우리는 ratiometric - pericam - MT 표현 구조와 함께 우리를 제공 아츠시 미야 와키 감사하고 싶습니다. 이 작품은 국립 보건원 (DB)에서 부여 GM081685에 의해 지원되었다.

1. Pericam - MT의 Transfection 및 세포 준비. <ol><li> 표준 6 자 문화 플레이트에 위치 멸균 유리 coverslips에 transfection 전에 플레이트 헬라 세포는 밤. </li><li> 제조 업체 (Invitrogen)에 의해 제안으로 DNA / Lipofectamine 2000 단지를 준비합니다. 우리는 ratiometric - pericam - MT 발현 벡터 4 μg 각 잘 대해 Opti - 멤 0.5 ML에 희석 Lipofectamine 2000 10 μL를 사용합니다. 세포는 transfection 전에 ~ 70 %의 합류해야합니다. </li><li> 항생제없이 동일한 미디어 1.5 ML 각 우물에 헬라 문화 미디어 (Dulbecco의 수정 독수리 중간 10 % FBS, 페니실린 / 스트렙토 마이신) 교체합니다. </li><li> 각 잘 transfected 수에 DNA / Lipofectamine 단지를 추가합니다. 37 O C 5 % CO 2에서 세포 문화 인큐베이터에서 4 시간 동안 신나게 부화와 함께 부드럽게 섞는다. </li><li> 항생제와 문화 매체와 항생제없이 미디어를 교체합니다. 세포 이미징 전에 1-2일에 대한 ratiometric pericam을 표현하도록 허용합니다. </li></ol> 2. 현미경 설정 및 이미지 수집 <ol><li> 107mM NaCl, 7.2mM KCl, 1.2mM MgCl 2, 1mM CaCl 2, 11.5mM 포도당, 0.1 % 소 혈청 알부민 및 20mM HEPES 7.2 : 이미징 솔루션을 적절한 이미지 챔버에 헬라 세포와 coverslip를 놓습니다. 저희 연구실은 Invitrogen에서 Attofluor 셀 챔버를 사용합니다. 거꾸로 현미경 단계에있는 이미징 챔버를 탑재합니다. </li><li> 40x (또는 이상) 기름 침지 목표를 사용하여 ratiometric pericam을 표현하는 하나 이상의 세포를 포함하는 관심 영역을 찾아보십시오. 우리는 ratiometric pericam은 380 nm의에서 photobleaching 적은 강한 것으로 나타났습니다, 그래서 우리는 적당한 세포를 식별이 파장을 사용합니다. 그림 1에서 가져온 이미지의 경우, 1.3 조리개 수치와 니콘 Superfluor 40X 목적은 사용되었다. 높은 배율의 목표는 (60 - 100x) 적절한 것입니다. 사용 여기 필터는 채도 기술 필터 D380/30x했다 (380 + / - 30 nm의)과 D495/10 (495 + / - 5 nm의), beamsplitter가 505DCXR (505nm longpass)이었고, 방사 필터 HQ535/50m (535도 + /되었습니다 - 25nm). 그림 1의 이미지를 획득하는 데 사용되는 우리의 현미경 로퍼 과학 Coolsnap 사령부 CCD 카메라, Ludl MAC6000 빠른 필터 휠과 체인저 및 MetaFluor 데이터 수집 및 분석 소프트웨어를 냉각이 장착된 니콘 TE2000 거꾸로 현미경으로 구성되어 있습니다. 이 프로토콜은 적절한 필터와 ratiometric 이미지를 캡처하고 처리할 소프트웨어를 사용하여 모든 표준 와이드 필드 현미경을 적용할 수 있습니다. </li><li> 495 및 380 nm의 필터를 사용하여 이중 여기에 대한 소프트웨어를 설정합니다. ratiometric pericam에 바인딩 칼슘은 495 nm의 흡광도에서 증가, 따라서 비율은 495 nm의 nm/380로 설정해야합니다. Ratiometric pericam은 415 nm의 4 칼슘 무료 여기 피크 있습니다. 그것이 대부분의 칼슘 이미징 연구소에서 유비 쿼터스 경우에만하기 때문에이 프로토콜에서 우리는 380 nm의 필터를 사용하는 것이 좋습니다. 410 NM 필터를 사용할 경우, 그것은 380 nm의 필터 것이 바람직합니다. </li><li> 495 및 380 nm의에 순차적으로 또는 흥미로운 이미지를 취득. 