이 작품은 독일 Forschungsgemeinschaft (DFG) BL567/3-1와 프리드리히 바우어 - 재단의 보조금에 의해 지원되었다. 우리 태그 - RFP-T2를 저희에게 제공하기 위해 캘리포니아 샌디에고 대학의 로저 Y. 첸 교수, 하워드 휴즈 의학 연구소의 연구소에 감사드립니다. 우리는 다행히도 우리에게 렌티 바이러스 플라스미드 FUGW를 제공하고 psPAX2/pMD2.G 데이비드 볼티모어, 캘리포니아 공과 대학교, 패서 디나, 그리고 디디에 Trono, 제네바, 제네바, 대학을 인정합니다.

<ol>
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저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

TED 방법의 장점과 단점은 최근의 출판물 34,35에서 광범위하게 논의되었다. 위에서 설명한 다른 방법에 비해, TED는 세포를 방해하고 관류의 문제를 우회. 또한, 두 가지 측면이 강조 될 필요가있다. ER 칼슘 이미징을위한 TED의 원리는 TED 벡터 구조의 도움과 낮은 친화력 합성 AM-에스테르 (2). 효율적이고 특혜 릴리스 ER 루멘의 활성 carboxylesterase (1). 대상으로 표현해야합니다 ER 루멘 칼슘 염료...

