이 논문에 제시된 연구 작업은 캐서린 샌드버그에 부여 R01 HL121456-01A1에 의해 지원되었다. 이 작품은 부분적으로 미국의 국립 보건원 (National Institutes of Health)에서 P30-CA051008에 의해 자금 지원 조지 타운 대학 의료 센터에서 현미경 및 이미지 공유 자원 (MISR)에 라이카 SP8 AOBS의 가용성에 의해 추가로 지원되었다. 우리는 기꺼이 라이카 SP8 영상 피터 존슨 안내, 칼 자이스 현미경 LLC의 알마 아놀드가 제공하는 영상 지원과 조지 타운 대학의 토마스 Coate, 생물학과의 실험실에서 자이스 혈구 LSM880의 사용을 인정합니다. 이 원고의 내용은 전적으로 저자의 책임이며 반드시 국립 보건원의 공식 견해를 대변하지 않습니다.

<ol>
	<li>Dale, L. B., Seachrist, J. L., Babwah, A. V., Ferguson, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+angiotensin+II+type+1A+receptor+intracellular+retention,+degradation,+and+recycling+by+Rab5,+Rab7,+and+Rab11+GTPases.">Regulation of angiotensin II type 1A receptor intracellular retention, degradation, and recycling by Rab5, Rab7, and Rab11 GTPases.</a> J Biol Chem. 279 (13), 13110-13118 (2004).</li><li>Hein, L., Meinel, L., Pratt, R. E., Dzau, V. J., Kobilka, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+trafficking+of+angiotensin+II+and+its+AT1+and+AT2+receptors:+evidence+for+selective+sorting+of+receptor+and+ligand.">Intracellular trafficking of angiotensin II and its AT1 and AT2 receptors: evidence for selective sorting of receptor and ligand.</a> Mol Endocrinol. 11 (9), 1266-1277 (1997).</li><li>Hunyady, L., 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=Differential+PI+3-kinase+dependence+of+early+and+late+phases+of+recycling+of+the+internalized+AT1+angiotensin+receptor.">Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor.</a> J Cell Biol. 157 (7), 1211-1222 (2002).</li><li>Lazari, M. F., Porto, C. S., Freymuller, E., Abreu, L. C., Picarelli, Z. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-mediated+endocytosis+of+angiotensin+II+in+rat+myometrial+cells.">Receptor-mediated endocytosis of angiotensin II in rat myometrial cells.</a> Biochem Pharmacol. 54 (3), 399-408 (1997).</li><li>Li, H., Li, H. F., Felder, R. A., Periasamy, A., Jose, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab4+and+Rab11+coordinately+regulate+the+recycling+of+angiotensin+II+type+I+receptor+as+demonstrated+by+fluorescence+resonance+energy+transfer+microscopy.">Rab4 and Rab11 coordinately regulate the recycling of angiotensin II type I receptor as demonstrated by fluorescence resonance energy transfer microscopy.</a> J Biomed Opt. 13 (3), 031206 (2008).</li><li>Li, H., 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=Actin+cytoskeleton-dependent+Rab+GTPase-regulated+angiotensin+type+I+receptor+lysosomal+degradation+studied+by+fluorescence+lifetime+imaging+microscopy.">Actin cytoskeleton-dependent Rab GTPase-regulated angiotensin type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy.</a> J Biomed Opt. 15 (5), 056003 (2010).</li><li>Yamamoto, A., 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=Bafilomycin+A1+prevents+maturation+of+autophagic+vacuoles+by+inhibiting+fusion+between+autophagosomes+and+lysosomes+in+rat+hepatoma+cell+line,+H-4-II-E+cells.">Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells.</a> Cell Struct Funct. 23 (1), 33-42 (1998).</li><li>Deliu, E., Tica, A. A., Motoc, D., Brailoiu, G. C., Brailoiu, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+angiotensin+II+activates+rat+myometrium.">Intracellular angiotensin II activates rat myometrium.</a> Am J Physiol Cell Physiol. 301 (3), C559-C565 (2011).</li><li>Fortian, A., Sorkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+fluorescence+imaging+reveals+high+stoichiometry+of+Grb2+binding+to+the+EGF+receptor+sustained+during+endocytosis.">Live-cell fluorescence imaging reveals high stoichiometry of Grb2 binding to the EGF receptor sustained during endocytosis.</a> J Cell Sci. 127 (Pt 2), 432-444 (2014).</li><li>Aymerich, M. S., 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=Real-time+G-protein-coupled+receptor+imaging+to+understand+and+quantify+receptor+dynamics.">Real-time G-protein-coupled receptor imaging to understand and quantify receptor dynamics.</a> ScientificWorldJournal. 11, 1995-2010 (2011).</li><li>Jaeger, W. C., Armstrong, S. P., Hill, S. J., Pfleger, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+Detection+of+Diversity+and+Bias+in+GPCR+Function.">Biophysical Detection of Diversity and Bias in GPCR Function.</a> Front Endocrinol (Lausanne). 5, 26 (2014).</li><li>Inuzuka, T., 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=Attenuation+of+ligand-induced+activation+of+angiotensin+II+type+1+receptor+signaling+by+the+type+2+receptor+via+protein+kinase.">Attenuation of ligand-induced activation of angiotensin II type 1 receptor signaling by the type 2 receptor via protein kinase.</a> C. Sci Rep. 6, 21613 (2016).</li><li>Liu, 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=Selective+inhibition+of+angiotensin+receptor+signaling+through+Erk1/2+pathway+by+a+novel+peptide.">Selective inhibition of angiotensin receptor signaling through Erk1/2 pathway by a novel peptide.</a> Am J Physiol Regul Integr Comp Physiol. 306 (8), R619-R626 (2014).</li><li>North, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+is+believing?+A+beginners'+guide+to+practical+pitfalls+in+image+acquisition.">Seeing is believing? A beginners' guide to practical pitfalls in image acquisition.</a> J Cell Biol. 172 (1), 9-18 (2006).</li><li>Mueller, S. C., Hubbard, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-mediated+endocytosis+of+asialoglycoproteins+by+rat+hepatocytes:+receptor-positive+and+receptor-negative+endosomes.">Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes.</a> J Cell Biol. 102 (3), 932-942 (1986).</li><li>Costantini, L. 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=A+palette+of+fluorescent+proteins+optimized+for+diverse+cellular+environments.">A palette of fluorescent proteins optimized for diverse cellular environments.</a> Nat Commun. 6, 7670 (2015).</li><li>Freundt, E. C., Czapiga, M., Lenardo, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoconversion+of+Lysotracker+Red+to+a+green+fluorescent+molecule.">Photoconversion of Lysotracker Red to a green fluorescent molecule.</a> Cell Res. 17 (11), 956-958 (2007).</li><li>Yoshimori, T., Yamamoto, A., Moriyama, Y., Futai, M., Tashiro, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bafilomycin+A1,+a+specific+inhibitor+of+vacuolar-type+H(+)-ATPase,+inhibits+acidification+and+protein+degradation+in+lysosomes+of+cultured+cells.">Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells.</a> J Biol Chem. 266 (26), 17707-17712 (1991).</li></ol>