재관류 장치를 통해 작용제의 응용 프로그램을하기 전에 최소한 30 s에 대한 기준 칼슘 수준의 이미지를 취득. 각 작용제에 대한 추가 시간을 표시합니다. 노출 시간 및 수집 간격은 여전히​​ 충분한 시간적 resolution.For 예제를 허용하면서 작용제의 자극에 대한 응답으로 모니터링 세미 신속한 칼슘 역학 위해 photobleaching 방지하기 위해 최적화해야 이미지가 인물 1B와 C. 더 빨리 획득 속도의 모든 이초을 인수했다 또한 6 가능합니다. staurosporine 관리 후 장기 영상은 인수 사이에 긴 간격 (30) (그림 1 D 및 E)를 함께 인수되었다. </li></ol> 3. 이미지 처리 및 분석 <ol><li> 실험이 완료되면 얻은 이미지는 오프라인 분석하실 수 있습니다. 당신이 칼슘 수준의 변화를 모니터하고자하는 관심 영역 (ROI)을 정의합니다. 필요한 경우 배경을 뺄 분야의 빈 영역 내에서 선택한 투자 수익 (ROI)을 사용합니다. 그것은 mitochondria 꽤 운동성이있는 및 동적 것을 지적하고, 연장 기간 동안 하나의 mitochondrion에을 바탕 이벤트는 항상 그렇지는 않습니다하여야한다. 따라서, 몇몇 세포 유형이 organelle의 높은 운동성을 고려, 투자 수익 (ROI)의 선택은 신중하게 모니터링해야합니다. 또한, 그림 1, 다양한 자극에 heterogeneously mitochondria 대응 입증. 데이터를 오프라인으로 분석하고 프레임으로 프레임 단위로 RO1 배치를 모니터링하는 것이 중요하다. 높은 운동성이있는 mitochondria에 mitochondrial 칼슘을 측정하는 상세한 설명이 다른 일곱 설명되었습니다. </li><li> 투자 수익 (ROI)에서 얻은 비율 측정은 Excel 또는 이와 유사한 graphingsoftware로 가져올 수 있습니다. 데이터는 단일 셀 또는 단일 mitochondrion에서 mitochondrial 칼슘 수준의 변화를 나타내는 성분으로 제시하거나, FL 평균 수여러 투자 수익 (ROI)에서 uorescence 신호. 우리는 또한 파 리간드 8 시까지 유도된 apoptosis를 겪고 lymphocytes의 인구의 평균 mitochondrial 칼슘 수준을 측정하는이 기술을 사용해 왔습니다. </li></ol> 4. 대표 결과 : <img src="/files/ftp_upload/2579/2579fig1.jpg" alt="그림 1" /> 그림 1. ratiometric - pericam - 후지산 (A) Subcellular 지방화. 첫 번째 이미지는 ratiometric - pericam - MT을 표현 라이브 헬라 세포를 보여줍니다. 두 번째 이미지는 mitochondrion - 선택적 염료의 MitoTracker 레드 CMXRos과 형광 염색법을 보여줍니다. 병합된 이미지의 노란색 형광은 ratiometric - pericam - MT 및 MitoTracker의 형광의 공동 지방화를 보여줍니다. (B) pseudocolor 비율 (495:380 NM) 10 MM ATP 취급 MT - ratiometric pericam을 표현하는 4 헬라 세포의 이미지 일련의 . (C) 관심있는 영역 (ROI)에 mitochondrial 칼슘 수준의 변화 부량는 pseudocolor 비율 (495:380 NM) 후지산을 표현 헬라 세포의 이미지 (B) 10 MM ATP 취급. (D) 시리즈에 표시된 - ratiometric pericam 0.5 μm의의 staurosporine과 치료. 개인 mitochondria에있는 칼슘 응답에 이질은 분명뿐만 아니라 60분하여 mitochondria의 중요한 조각이다. 다른 칼슘 수준에서 큰 변화 (노란색 화살표 머리)를 표시하지 않습니다 반면 일부 mitochondria는, 칼슘 (흰색 화살표 머리)에 oscillatory 증가했습니다.와 apoptosis의 유도 후 하나의 헬라 세포의 글로벌 mitochondrial 칼슘 수준 증가 (E) 부량 0.5 μm의의 staurosporine.