ER의 생리적 칼슘 반응을 해결하기 위해, 우리는 ER로 합성 칼슘에 민감한 염료의 트래핑을 개선하기 위해 새로운 전략을 개발했다. 이 방법은 세포 외 칼슘의 면전에서 무료 ER 칼슘의 직접, 무중단 실시간으로 모니터링 할 수 있습니다. ER 칼슘의 기능 및 신호 칼슘 신호는 다른 세포 유형, 예를 들면 근육 세포, 뉴런과 학습과 기억 1,2에서 시냅스 전달에 관여로 근육 수축을 중재에서 glial 세포와 그 기능 범위에 있습니다. 칼슘은 유전자 전사, 세포 증식, 신경 흥분, 세포 죽음 및 이벤트 1-7 신호를 다른 세포의 조절에 관여되어 있기 때문에 무료로 칼슘 농도의 변화는 높은 과학적 관심이다. 이러한 모든 세포 칼슘 신호는 기능적으로 연결되어 있고 칼슘을 세포 내에 기여하는매장 역학 8-10. 모든 칼슘 신호 사이의 일반적인 기능은 세포 공간과 칼슘의 흐름, 세포질 및 세포 소기관, 주로 ER 및 미토콘드리아이다. 이 서로 다른 신호 구성 요소에 의해 감지되는 이러한 세포 소기관 내 칼슘 농도의 동적 변경을 발생합니다. 일반적으로 100-800 μM 사이 ER 범위에서 칼슘 농도가 세포질에 칼슘 농도에 가까운 100 nm의, 그리고 세포 공간에 농도가 1 ~ 2 밀리미터 정도입니다. 따라서, 세포질 2,9,10으로 칼슘의 흐름에 대한 높은 화학 원동력이있다. 가장 일반적으로 조사 ER-파생 된 칼슘 신호는 포스 C (PLC)를 활성화 G-단백질 결합 수용체 (GPCR)의 자극에 따라 달라집니다. 차례 PLC는 이노시톨 1,4,5-trisphosphate (IP 3) 1을 생산하고 있습니다. 그 REC에 IP 3의 바인딩에 따라eptor이 (IP 3 녹화, 그림 1) ER 막에 칼슘 이온은 ER 루멘에서 해제됩니다. 역사적으로, ER에서 IP 3 매개 칼슘 방출은 처음 - 비록 간접적 - Streb 등으로 포상 췌장 세포에서 측정 된 1983 십일인치. 이 책은 처음으로 제안 아세틸 콜린, 포스 C 및 IP 3를 포함하는 신호 폭포. 칼슘 방출이 방법은 일반적으로 IP 3에 의한 칼슘 방출 (IiCR) (그림 1)이라고합니다. 수용체 tyrosin 키나제 링크 IP 3 높이 12 후 신호 ER의 칼슘 성장 인자와 신경 성장 인자의 작용에 의해 포스의 Cγ의 키나제에 의존 활성화. IiCR뿐만 아니라, 칼슘 상승은 ryanodine 재에 의해 전압 게이트 칼슘 채널 (칼슘 V), 및 후속 칼슘에 의한 칼슘 방출 (CiCR)를 통해 예를 들어, ionotropic 칼슘 항목에 의해 중재 될 수있다ceptors (RYR). IiCR 및 CiCR은 생리 학적으로 매장 운영 칼슘 항목 (SOCE)에 연결되어 있습니다. SOCE는 ER의 칼슘 릴리스에 대한 센서 STIM (기질의 상호 작용 분자)의 활동을 포함한다. STIM은 일시적인 수용체 잠재적 인 채널 (Trp의) 13 Orai 칼슘 채널 14도 전압 게이트 칼슘 채널 15 (그림 1)를 통해 세포 칼슘 항목을 자극하기 위해 표시되었습니다. ER 칼슘의 손실은 동적 적극적으로 펌프 칼슘 다시 ER로 사코 - 소포체 칼슘 ATPase의 (SERCA)의 작용에 의해 구출된다. 같은 쌥시 가르 긴 등의 약물 SERCA를 차단하면 세포질 구획 ER 칼슘의 지속적인 손실을 발표했다. 이 ER 칼슘 &quot;누수&quot;은 Sec61 단백질 복합체 16,17 (그림 1) 응급실 intramembrane 기공 복합체에 의해 발생합니다. 1998 년 Berridge는 모델, &quot;신경 세포 모델에서 신경&quot;, 어느 것이를 발표H는 신경 세포의 칼슘는 5 통합에서 ER의 원리 생리적 역할을 제안합니다. 이 모델은 신경 세포막 5의 세포 내 &quot;이미지&quot;를 형성하는 연속 ER 막 시스템의 존재를 고려합니다. 이 바이너리 진핵 세포 멤브레인 시스템은 뉴런 빠르고 느린 칼슘 신호의 시간적 및 공간적 통합을위한 기본 전제 조건으로 주장했다. 같은 신경 세포의 다른 쪽이 나 돌기의 병용 또는 이후에 하나 발생할 칼슘 신호는 세포의 세포체 또는 그들이 5-18을 표현하는 ER을 통해 핵을 수여한다. 그런 다음, 그 합은 신호 폭포의 유전자 전사 또는 통합의 신경 규제의 흥분에 영향을 미칠 수 있습니다. 따라서, ER 칼슘 신호의 통합을 지원합니다. 이 개념에 대한 하나의 전제 조건은 여러 연구에 의해 주장되었으며 적어도 somato-D에 대한 입증 된 어느 하나의 세포에서 ER의 연속성이다endritic 지역과 가까운 거리에 축삭 돌기 19-21. 긴 축삭 돌기에서 ER 연속성이 있는지 여부는 논쟁의 문제입니다. ER 막에 무료로 칼슘의 흐름을 측정하는 전략 칼슘 신호는 가장 자주 세포질 22,23으로 모니터링됩니다. 따라서, 그것은 쉽게 칼슘 2 + 세포에서 또는 6,24 세포 내 상점에서 세포질로 유입 여부를 구별 할 수 없습니다. 이 제한을 극복하기 위해, 직접 ER의 칼슘 이미징을위한 방법 론적 전략이 개발되었다. 