저자는 더 경쟁 재정적 이해 관계가 없음을 선언합니다.

여기에 제시된 예비 실험은 24 분의 비교적 짧은 시간 과정 동안, 리소좀에 AT의 1A R-GFP의 전달에는 상당한 변화가 발생하지 않았 음을 보여줍니다. 일반적으로 리소좀에 내면화 수용체의 전달은 빠르면 15 ~ 20 분으로 발생할 수 있습니다. 이 실험은 1A의 R AT 내면화의 대부분이 큰, 핵 주변 엔도 좀을 대상으로 가설과 일치한다. R은 리소좀에 도착 1A AT 적어도 일부는 리 등...

전반적인 목표는 수용체 작용제 치료 후 시간과 특정 세포 내 소기관과 수용체의 colocalization을의 공간에서 정량적 증거를 획득하는 것입니다. 따라서, 여기에 즉시 목표 경과 촛점 리소좀의 이미지와 인간 배아 신장 세포에서 수용체 (HEK) 정량적으로 수용체의 전달을 측정하기 위하여 3 차원의 화상 데이터를 형질 다음이어서 조립 분석 걸친 트랜스를 캡처하는 리소좀. 횡단 수용체가 생리 학적 및 병태 생리 학적 조건에 의해 규제되는 방법을 결정하는 데 도움이 수용체 리간드에 의해 활성화되면 세포 내 이입 동안 리소좀 또는 다른 세포 소기관이있는 횡단 수용체의 동시 인신 매매를 조사. 1A AT R 주로 HEK293 촬영 한 대부분 있지만, 모델 전지 시스템의 중앙 II로 처리 다음 리소좀 저하 될 것으로 추정된다리간드, 앙 II의 대량 리소좀 2, 3, 4, 5의 열화 상태 eptors 주로 세포막 나중에 재활용 multivesicular 재활용 엔도 좀에 편재되어있다. 최근 리튬 등. 포스터 공명 에너지 전이 (FRET) 및 형광 수명 이미징 현미경 (FLIM) 기술을 이용하여 증명하는 AT 1A는 R의 colocalizes LAMP1으로 (~ 10 nm의 척도), 리셉터의 적어도 일부에 타겟팅 및 열화되는 것을 제안 리소좀 막 단백질 리소좀 (6)에 의해. 이 저자는 리소좀 억제제 클로로퀸은 클로로퀸의 효과가 리소좀 늦은 엔도 좀 또는 autophagosomes의 융합을 차단하는 것을 제안 이전 보고서와 일치 1A R-램프 1 협회에서 차단 지적했다. 기타 간접 접근 (1A)의 R 리터 AT 결정리소좀에서 ocalization는 리소좀 3, 4, 5, 6, 7, 8 (1A)의 R의 앞에서 추론 리소좀 억제제 bafilomycin 이용했다. 라이브 셀 이미징은 세포 부피 및 GFP-키메라 수용체의 손실 / 디밍 / 재배포에 고정의 잠재적 영향을 방지 할 수 있습니다. 이전 연구 colocalization을하거나 또는 리포좀 등의 수용체 - 결합 대용 표지 생균 사용에 수용체의 동시 발생을 결정 고정 세포 의존 한 β-아레스 1, 2, 3, 4, 5, 6. 회전 디스크 공 촛점 또는 레이저 포인트 스캐닝의 conf를 사용하여 다른 된 GPCR의 라이브 셀 이미징하나로 이루어질 수 또는 HYD 탐지기가 장착 OCAL 현미경은 30 초 간격으로 9, 10 Z-스택에 몇 군데 개별 세포 수용체의 내면화와 인신 매매를 관찰하는 연구자 수있었습니다. 라이브 세포 Z - 스택이 빠르게 수집 그러나 경우에도, 분석은 2D (10)에 즉 각 스택 내에서 단일 평면 선택 후 발생할 수 있습니다. 