<ol>
	<li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signalling.">The versatility and universality of calcium signalling.</a> Nat Rev Mol Cell Biol. 1, 11-21 (2000).</li><li>Hajnoczky, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+calcium+signalling+and+cell+death:+approaches+for+assessing+the+role+of+mitochondrial+Ca2++uptake+in+apoptosis.">Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis.</a> Cell Calcium. 40, 553-560 (2006).</li><li>Nagai, T., Sawano, A., Park, E. S., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circularly+permuted+green+fluorescent+proteins+engineered+to+sense+Ca2.">Circularly permuted green fluorescent proteins engineered to sense Ca2.</a> Proc Natl Acad Sci U S A. 98, 3197-3202 (2001).</li><li>Rizzuto, R., Simpson, A. W., Brini, M., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+changes+of+mitochondrial+Ca2++revealed+by+specifically+targeted+recombinant+aequorin.">Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin.</a> Nature. 358, 325-327 (1992).</li><li>Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+indicators+for+Ca2++based+on+green+fluorescent+proteins+and+calmodulin.">Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.</a> Nature. 388, 882-887 (1997).</li><li>Shimozono, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+imaging+of+subcellular+Ca2++concentrations+using+a+dual-excitation+ratiometric+indicator+based+on+green+fluorescent+protein.">Confocal imaging of subcellular Ca2+ concentrations using a dual-excitation ratiometric indicator based on green fluorescent protein.</a> Sci STKE. , pl4-pl4 (2002).</li><li>Wozniak, 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=Requirement+of+biphasic+calcium+release+from+the+endoplasmic+reticulum+for+Fas-mediated+apoptosis.">Requirement of biphasic calcium release from the endoplasmic reticulum for Fas-mediated apoptosis.</a> J Cell Biol. 175, 709-714 (2006).</li><li>Boehning, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+c+binds+to+inositol+(1,4,5)+trisphosphate+receptors,+amplifying+calcium-dependent+apoptosis.">Cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis.</a> Nat Cell Biol. 5, 1051-1061 (2003).</li><li>Csordas, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+interorganelle+contacts+and+local+calcium+dynamics+at+the+ER-mitochondrial+interface.">Imaging interorganelle contacts and local calcium dynamics at the ER-mitochondrial interface.</a> Mol Cell. 39, 121-132 (2010).</li><li>Matsuyama, S., Llopis, J., Deveraux, Q. L., Tsien, R. Y., Reed, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+intramitochondrial+and+cytosolic+pH:+early+events+that+modulate+caspase+activation+during+apoptosis.">Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis.</a> Nat Cell Biol. 2, 318-325 (2000).