요약하면, 다음과 같은 전략이 사용됩니다. (1) 유전자 조작 단백질 지표 25-27 단백질 기반의 낮은 친화력 칼슘 2 + 지표 칼슘 감지 단백질과 결합 된 발광 단백질 aequorin 또는 GFP를 사용하여 ER-대상. 이러한 유전자 조작 칼슘 2 + 지표 (GECIs)는 수신호 펩타이드의 도움으로 응급실을 대상으로 적극적으로 보존 및 검색 모티브를 사용하여 ER에 보관됩니다. 카멜레온 분할 YC7.3ER 29, 그리고 카멜레온의 D1 30, 그리고 카멜레온 원리에 대한 일반적인 ER 칼슘 2 + 지표베이스는 카멜레온 YC4.3 26,28입니다. AM-에스테르 낮은 친화력 칼슘 2 (2) 직접 에스테르 가수 분해 효소 기반의 염료 로딩 + 지표 31,32가. 표시기 염료 (크기 - Fura2-AM, 매기 - Fluo4-AM 또는 Fluo5N-AM) 파생 오전을 통과 친 유성, 칼슘 구분 상태에서 생체막. 그런 다음, 세포질뿐 아니라 ER에서, 내생 에스 테라 AM-에스테르 그룹의 다니엘과 세포질에 적극적으로 염료의 일정 금액 뒤에와 ER에두고, 칼슘 2 + 표시를 놓습니다. 세포질 칼슘 농도가 잘 드 아래에 남아 그러므로,이 방법은 오래 ER에서 높은 칼슘 농도 조건에서 유용하다특히 특징적인 칼슘 신호 (낮은 μM로 예를 들면 NM) 동안 낮은 선호도 지표의 방호 한계. 플라즈마 멤브레인 permeabilization 32와 함께 (3) AM-에스테르 로딩. 모든 나머지 세포질 칼슘 2 + 표시는 인공 세포 버퍼에 &quot;중성&quot;세제 (예 : 사포닌)의 소량의 플라즈마 멤브레인 permeabilization에 의해 제거됩니다. 따라서, 세포 세포막에 직접 세포막의 &quot;구멍&quot;을 통해 세포 버퍼에 IP 3에 예를 들어, 자극 할 수있다. (4) 칼슘의 전체 셀 구성 및 동시 측정에 따라 세포질의 투석 + ER 루멘 및 세포질 32,33 인치 세포가 먼저 낮은 친화력 칼슘 2 + 지표 (예를 들어 크기 - Fura2 - 함께로드됩니다 AM), 비례 UV 빛. 그 후, 패치 피펫, 남아있는 CYT의 도움으로osolic 낮은 친화력 칼슘 2 + 지표는 높은 친화력 칼슘 2 + 지표 (예를 들면 플루오-3, 가시 광선)를 포함하는 버퍼 세포질에서 dialysed 있습니다. 이 전략은 세포질과 ER 유도 신호의 동시 녹음을 할 수 있습니다. (5) 대상 - 에스테르 가수 분해 효소에 의한 염료 로딩 8,34. Carboxylesterase은 (CES) ER의 내강을 대상으로 낮은 친화력 칼슘 2 + 지표 AM-에스테르 형태로 효율적으로 트래핑 높은 에스테르 가수 분해 효소 활동을 제공합니다. 대상 - 에스테르 가수 분해 효소 유도 염료로드 (TED) ER 루멘 낮은 친화력 칼슘 2 + 지표를 대상으로 개선하기 위해 TED가 개발되었다. TED는 식 구조를 통해 달성된다 ER 대상 마우스 carboxylesterase (CES2) (그림 2)의 발현을 필요로한다. 재조합 CES-구조를 표현 세포에서 칼슘의 AM-에스테르 양식을 배양하는식일 염료 (Fluo5N-AM, 그림 2). 그런 다음, ER에서, 염료 칼슘 2로 변환 + 민감한 막 투과성 칼슘 따라서 ER 루멘 8 높은 농도에서 염료를 트래핑 높은 에스테르 가수 분해 효소 활동에 의해 + 표시 복잡 (Fluo5N/Ca 2 +), 34. 방법은 SERCA 17의 봉쇄 한 후 예를 들어 직접 &quot;누설 채널&quot;을 통해 대사성, 퓨린 또는 글루타민산 염 수용체 8,34를 통해 인스턴스에 대한 IiCR - 경로를 통해 ER 칼슘 방출을 조사하고, ER의 칼슘 고갈을 시각화하는 것이 특히 유용합니다 34. 우리의 경험에 낮은 친화력 칼슘 2 + 지표 Fluo5N-AM 현재 TED 염료로드 전략을 사용하는 가장 좋은 가능한 지표입니다. Fluo5N-AM은 칼슘 2 +에 대한 낮은 친화력이 (해리 상수 K D ~ 90 μM, 그림 2), 거의 비 형광의 AM-형태이지만, calciu에 높은 형광 방출을 제공합니다M 바인딩 8,34. Fluo5N/Ca 2 + 단지는 같은 FITC, 알렉사 488, 또는 EGFP와 같은 표준 염료에 해당 ~ 490 ㎚의 표준 광원으로 흥분 할 수 있습니다. 세포질 칼슘 신호는 거의 낮은 μM 범위에서 농도에 도달하지 않으므로 거의 세포질 35 Fluo5N에 의해 감지됩니다. TED의 성능을 향상시키기 위해 몇 가지 재조합 벡터의 구조는 (그림 3)을 개발 하였다. 원래 CES2의 코딩 서열 (Refseq의 기탁 번호 NM_145603, CES2c)과 최고의 TED의 성능에 따라 TED 벡터는 CES의 구조를 안정적으로 발현으로 관찰된다. 새로운 TED 벡터는 빨간색 형광 단백질 TagRFP-T2 36 융합 CES2의 핵심 요소를 표현한다. 이러한 벡터들은 세포를 형질 확인하고 Fluo5N/Ca 2의 변화의 정상화를위한 내부 통제로 적색 형광을 사용하는 데 사용할 수 있다는 장점을 가지고 + 형광. 빨간색 형광은 자극 조건 하에서 ER과 ER 역학의 변화의 구조적 분포를 시각화 할 수있는 가능성을 제공합니다.