이 문제의 내재화 GPCR 또는 티로신 키나제 수용체 공부 충분할 수도 있지만, AT 1A는 루피는 시간의 크기와 위치를 변화함으로써 본질적으로 적절하게 각 시점에서 대표적인 단일 슬라이스를 이용하여 시료에 더 어렵 소체에 축적한다. 철저히 셀 통해 크기 및 위치에 다른 소포 각 모집단을 검출하는 1A의 R AT 분류의 경로를 결정하기 위해, 3 차원 영상 및 3 차원 분석을위한 방법은 이러한 변화를 추적하고되도록 고안<html> 이러한 리소좀 등 다양한 세포 내 구획의 표시와 함께 사용. 수사관들은 직접 셀과 중앙 II 자극 다음 세포 내 구획에 그 이전에 AT 1A는 R의 움직임을 시각화하는 라이브 세포 이미징에 대해이 프로토콜을 사용할 수 있습니다. 세포 내 구획 형광 단백질 키메라 또는 다른 형광 마커 태그. 이 프로토콜은 또한 야생형 수용체 대 비교하는 돌연변이 또는 약물 치료를 다음과 같이 변경을 검출하기 위해 200 nm의 최소 해상도 세포 내 소기관의 수용체를 지역화하는 첫 번째 방법으로 사용될 수있다. 이 라이브 셀 이미징 구비하는 공 초점 현미경에 대해 수행 될 수 있기 때문에이 기술은 액세스. 이 방법의 상대적인 용이성이 증가 전문성과 상호 작용 분자 (6)을 검출 FRET / FLIM / BRET 기술을 수행하는 데 필요한 장비와 대조&quot;외부 참조&quot;&gt; 11. 이러한 측정은 높은 해상도 (~ 10 ㎚)과 세포 내 소기관 내 위치 파악이 유추됩니다에서 단백질 - 단백질 상호 작용을 정의합니다. 이러한 고급 기술은 후속 또한 분자 관심 작용보다는 세포 내 구획을 통해 수용체 통로를 정의하는 데 사용되며, 이들은 직접 셀 (11) 내에서 특정 시간과 장소에서 단백질 - 단백질 상호 작용을 보여준다. 화상 취득이 느리기 때문에 FLIM, 억 셉터 농도에 독립적 인 FRET 기술은 주로 고정 된 샘플들에 수행된다. 대조적으로, 비 FRET 빠른 영상과 상호 작용하는 단백질의 고해상도 colocalization을을 제공한다. 비 FRET의 단점은 이미징 위드 에피 형광 이미징 속도를 얻기 위해 감소 해상도 및 세포 기관 (12)의 대조 결과 전체 셀을 포함를 활용한다. 생물 발광 공명 에너지 전달 (BRET)는 다른 인시간 (11)를 통해 분자 근접의 인수를 측정하기 된 GPCR에 적용된 고급 기술. 이 기술에서, 단백질 형광 분자는 분할되고, 각 반 당해 두 단백질 중 하나에 연결. 되면 관심 바인드 두 단백질 서로 키메라 태그의 부분은 형광 시간 동안 정량 증가 형광 취득 조립 재. 여기서 우리는 중앙 II 자극과 중앙 II 자극을 다음과 리소좀에 1A의 R AT 내면화 전달의 전위 변화에 따라 셀에 AT 1A는 R의 인신 매매를 연구하는 이미지 정량화와 결합 라이브 세포 이미징을위한 간단한 방법을 제시한다. 이 기술의 궁극적 인 사용은 야생형과 돌연변이 수용체를 비교 막 인신 매매의 차이를 특성화하는 것입니다. 이러한 차이는 AT 1A는 R leadi는을의 생리 활성 메커니즘 (들)에 영향을 파악하는 데 도움이 될 것입니다혈압 조절 ng 내지.