</li><li>Jiang, D., Zhao, L., Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screen+identifies+Letm1+as+a+mitochondrial+Ca2+/H++antiporter.">Genome-wide RNAi screen identifies Letm1 as a mitochondrial Ca2+/H+ antiporter.</a> Science. 326, 144-147 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기 mitochondrial 타겟 ratiometric pericam를 사용하여 mitochondrial 칼슘을 측정하는 매우 간단한 방법을 제시한다. 것처럼 쉽게 허용 신호 대 잡음 비율과 헬라 세포에서 개별 mitochondria를 볼 수 있습니다 없음 deconvolution 표준 위드 광학을 사용하여 그림 1A에 표시됩니다. 공촛점 현미경 또는 기타 전문 장비에 대한 필요성을 obviating 때 자기편 헬라 세포는 문화에 대부분의 세포처럼 크게 버리고 있기 때문입니다. 우리는 폴리 - 리신 코팅 coverslips 8 준수 Jurkat 세포에서 유사한 결과를 발견했다. 염료를 사용하는 방법을 대조적으로, 그것은 비 invasively 같은 ratiometric pericam (그림 1E)로 인코딩된 유전자 칼슘 표시기를 사용하여 시간 mitochondrial 칼슘 수준을 모니터링하는 것도 가능합니다. 세포 죽음 동안 mitochondrial 칼슘을 분석 때 특히 중요합니다. 또한, 그것은 apoptotic 과정에서 분열 / mitochondria의 조각을 시각화하는 것도 가능합니다. 와 같은 인물 1D와 E에 나타난 staurosporine 치료는 20 분 mitochondria있는 봉우리를 선택 subpopulations에서 칼슘 느린 증가됩니다. 칼슘 수준은 다시 이시간 치료 후 다시 올라가기 전에 mitochondrial 조각과 함께 수반하는 내려가. 이것은 Fura - 2 9를 사용하여 측정 staurosporine 처리에 의해 유도된 cytosolic 칼슘 느린 파도와 일치합니다. 따라서, 중요한 운동 정보가있는 정적 측정을 고용하는 방법으로 가능하지 않다 얻을 수 있습니다. 우리가 비율과 그림 1에없는 칼슘 농도를 제시했지만, 그것은 절대 칼슘 레벨 4, 10를 계산하기 위해 현장에서 센서를 보정하는 것이 가능합니다. 고려해야 할 한 가지 중요한 주의해야 할 점은 ratiometric pericam는 산도 4, 10 민감한 때문입니다. 모두 cytosolic 및 mitochondrial 산도 수준은 apoptosis 11 동안 극적으로 변화 수 있듯이, 이것은 중요한 고려 사항이다. 절연 mitochondria 표현 pericam에서 산도의 적정가 입증되는 증가 [H +] 증가 410 nm의에서 여기, 동시에 mitochondrial 칼슘을 모두 측정하는 악용되어 속성에 의해 자극 방출에 약간의 영향으로 495 nm의에서 흥분 pericam의 방출 및 산도 12. 따라서, 선택 410 나노미터 여기와 방출을 모니터링하는 것은 산도에 대한 감소 우려와 비 ratiometrically 모니터 mitochondrial 칼슘하기위한 수단을 제공합니다. 마지막으로, 여기에 제시된 프로토콜은 따라서 대부분의 실험실로이 기술을 이용할 수 있도록, 광범위하게 사용 가능한 필터와 표준 epifluorescent 현미경에 액세스할 수 있어야합니다.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Inverted wide-field fluorescent calcium imaging microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>MEA53310</td>
<td>Any wide-field inverted microscope equipped with 380 nm/495 nm excitation filters and appropriate longpass and emission filter can be easily adapted to use this protocol.</td>
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<tr>
<td>Imaging Software</td>
<td>&nbsp;</td>
<td>Molecular Devices, Inc</td>
<td>Metafluor</td>
<td>&nbsp;</td>
<tr>
<td>Attofluor cell chamber</td>
<td>&nbsp;</td>
<td>Invitrogen Corporation</td>
<td>A-7816</td>
<td>Any imaging chamber for an inverted microscope which allows perfusion would suffice.</td>
</tr>
<tr>
<td>Lipofectamine 2000</td>
<td>&nbsp;</td>
<td>Invitrogen Corporation</td>
<td>11668-019</td>
<td>Most transfection reagents/protocols would be appropriate for these studies </td>
</tr>
<tr>
<td>MitoTracker Red CMXRos</td>
<td>&nbsp;</td>
<td>Invitrogen Corporation</td>
<td>M7512</td>
<td>&nbsp;</td>		</tbody>
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monitoring dynamic changes in mitochondrial calcium levels during apoptosis using a genetically encoded calcium sensor