<table border="1">
	 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>(S)-3,5-DHPG (Dihydroxyphenylglycine)</td>
 <td>Tocris</td>
 <td>0805</td>
 <td></td>
 </tr>
 <tr>
 <td>5';-ATP-Na2, ATP disodium salt hydrate</td>
 <td>Sigma</td>
 <td>A26209-1G</td>
 <td></td>
 </tr>
 <tr>
 <td>B-27 supplement,50x</td>
 <td>Invitrogen</td>
 <td>17504-044</td>
 <td></td>
 </tr>
 <tr>
 <td>Carbamoylcholine chloride (Charbachol)</td>
 <td>Sigma</td>
 <td>C4382-1g</td>
 <td></td>
 </tr>
 <tr>
 <td>Cyclopiazonic acid (CPA)</td>
 <td>Ascent scientific</td>
 <td>Asc-300</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethylsulfoxide (DMSO)</td>
 <td>Sigma</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM with Glutamax</td>
 <td>Invitrogen-Gibco</td>
 <td>31966-047</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's PBS without Ca/Mg 1x</td>
 <td>PAA</td>
 <td>H15-002</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal calf serum</td>
 <td>Invitrogen-Gibco</td>
 <td>10270-106</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluo-5N-AM, 10 x 50 &mu;g</td>
 <td>Invitrogen</td>
 <td>F14204</td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamate</td>
 <td>Ascent scientific</td>
 <td>Asc-049</td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamax</td>
 <td>Invitrogen</td>
 <td>35050-038</td>
 <td></td>
 </tr>
 <tr>
 <td>Ham's F12 nutrients mixture</td>
 <td>Invitrogen-Gibco</td>
 <td>21765-029</td>
 <td></td>
 </tr>
 <tr>
 <td>Hank's BSS(1x) without Ca,without Mg, with Phenol Red, HBSS</td>
 <td>PAA Laboratories</td>
 <td>H15-010</td>
 <td></td>
 </tr>
 <tr>
 <td>Horse serum</td>
 <td>SHD3250YK</td>
 <td>Linearis</td>
 <td></td>
 </tr>
 <tr>
 <td>Ionomycin Ca2+ salt</td>
 <td>Ascent scientific</td>
 <td>Asc-116</td>
 <td></td>
 </tr>
 <tr>
 <td>murine EGF </td>
 <td>PreproTech</td>
 <td>315-09</td>
 <td></td>
 </tr>
 <tr>
 <td>N2 supplement 100x</td>
 <td>Invitrogen</td>
 <td>17502-048</td>
 <td></td>
 </tr>
 <tr>
 <td>Neurobasal medium</td>
 <td>Invitrogen-Gibco</td>
 <td>21103-049</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F127, low UV absorbance,2g</td>
 <td>Invitrogen</td>
 <td>P6867</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-DL-ornithine hydrobromide PORN</td>
 <td>Sigma</td>
 <td>P8638</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-D-lysine hydrobromide</td>
 <td>Sigma</td>
 <td>P7280</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-L-Lysin</td>
 <td>Sigma</td>
 <td>P2636</td>
 <td></td>
 </tr>
 <tr>
 <td>Thapsigargin</td>
 <td>Calbiochem</td>
 <td>586005-1mg</td>
 <td></td>
 </tr>
 <tr>
 <td>TryLE Express</td>
 <td>Invitrogen</td>
 <td>12605-028</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Worthington</td>
 <td>LS-003707</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsininhibitor from Glycine MAX (soybean)</td>
 <td>Sigma</td>
 <td>T6522</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Materials</td>
 </tr>
 <tr>
 <td>Confocal system: inverted laser scanning microscope: FV1000 with IX81</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Confocal system: laser combiner FV10 and diode lasers (405, 473, 559, 635)</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Confocal system: scan head FV10-SPD with spectraldetector</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Heater controller TC-344B</td>
 <td>Warner Instruments</td>
 <td>64-0101</td>
 <td>to control in line solution heater</td>
 </tr>
 <tr>
 <td>NIH ImageJ software</td>
 <td>WS Rasband, ImageJ, US National Institutes of Health, <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>, 1997--2006</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UAPON20XW340 NA 0,7 WD 0,35</td>
 <td>Olympus</td>
 <td>N2709100</td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UPLFLN40XO</td>
 <td>Olympus</td>
 <td>N1478700</td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UPLSAPO60XO/1.35, WD=0.15</td>
 <td>Olympus</td>
 <td>N1480700</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber, inverse</td>
 <td>custom-made</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber, RC-49FS</td>
 <td>Warner Instruments</td>
 <td>W4 64-1709 </td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion tube: PT-49</td>
 <td>Warner Instruments</td>
 <td>64-1730</td>
 <td>for perfusion</td>
 </tr>
 <tr>
 <td>Peristaltic pump MiniPlus 3 (4 channels)</td>
 <td>Gilson</td>
 <td>n.a. </td>
 <td>perfusion pump</td>
 </tr>
 <tr>
 <td>Single inline solution heater SH-27B</td>
 <td>Warner Instruments</td>
 <td>64-0102</td>
 <td>controlled by heater controller</td>
 </tr>
 <tr>
 <td>Suction tubes: ST3R</td>
 <td>Warner Instruments</td>
 <td>64-1407</td>
 <td>for perfusion</td>
 </tr>
 <tr>
 <td>Heating insert: Tempocontrol 37-2 digital 2-channel</td>
 <td>Pecon</td>
 <td>n.a. </td>
 <td></td>
 </tr>
	 </tbody>
 </table>

direct imaging of er calcium with targeted-esterase induced dye loading (ted)

칼슘 역학의 시각화 세포 생리학에서 칼슘의 역할을 이해하는 것이 중요합니다. 칼슘 역학을 조사하기 위해, 합성 형광 칼슘 2 +의 indictors가 인기를 끌고있다. 여기에 우리가 TED (= 대상 - 에스테르 가수 분해 효소 유도 염료로드), 칼슘의 방출을 개선하는 방법을 보여 + 다른 세포 유형의 ER 루멘의 표시기 염료. 지금까지 TED는 세포 라인, glial 세포 및 생체 뉴런에 사용되었다. 벡터 구조를 사용하여 ER 루멘에 높은 carboxylesterase 활동의 대상으로 효율적인 재조합에 TED 기지가 표현 Carboxylesterases (CES). 최근 TED 벡터 따라서 동시에 두 색 이미징을 가능하게 빨간색 형광 단백질에 융합 CES2의 핵심 요소가 포함되어 있습니다. 해당 ER 구조가 빨간색으로 표시되는 동안 ER 무료 칼슘의 역학은 하나의 색상에서 몇 군데 있습니다. 프로 시저의 시작 부분에서, 세포 렌티 바이러스로 형질 있습니다. 그 후, 감염된 세포다시 마지막 라이브 셀 이미징을 사용하는 coverslips를에 놓는. 그런 다음, 살아있는 세포가 아세 톡시 메틸 에스테르 (AM-에스테르) 낮은 친화력 칼슘의 형태로 배양하는 + 지표, 예를 들어 Fluo5N-AM, 매기 - Fluo4-AM, 또는 매기 - Fura2-AM. 칼슘 2 + 지표 및 친수성 형광 염료 / CA 2 + 복합 AM 양식에서 소수성 측쇄 오프 응급실 클리브의 에스테르 가수 분해 효소 활동이 형성된 ER 루멘에 갇혀있다. 염료로드 후, 세포는 거꾸로 공 촛점 레이저 스캐닝 현미경에서 분석됩니다. 세포는 지속적으로 벨소리와 같은 솔루션을 관류와 ER의 칼슘 역학 시간 경과 영상으로 직접 시각화 수 있습니다. ER의 칼슘 저장소의 보충이 형광 강도의 증가를 생산하는 반면, ER에서 칼슘 방출은 관심 영역에서 형광 강도의 감소에 의해 식별됩니다. 마지막으로, 시간이 지남에 따라 형광 강도의 변화는 ΔF / F 0의 계산에 의해 결정됩니다.

