵 검출되었다. 도 3은 우리의 이전에 발행 된 결과 (15)와 일치하는 HEK293 세포에서 S. 자포니카 GnRHR에서의 세포 내 위치 파악을 나타낸다. 때 CLSM에서 에스 GnRHR (에스...

Watch this Scientific Journal Video about Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy at JoVE.com

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

이 프로토콜은 포유 동물 세포에서 G 단백질 연결 수용체 (GPCR)의 내재화 초점 현미경 검출을 설명한다. 이것은 기본적인 세포 배양, 형질 전환, 및 공 초점 현미경 절차를 포함하는 융합 발현 GPCR의 세포 내 위치 파악 및 내재화를 검출하는 효율적이고 쉽게 해석 방법을 제공한다.

공 초점 현미경으로 리간드 - 활성화 된 G 단백질 결합 수용체 내재화 검출

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...

detection-ligand-activated-g-protein-coupled-receptor-internalization

Research

JoVE Journal

Biology

10.6K 조회수.  Zhejiang Ocean University.  이 프로토콜은 포유 동물 세포에서 G 단백질 연결 수용체 (GPCR)의 내재화 초점 현미경 검출을 설명한다. 이것은 기본적인 세포 배양, 형질 전환, 및 공 초점 현미경 절차를 포함하는 융합 발현 GPCR의 세포 내 위치 파악 및 내재화를 검출하는 효율적이고 쉽게 해석 방법을 제공한다. 

비디오: Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

공촛점 현미경으로 Zebrafish 배아 뇌 영상 라이브

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

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생물학

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 ...

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

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

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

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

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 ...

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 ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

라이브 세포의 저장소로 작동하는 칼슘 항목의 형광 기반의 측정 : 교양 암 세포에서 골격근 섬유에

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

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 ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

공 초점 및 다중 광자 현미경을 사용하여 다섯 형광 단백질에 의해 표시됨 조혈 줄기 및 전구 세포의 생체 내 클론 추적에서

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 ...