다양한 자극에 대한 응답으로 세포 내 칼슘 농도의 동적 변화는 확산, 분화 및 apoptosis 한 많은 세포 과정을 조절. mitochondria에서 apoptosis, 칼슘 축적하는 동안 프로 apoptotic 요소의 릴리스는 cytosol이에 mitochondria에서 추진하고 있습니다. 직접 apoptosis 동안 현장에서 살아있는 세포에서 mitochondrial 칼슘을 측정하기 위해 관심을 따라서이다. 듀얼 여기 ratiometric 및 비 ratiometric 합성 칼슘 지표 염료와 함께 로드된 세포의 고해상도 형광 이미징은 세포내 칼슘 신호의 다양한 측면을 연구하기위한 신뢰성과 다양한 도구로 입증되었습니다. 이러한 기술을 사용 cytosolic 칼슘 fluxes을 측정하는 것은 비교적 간단합니다. 그러나,로드 &#39;- 2 및로드&#39;- FF로 합성 칼슘 지표를 사용하여 손상 세포 intramitochondrial 칼슘 수준을 측정하는 것은 더욱 어려운 것입니다. mitochondria를 타겟으로 합성 지표 mitochondrial 칼슘 반복 증가에 대한 답변을 무딘하고, mitochondrial 형태학에게 3 중단합니다. 또한, 합성 지표는 장기적인 실험들이 적합하게 몇 시간 동안 mitochondria 밖으로 누설하는 경향이있다. 따라서, mitochondria를 타겟으로하는 녹색 형광 단백질 (GFP) 4 aequorin 5를 기반으로 유전자 인코딩 칼슘 지표는 크게 mitochondrial 칼슘 역학의 측정을 촉진합니다. 여기, 우리는 다른 자극에 대한 응답으로 mitochondrial 칼슘 fluxes의 실시간 측정을위한 간단한 방법을 설명합니다. 방법을 선택 mitochondria을 타겟으로하는 &#39;ratiometric - pericam&#39;의 형광 현미경을 기반으로합니다. Ratiometric pericam는 circularly permuted 노란색 형광 단백질과 calmodulin 4 핵융합 기반으로 칼슘 표시기입니다. 방출 스펙트럼, 봉우리 주변에 515 nm의은 변함이 동안 ratiometric pericam에 칼슘의 바인딩은 415 nm의에 494 nm의 여기로부터 피크의 변화가 발생합니다. Ratiometric pericam는 ~ 1.7 μm의 4 체외에서 상수를 분리하여 하나의 칼슘 이온을 바인딩합니다. ratiometric pericam 이러한 특성 mitochondrial 칼슘 농도에 신속하고 장기적인 변화의 부량을 허용합니다. 또한, 우리는 일반적으로 사용 가능한 필터 세트와 표준 넓은 분야 칼슘 이미징 현미경이 방법의 적응을 설명합니다. 별개의 두 agonists, purinergic 작용제 ATP와 apoptosis - 유도하는 약물 staurosporine을 사용하여, 우리는이 방법은 시간이 초 시간적 해상도 mitochondrial 칼슘 농도의 변화를 모니터링에 적합한 것을 보여줍니다. 또한, 우리는 ratiometric pericam 또한 apoptosis 동안 mitochondrial 분열 / 조각을 측정하는 데 유용 것을 보여줍니다. 따라서 ratiometric pericam은 특히 apoptosis 동안 mitochondrial 칼슘 역학의 지속적인 장기 측정에 가장 적합합니다.