이 프로토콜은 무료 ER 칼슘의 직접 이미징을위한 무중단 방식을 제공합니다. 낮은 친화력 합성 칼슘 2 + 지표 발표하고 대상 ER의 도움으로 재조합 에스테르 가수 분해 효소 효소의 활동과 ER 루멘에 갇혀있다. ER 루멘 칼슘 2 + 지표 염료의 향상된로드는 ER의 칼슘 저장소 역학의 직접적이고 빠른 영상을 가능하게합니다. TED에 대한 세포 유형 ...

Watch this Scientific Journal Video about Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED at JoVE.com

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

대상 - 에스테르 가수 분해 효소 유도 염료로드 (TED)는 형광 이미징에 의해 세포 내 칼슘 저장소 역학의 분석을 지원합니다. 방법은 합성 낮은 친화력 CA의 로컬 폭로을 향상 소포체 (ER)로 재조합 Carboxylesterase의 대상에 기지<sup&gt; 2 +</supER 루멘&gt;에 표시 염료.

대상 - 에스테르 가수 분해 효소와 ER 칼슘의 직접 이미징 유발 염료 로딩 (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

direct-imaging-er-calcium-with-targeted-esterase-induced-dye-loading

Research

JoVE Journal

Biology

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

19.1K 조회수.  University of Wuerzburg. 대상 - 에스테르 가수 분해 효소 유도 염료로드 (TED)는 형광 이미징에 의해 세포 내 칼슘 저장소 역학의 분석을 지원합니다. 방법은 합성 낮은 친화력 CA의 로컬 폭로을 향상 소포체 (ER)로 재조합 Carboxylesterase의 대상에 기지 2 +에 표시 염료.

비디오: Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

칼슘 지표로 로딩 Drosophila 신경 터미널

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

연구

생물학

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

레이어의 VIVO에서 2 광자 칼슘 이미징 마우스의 2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

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 ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

조립, 로딩 및 분석 초원 심 분리기 샘플 셀 정렬

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

siRNA의 중재 진 입을와 결합 라이브 세포 칼슘 영상은 CA를 나타냅니다 2 + ER 막의 누출 채널과 규제 메커니즘

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Expression and Purification of Virus-like Particles for Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user&#39;s microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

표현과 예방 접종에 대한 바이러스 입자의 정제

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

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 ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

중간 필 라 멘 트와 2 차원 직접 확률적 광학 재건 현미경으로 Microtubules 이미징

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

We describe a comprehensive and practical protocol for fluorescence recovery after photobleaching experiments with live cells. Although the protocol was used to measure the mobility of yellow fluorescent protein-tagged p62 in aggresome-like induced structures, it can be applied to a variety of microscopy systems and fluorescent proteins.

Aggresome 같은 유도 구조에서 Photobleaching의 황색 형광 단백질 태그 p62 후 형광 복구

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

마우스 췌도 세포의 하위 집단에 있는 화상 진찰 칼슘 역학

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...