<table><tbody><tr><td>Angiotensin II (Ang II)</td><td>Sigma-Aldrich</td><td>A9525&amp;nbsp;</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle medium (DMEM 1x)</td><td>Gibco -ThermoFisher Scientific</td><td>11960-044</td><td>Warm in 37 &amp;deg;C water bath before use&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>F2442</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Penicillin Streptomycin (Pen Strep)</td><td>Gibco -ThermoFisher Scientific</td><td>15140-122</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>L-Glutamine 200 mM (100x)</td><td>Gibco (life technologies)</td><td>25030-081</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Human Embryonic Kidney (HEK)-293 cells</td><td>American Type Culture Collection (ATCC)&amp;nbsp;</td><td>CRL-1573</td><td>Passage number 5 - 12</td></tr><tr><td>Nunc Lab-Tek II chambered 1.5 German Coverglass System</td><td>Sigma-Aldrich</td><td>155409</td><td></td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>ThermoFisher Scientific</td><td>11668019</td><td>Store at 4 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Opti-MEM, Reduced Serum Medium</td><td>Gibco- ThermoFisher Scientific</td><td>31985-070</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>PBS 10x&amp;nbsp;</td><td>Fisher BioReagents</td><td>BP3994</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Dulbecco&#039;s Modified Eagle medium (DMEM 1x)&amp;nbsp;</td><td>Gibco -ThermoFisher Scientific</td><td>31053-028</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>LysoTracker Red DND-99</td><td>molecular probes -ThermoFisher Scientific</td><td>L7528</td><td>Store aliquotes in dark at -20 &amp;deg;C .&amp;nbsp;</td></tr><tr><td>Volocity Ver. 6.3 Image Analysis software</td><td>PerkinElmer</td><td>Visualization and Quantitation Modules</td><td>For 3D image analysis of image stacks</td></tr><tr><td>Leica SP8 AOBS</td><td>Leica Microsystems</td><td>laser scanning confocal microscope</td><td>For image collection</td></tr><tr><td>Zeiss LSM 880</td><td>Carl Zeiss</td><td>laser scanning confocal microscope</td><td>For image collection</td></tr></tbody></table>

live cell imaging and 3d analysis of angiotensin receptor type 1a trafficking in transfected human embryonic kidney cells using confocal microscopy

라이브 셀 이미징 동시에 안지오텐신 II (중앙 II) 자극 다음 형질 전환 된 인간 배아 신장 293 (HEK) 세포에서 안지오텐신 1a 형의 (1A의 R AT) 수용체 및 세포 내 구획의 시간 경과 이미지를 캡처하는 데 사용됩니다. HEK 세포를 일시적으로 플라스미드 DNA 향상된 녹색 형광 단백질 (EGFP) 태그 (1A)의 R AT 함유 형질. 리소좀은 빨간색 형광 염료로 식별됩니다. 라이브 셀 이미지는 중앙 II 자극 후 레이저 스캐닝 공 초점 현미경에 포착 시간이 지남에 따라 세 가지 차원 (3D, 복셀)에서 소프트웨어로 분석된다. 라이브 세포 이미징 수용체 피킹으로 조사 할 수 있으며 정착과 관련된 교란을 방지하고, 특히, EGFP 태그가 막 수용체의 손실 또는 artefactual 변위. 각 셀은 시간을 추적 같이 따라서, 수용체의 세포 내 지역화 묘화하고 측정 할 수있다. 이미지는 sufficientl을 취득해야합니다Y는 빠른 속도로 빠른 소포의 움직임을 촬영합니다. 그러나, 빠른 속도로 영상을 수집 된 광자의 수는 감소된다. 절충안은 이미징 속도를 얻기 위해 같은 복셀 사이즈 촬상 파라미터의 선택에서 이루어져야한다. 라이브 세포 이미징의 중요한 응용 프로그램 이름하지만 몇 가지로 단백질 인신 매매, 이동, 증식, 세포주기, 세포 사멸, 자식 작용 및 단백질 - 단백질 상호 작용과 역학을 공부한다.

이 실험의 대표 세트에, HEK 세포를 N2 및 pEGFP-13 플라스미드로 클로닝 (1A R-GFP AT) 안지오텐신 계 1A 수용체 구성 (E1,2,3-AT 1A는 R)로 형질 감염시켰다. HEK의 시간 경과 이미지 획득 및 동영상 (라이브 셀 이미징 1 라이브 셀 이미징이 동영상 참조)로 연주 하였다. 영화와 스틸 리간드 자극 후 1A AT R-EGFPs는 세포막...

Watch this Scientific Journal Video about Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy at JoVE.co

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

여기에서 우리는 안지오텐신 II 치료에 의해 시작 엔도 시토 시스 동안 녹색 형광 단백질 태그 안지오텐신 형 1A 수용체를 발현하는 이미지 세포에 대한 프로토콜을 제시한다. 이 기술은 시간이 지남에 따라 입체적 수용체 리소좀의 공동 현지화를 분석하는 소프트웨어를 이용하여 다음 라벨링 제 형광 마커 리소좀 등을 포함한다.

공 초점 현미경을 사용하여 트랜 인간 배아 신장 세포의 라이브 셀 이미징 및 3D 분석 안지오텐신 수용체의 유형 (1A)의 인신 매매

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

live-cell-imaging-3d-analysis-angiotensin-receptor-type-1a

Research

JoVE Journal

Biology

9.6K 조회수.  Georgetown University Medical Center.  여기에서 우리는 안지오텐신 II 치료에 의해 시작 엔도 시토 시스 동안 녹색 형광 단백질 태그 안지오텐신 형 1A 수용체를 발현하는 이미지 세포에 대한 프로토콜을 제시한다. 이 기술은 시간이 지남에 따라 입체적 수용체 리소좀의 공동 현지화를 분석하는 소프트웨어를 이용하여 다음 라벨링 제 형광 마커 리소좀 등을 포함한다. 

비디오: Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

Leukotriene B의 실시간 영상 4 중재 셀 마이 그 레이션 및 β - arrestin로 BLT1 상호 작용

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

연구

면역학 및 감염병학

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

3 차원 매트릭스에서 찬란 태그가 추가된 단백질을 표현 마이 그 레이션 전지의 라이브 세포 이미징

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

생체공학

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

분석<em&gt; 생체</em&gt; 셀 마이그레이션 Zebrafish의 배아에서 세포 이식 및 시간 경과 이미징을 사용하여

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

발생학

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

공촛점 이미징에 대한 분리 및 성인 심장 Myocytes의 유전자 조작

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

생물학

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

안지오텐신 II 수용체의 현지화 시각화에 대한 수용체자가 방사선 프로토콜

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...

신경과학

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.
The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium.

백혈구 롤링 Angiotensin II-주입 마우스에서 접착 시각화: 기술 및 함정

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

3 차원 형광 이미지에서 세포 형태학의 변경 사항 단정 지을 분석 도구

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

배양 된 세포의 소포체 하위 도메인의 시각화

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

차 쥐 신생아 심근 라이브 셀 이미징은 아데노 바이러스 다음과 렌티 바이러스 형질 도입은 공 초점 현미경 디스크를 회전시키는 사용

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

Colocalizational 분석에 의한 수용체 후 국제화 인신 매매에 약물 유발의 이동을 추적

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...