Watch this Scientific Journal Video about Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor at JoVE.com

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

이 프로토콜은 형광 이미징에 의해 mitochondrial 칼슘 fluxes의 실시간 측정을위한 방법을 설명합니다. 이 메서드는 선택 mitochondria로 표현 circularly permutated YFP 기반 듀얼 여기 ratiometric 칼슘 센서 (ratiometric pericam - MT)을 활용합니다.

유전자 변형으로 인코딩된 칼슘 센서를 사용하여 Apoptosis 동안 Mitochondrial 칼슘 레벨에서 동적 변경 사항 모니터링

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

monitoring-dynamic-changes-mitochondrial-calcium-levels-during

Research

JoVE Journal

Biology

23.8K 조회수.  University of Texas Medical Branch. 이 프로토콜은 형광 이미징에 의해 mitochondrial 칼슘 fluxes의 실시간 측정을위한 방법을 설명합니다. 이 메서드는 선택 mitochondria로 표현 circularly permutated YFP 기반 듀얼 여기 ratiometric 칼슘 센서 (ratiometric pericam - MT)을 활용합니다.

비디오: Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

이중층 탄성 등록 정보 모니터링 변경에 대한 단일 분자 방법

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

연구

생물학

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths.  The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding.  Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

Fura - 2 오전 사용하여 두피 뉴런의 칼슘 이미징

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated Calcium (CRAC) channels. CRAC channels play important role in regulation of T cell proliferation and gene expression. Abnormal CRAC channel function in T cells has been linked to severe combined immunodeficiency and autoimmune diseases 1, 2 . Studying CRAC channel function in human T cells may uncover new molecular mechanisms regulating normal immune responses and unravel the causes of related human diseases. Electrophysiological recordings of membrane currents provide the most accurate assessment of functional channel properties and their regulation. Electrophysiological assessment of CRAC channel currents in Jurkat T cells, a human leukemia T cell line, was first performed more than 20 years ago 3, however, CRAC current measurements in normal human T cells remains a challenging task. The difficulties in recording CRAC channel currents in normal T cells are compounded by the fact that blood-derived T lymphocytes are much smaller in size than Jurkat T cells and, therefore, the endogenous whole-cell CRAC currents are very low in amplitude. Here, we give a step-by-step procedure that we routinely use to record the Ca2+ or Na+ currents via CRAC channels in resting human T cells isolated from the peripheral blood of healthy volunteers. The method described here was adopted from the procedures used for recording the CRAC currents in Jurkat T cells and activated human T cells 4-8.

Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

We provide a step-by-step protocol for whole-cell patch clamp recording of Calcium Release-Activated Calcium (CRAC) currents in peripheral blood mononuclear cell-derived human T lymphocytes.

인간의 T Lymphocytes에서 칼슘 출시가 활성화된 칼슘의 전체 세포 기록 (CRAC) 해류

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes의 빠른 칼슘 Fluxes 측정

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response 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 ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

사용 소포체 칼슘 항상성 모니터링<em&gt; Gaussia</em&gt; 루시 페라 제 SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

형광 현미경으로 살아있는 세포의 잠재적 인 미토콘드리아의 칼슘과 미토콘드리아 막의 동시 측정

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.

표적화 된 Ca를 이용한 ER / SR 칼슘 방출 모니터링<sup&gt; 2+</sup&gt; 센서 캐쳐<sup&gt; +</sup

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

실시간 Vivo에서 녹음 애기 칼슘 신호의 곤충 먹이 형광 바이오 센서를 사용 하 여 중

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis of the screen is to generate a mutant population that can be screened with a phenotype of interest. EMS (ethyl methane sulfonate) is a commonly used alkylating agent for inducing random mutation in a classical forward genetic screen to identify multiple genes involved in any given process. Cytosolic calcium (Ca2+) elevation is a key early signaling pathway that is activated upon stress perception. However the identity of receptors, channels, pumps and transporters of Ca2+ is still elusive in many study systems. Aequorin is a cellular calcium reporter protein isolated from Aequorea victoria and stably expressed in Arabidopsis. Exploiting this, we designed a forward genetic screen in which we EMS-mutagenized the aequorin transgenic. The seeds from the mutant plants were collected (M1) and screening for the phenotype of interest was carried out in the segregating (M2) population. Using a 96-well high-throughput Ca2+ measurement protocol, several novel mutants can be identified that have a varying calcium response and are measured in real time. The mutants with the phenotype of interest are rescued and propagated till a homozygous mutant plant population is obtained. This protocol provides a method for forward genetic screens in Ca2+ reporter background and identify novel Ca2+ regulated targets.

A forward genetic screen based on Ca2+ elevation as a read-out leads to identification of genetic components involved in calcium dependent signaling pathways in plants.

칼슘 신호의 새로운 표적을 확인하기 위해 트랜스제닉 칼슘 리포터 Aequorin을 사용하여 유전자 화면을 전달

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis ...