Name: subcellular fractionation for erk activation upon mitochondrial-derived peptide treatment
Uploaded: 2017-09-25T15:00:00.000+00:00
Duration: 7 min 55 s
Description: Watch this Scientific Journal Video about Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment at JoVE.com

이 일 권, 노화 연구 프로그램에 엘리슨/멀리 박사 친교에 의해 지원 되었다 그리고 글렌 재단 수상 및 NIH PC에 부여 (1P01AG034906, 1R01GM 1R01ES 090311 020812). 모든 저자는 영화에 표시 됩니다.

1. 셀에 펩 티 드 치료 <ol><li>2 백만 SH-SY5Y 접시 또는 10 cm로 HEK293 세포 (2 x 10 6) 요리와 2 일 동안 그들을 성장</li> <li>(선택 사항) 다음 날, 셀 세럼 무료 Dulbecco 씻어 &#39; s 수정 매체 (DMEM) 미디어를 한 번 독수리 및 혈 청 치료 혈 청 무료 미디어에서 일을 해야 하는 경우 무료 DMEM 하룻밤 함께 품 어.</li>
	<li>3 일에 0.2 µ m 필터링, 증류수, 고 1 m m 재고 솔루션으로 그들을 다시 구성에서 S14G humanin 펩 티 드를 분해. 
	참고:는 펩 티 드 (예를 들어, 전체 충전) 펩 티 드의 시퀀스 특성에 의해 결정 될 수 있다 고는 펩 티 드 상업적으로 사용할 수 있는 경우는 제조 업체에 의해 제공 될 수 있습니다 적절 한 용 매에 녹아 해야 합니다. 예를 들어, S14G humanin, SHLP6, SHLP2, 및 태그 c 물에 용 해. Aliquot 동결을 방지 하 고 주기를 해빙 하는 적절 한 볼륨 (예를 들어, 50 μ)에서 펩 티 드. 일단 해 동, 사용 하지 않는 다시.</li>
	<li>미리 데워 진 혈 청 포함 약 수 또는 혈 청-무료 미디어 50 ml 원뿔 튜브 및 실내 온도 1 m m 재고 솔루션 작업 농도 (예를 들어, 1 µ M, 10 μ M)에 그것을 만들기 위해 추가.</li>
	<li>6 mL 펩 티 드 솔루션 단계 1.4에서에서 미디어 바꿉니다. 시간의 적절 한 금액에 대 한 셀을 품 어 고. 
	참고: 선택 ERK 활성화 상태에 따라 보육 시간.</li>
</ol> 2. Cytosol와 핵 subcellular 분별 <ol><li>얼음 처럼 차가운 PBS의 10 mL로 두 번 셀을 씻고, 얼음 처럼 차가운 PBS의 5 ml에서 접시에서 셀 긁 고 15 mL 원뿔 튜브에 세포 현 탁 액을 전송.</li>
	<li>4에서 5 분 동안 500 x g에서 세포를 원심 ° c.</li>
	<li>PBS를 발음 하 고 다시 얼음 처럼 차가운, 분별 버퍼의 200 µ L에 펠 릿을 일시 중단 (10 mM HEPES pH 7.6, 3 m m MgCl 2, 10 m m KCl, 5% (v/v) 글리세롤 1% 비 이온 계면 활성 제 (C 13 H 22 O (C 2 H 4 O) = n), 효소/인산 가수분해 효소 억제제). 
	참고: 유지 하기 위해 단백질의 인 산화 상태에, 인산 가수분해 효소 억제 물 뿐만 아니라 프로 테아 제 억제 물 갓 추가 되어야 하 고 샘플 지켜져야 한다 얼음에 모든 시간. 동결과 해 동을 반복 샘플의 주기를 피해 야 한다. -80에 그들을 동결 하기 전에 작은 금액 (예: 50 μ g)으로 aliquot 샘플 ° c.</li>
	<li>다음 4에 5 분 동안 250 x g에서 원심 고 15 분 동안 얼음에 다시 일시 중단 된 펠 릿을 품 어 ° c.</li>
	<li>세포질 분수 상쾌한 수집 하 고 핵 부분을 위해 펠 릿을 유지.</li>
	<li>원심 18000 x g 4 ° C에서 10 분 동안에 다른 오염 물질과 세포 파편을 제거 하 고 새로운 microcentrifuge 튜브에는 상쾌한 전송에 상쾌한. 이것은 cytosol 분수.</li>
	<li>200 μ 차가운 워시 버퍼에 펠 릿을 resuspend (10 mM HEPES pH 7.6, 1.5 m m MgCl 2, 420 mM NaCl, 25% (v/v) 글리세롤, 0.2 m m EDTA, 효소/인산 가수분해 효소 저 해제 =) 및 4 ° C에서 5 분 동안 250 x g에서 원심 분리기</li>
	<li>는 상쾌한 제거 및 100 μ 얼음 처럼 차가운, 핵 추출 버퍼에 펠 릿을 resuspend (20 mM HEPES pH 7.6, 1.5 m m MgCl 2, 420 mM NaCl, 25% (v/v) 글리세롤, 0.2 m m EDTA, 효소/인산 가수분해 효소 억제제 =)와 10 번을 sonicate (5 초, 10 s , 30% 앰프.) 얼음에</li>
	<li>4 ° c.에서 10 분에 대 한 18000 x g에서 원심 분리기</li>
	<li>새로운 microcentrifuge 튜브는 상쾌한 전송. 이것은 핵 분수.</li>
	<li>계량 BCA 분석 결과 사용 하 여 단백질 금액</li></ol>
 3. 원유 미토 콘 드 리아 분수 <ol><li>얼음 처럼 차가운 PBS의 10 mL와 함께 각 10 cm 접시에 셀을 씻어 5 mL의 얼음 처럼 차가운 PBS, 추가 하 고 셀 스 크레이 퍼를 사용 하 여 셀을 분리. 
	참고: 서 럽에 대 한 미토 콘 드리 아의 좋은 수확량을 셀의 3 10 cm 요리 사용.</li>
	<li>15 mL 원뿔 튜브에 세포 현 탁 액을 전송 하 고 한 15 ml 원뿔 튜브에 세 10 cm 요리에서 모든 결합.</li>
	<li>4 시 10 분 600 x g에서 세포를 원심 ° c.</li>
	<li>PBS를 발음 하 고 다시 얼음 처럼 차가운 미토 콘 드리 아 절연 버퍼의 1 mL에 펠 릿을 일시 중지 (10 mM Tris MOPS, pH를 조정 하는 1mm EGTA/트리 스, 자당, 200mm = 7.4).</li>
	<li>얼음에 소계 코팅된 유 봉과 2 mL 균질 화기의 25 선 셀 균질 
	참고:이 단계는 미토 콘 드 리아 무결성을 유지 하 고 미토 콘 드리 아 분수의 수익률을 극대화 하는 중요 한. 획 수는 각 세포 유형에 최적화 되어야 합니다. 절차를 시작 하기 전에 균질 화기를 쿨.</li>
	<li>Microcentrifuge 튜브에는 homogenate를 전송 하 고 핵 및 손상 되지 않은 세포 제거 하 4 ° C에서 10 분 600 x g에서 원심.</li>
	<li>는 상쾌한 수집 하 고 새로운 microcentrifuge 관에 그것을 전송 하 고, 4에서 10 분 동안 7000 x g에서 원심 ° c. 
	참고: 펠 릿은 느슨한 관리와 함께 상쾌한을 수집 하 고는 펠 렛을 방해 하지 않으려고.</li>
	<li>는 상쾌한을 제거, 다시 차가운 미토 콘 드리 아 절연 버퍼의 200 μ와 펠 릿을 일시 중단 하 고 새로운 microcentrifuge 튜브에 솔루션을 전송. 4에서 10 분 동안 7000 x g에서 튜브를 원심 ° c.</li>
	<li>반복 단계 3.8 펠 릿을 세척입니다. 이 단계에서 새로운 microcentrifuge 튜브는 상쾌한 전송할 필요는 없습니다.</li>
	<li>는 상쾌한 씻어 펠 릿에서 제거 하 고 다시 RIPA 버퍼의 50 μ와 미토 콘 드리 아를 포함 하는 펠 릿을 일시 중지.</li>
	<li>품 10 분에 대 한 얼음에 서 스 펜 션</li>
	<li>4에서 16000 x g 15 분 동안에 현 탁 액을 원심 ° c.</li>
	<li>새로운 microcentrifuge 튜브는 상쾌한 전송. 이것은 미토 콘 드리 아 분수.</li>
	<li>BCA 분석 결과 사용 하 여 단백질 양을 계량.</li>
</ol> 4. 인 특정 단백질을 위한 서쪽 Blotting <ol><li>는 SDS polyacrylamide 젤 전기 이동 법 (8-16% 들어찬 젤)를 수행 하 고 전송 단백질 PVDF 막 4.</li>
	<li>TBST에 5 %BSA 막 품 어 (0.1% 폴 20) 배경 비 특정 바인딩 사이트 차단에 실 온에서 30 분. 
	참고: phosphorylated 단백질 검출을 위한 BSA 하지 우유와 막을 차단 합니다. 우유 카 세 인, 높은 일반적인 신호는 풍부한 phosphoprotein 포함.</li>
	<li>하룻밤 4 ° C에서 1 차 항 체 (안티-인-ERK1/2)와 막 품 어.</li>
	<li>다음 날 씻어 TBST 가진 멤브레인 (0.1% 폴 20) 세 번 실 온에서 5 분.</li>
	<li>품 어 실 온에서 1 h에 대 한 이차 항 체 (항 토끼 HRP)와 막.</li>
	<li>TBST 가진 막 세척 (0.1% 폴 20) 세 번 실 온에서 5 분.</li>
	<li>ECL 솔루션 막 품 어 및 이미지 분석기에서 막 이미지.</li>
	<li>스트립 스트립 실내 온도 및 세척 5 분에 대 한 세 번 TBST 가진 막에 15 분에 대 한 버퍼와 막</li>
	<li>4.2-4.7 안티 총 ERK1/2 항 체를 사용 하 여 단계를 반복.</li>
	<li>각 분수의 순도 확인, SDS 페이지를 실행 하 고 immunoblots (예: 핵, 미토 콘 드리 아에 대 한 TOM20 및 GAPDH cytosol에 대 한 대 한 Lamin B1) 각 구획에 대 한 마커를 인식 하는 항 체를 사용 하 여 수행.</li>
</ol>

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느 코헨은 주주 및 CohBar, Inc. 켈빈 엔 CohBar, i n c.에 대 한 컨설턴트 역임 했다에 대 한 컨설턴트

여기, 우리가 보여주는 두 가지 서로 다른 세포 유형, 발생 하는 humanin 펩 티 드-중재 ERK1/2 활성화 및 활성화 ERK1/2의 subcellular 지 방화 (예를 들면, 펩 티 드, 시간 및 셀의 조건에 따라 다를 수 있습니다. 입력) 합니다. 그것은 보였다 그 humanin 두 개의 다른 수용 체22,23, humanin의 다른 복용량에 대 한 요구 사항 뿐 아니라 두 셀 라인 신호에 차이점을 ?...

신흥 연구는 미토 콘 드리 아에서 파생 된 펩 티 드 (MDPs) cytoprotection 및 대사1,2,3중요 한 역할을 재생을 보여줍니다. 신호 변환 통로 MDPs의 존재 이해는 MDPs는 다양 한 기능을 조절 하는 메커니즘에 대 한 통찰력 제공. 첫 번째 확인 된 민주당 humanin, extracellular 신호 통제 키 1/2 (ERK1/2)을 증가 표시 되었습니다4,5바인딩 그것의 수용 체를 통해 인 산화. 그러나, ERK1/2 활성화의 다운스트림 효과 여전히 underexplored.
ERK1/2 캐스케이드 다양 한 확산, 셀 이동, 세포 물질 대사, 생존, 및 apoptosis6,,78을 포함 하 여 세포질 과정에에서 필수적인 중재자 역할을 합니다. 모든을 통합 하는 것을이 다른 세포질 과정, 활동 및 ERK1/2의 subcellular 지 방화는 단단히 통제 가수분해 및 비 계 단백질9,10. 포스트 번역 상 수정 뿐 아니라 그것의 신호 기능, 활동, 및 특이성11,12조절 또한 ERK1/2의 동적 였죠. ERK1/2 주로 cytosol13에서 지역화 됩니다. 고정 및 비 계 단백질의 세트, 세포의 표면 또는 세포질13확산에 cytoskeletal 요소에 ERK1/2을 유지 도움이. 자극, ERK1/2은 phosphorylated 고 핵, 미토 콘 드리 아, 골, 리소좀14, 등 다른 subcellular 구획에 ERK1/2의 전 수 있도록 그 정박 단백질에서 해리 된다 15 , 16.
비록 humanin는 ERK1/2 신호 전달 경로 활성화, ERK1/2의 활성화만 총 세포 lysates에서 관찰 됩니다. ERK1/2 subcellular 지 방화의 다운스트림 효과, 모두 subcellular 지 방화의 분석 및의 총 수준에 중요 한 역할 때문에 이전에 설명한 대로 phosphorylated ERK1/2는 포괄적인 이해를 제공 하는 데 필요한 ERK1/2 활성화 humanin 유도 및 다운스트림 목표의 활성화.
활성화 ERK1/2의 대상 세포를 이해 하려면 뒤에 phosphorylated ERK1/2 서 부 럽 subcellular 분류 수행 되었다. 이 방법은 표준 실험실 장비 및 시 약 사용으로 쉽게 구현할 수 있습니다. 격리 된 subcellular 구획 straightforwardly 해석 결과 허용 하는 고 순도의 있습니다. ERK1/2 Immunostaining의 비슷한 결과 얻을 수 있습니다. 그러나, 특정 subcellular 구획은 상대적으로 시각화 하 고 특별 한 고정 및 permeabilization 방법이 필요로 어렵습니다. ERK1/2 레벨 subcellular 구획에 변화 하 고 전체 세포 lysates 볼 때이 변화 거짓 긍정과 거짓 부정적인 신호를 발생할 수 있습니다. 따라서, 격리 된 subcellular 구획을 사용 하 여 한 immunoblot 우리가 ERK1/2 지역화의 더 나은 이해를 제공 합니다.
방법의 다양성에 다른 MDPs 또는 STAT3 같은 다른 신호 분자의 전 좌를 포함 하 여 다른 자극의 효과 조사 하는 프로토콜의 수정 수 있습니다. 최근 발견 된 작은 humanin와 같은 펩 티 드 (SHLPs) 16S rRNA 지구 humanin 인코딩 및 HN17에 비해 유사 하지만 개별 속성에서 인코딩됩니다. 예를 들어 SHLP2 및 SHLP3에 8 h humanin 검토 다른 펩 티 드에 대 한 응답에서 ERK1/2의 subcellular 지 방화 5 분 이내 ERK1/2를 활성화 하는 있지만 우리가 줄 것 이다 이러한 펩 티이 드의 생물학의 더 나은 이해 후 ERK1/2를 활성화 해야 합니다. 증거를 신흥 신호 분자의 subcellular 지 방화의 다운스트림 효과에 중요 한 역할을 담당 했다. 예를 들어, STAT3 전통적으로 주로 휴식 셀에 cytosol에서 지역화는 알려져 있으며 다음 응답 cytokines18으로 유전자 발현을 활성화 하는 핵으로 translocates. 또한 STAT3 미토 콘 드리 아를 translocates 하 고 TCA 주기 및 ATP 생산19. Autophagy 규제에 관한 STAT3의 다른 subcellular 지 방화 다양 한 방법으로20에서 autophagy를 조절 한다. 예를 들어 핵 STAT3 transcriptionally autophagy 관련 유전자를 조절 하 고 autophagy 변조기 역할. 세포질 STAT3 constitutively autophagy 신호 분자와 상호 작용 하 여 autophagy를 억제. 미토 콘 드 리아 STAT3 억제 하 고 산화 스트레스 유발 autophagy를 억제 하 여 mitophagy를 방지 합니다. 따라서,이 subcellular 구획 격리 방법은 다른 신호 분자로 ERK1/2의 역할을 이해 하기 위한 중요 한입니다.

<table><tbody><tr><td>p44/42 MAPK (ERK1/2)</td><td>Cell signaling</td><td>9102</td><td>Dilution 1:1,000</td></tr><tr><td>phospho-p44/42 MAPK (ERK1/2)(Thr202/Tyr204)</td><td>Cell signaling</td><td>4370</td><td>Dilution 1:1,000</td></tr><tr><td>Lamin B1</td><td>Cell signaling</td><td>12586</td><td>Nuclear Marker, Dilution 1:1,000</td></tr><tr><td>GAPDH</td><td>Cell signaling</td><td>5174</td><td>Cytoplasmic marker, Dilution 1:2,000</td></tr><tr><td>Tom20</td><td>Santa cruz</td><td>SC-17764</td><td>Mitochondria marker, Dilution 1:2,000</td></tr><tr><td>anti-Rabbit-HRP conjugated</td><td>Cell signaling</td><td>7074</td><td>Dilution 1:30,000</td></tr><tr><td>RIPA Lysis and Extraction Buffer</td><td>ThermoFisher SCIENTIFIC</td><td>89900</td><td></td></tr><tr><td>100 mm Culture Dish</td><td>ThermoFisher SCIENTIFIC</td><td>12556002</td><td></td></tr><tr><td>HNG peptide</td><td>Genescript</td><td></td><td></td></tr><tr><td>25mm sylinge filter</td><td>ThermoFisher SCIENTIFIC</td><td>09-719A</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>MgCl2</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G9012</td><td></td></tr><tr><td>Triton X-100</td><td>ThermoFisher SCIENTIFIC</td><td>BP151-100</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>3609</td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>M1254</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S7903</td><td></td></tr><tr><td>Tris-base</td><td>ThermoFisher SCIENTIFIC</td><td>BP152-1</td><td></td></tr><tr><td>HCL</td><td>Sigma</td><td>H1758</td><td></td></tr><tr><td>PBS</td><td>Lonza</td><td>17-512F</td><td></td></tr><tr><td>Cell Scraper</td><td>FALCON</td><td>353085</td><td></td></tr><tr><td>Halt&amp;trade; Protease and Phosphatase Inhibitor Cocktail (100X)</td><td>ThermoFisher SCIENTIFIC</td><td>78440</td><td></td></tr><tr><td>Thomas Pestle Tissue Grinder Assemblies with Smooth Pestles</td><td>Thomas Scientific</td><td>3432S90</td><td></td></tr><tr><td>Tween-20</td><td>ThermoFisher SCIENTIFIC</td><td>BP337-500</td><td></td></tr><tr><td>BSA</td><td>ThermoFisher SCIENTIFIC</td><td>BP1600-100</td><td></td></tr><tr><td>8-16% Mini-PROTEAN TGX Precast Protein Gels</td><td>BIO RAD</td><td>4561104</td><td></td></tr><tr><td>Mini Trans-Blot Module</td><td>BIO RAD</td><td>1658030</td><td></td></tr><tr><td>Trans-Blot Turbo Transfer System</td><td>BIO RAD</td><td>1704150</td><td></td></tr><tr><td>Trans-Blot Turbo RTA Mini PVDF Transfer Kit</td><td>BIO RAD</td><td>1704272</td><td></td></tr><tr><td>Clarity Western ECL Blotting Substrates</td><td>BIO RAD</td><td>1705060</td><td></td></tr><tr><td>Restore Western blot stripping buffer</td><td>ThermoFisher SCIENTIFIC</td><td>21059</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>ThermoFisher SCIENTIFIC</td><td>11965-092</td><td></td></tr><tr><td>Sonicator, Medel: FB120</td><td>ThermoFisher SCIENTIFIC</td><td>695320-07-12</td><td></td></tr></tbody></table>

subcellular fractionation for erk activation upon mitochondrial-derived peptide treatment

미토 콘 드리 아에서 파생 된 펩 티 드 (MDPs) 미토 콘 드리 아 게놈의 다른 알려진된 유전자 내의 작은 열려있는 독서 프레임으로 인코딩됩니다 펩 티 드의 새로운 클래스는. MDPs는 apoptosis에서 신경 세포를 보호, 개선 신진 대사 마커, 화학요법에서 세포를 보호 등 생물학적 효과의 다양 한이 있다. Humanin 발견 첫 번째 민주당 고 민주당 가족 중 가장 공부 펩 티 드 이다. 막 수용 체와 humanin의 하류 신호 경로 신중 하 게 특징 되었습니다. 태그 c와 SHLP1-6 등 추가 MDPs 최근에 발견 되었습니다 고 신호 메커니즘 아직 해명 될. 여기이 펩 티이 드의 기능을 결정 하는 셀 문화 기반 방법을 설명 합니다. 특히, 서 부 럽 함께에서 셀 분류 기법 활성화의 양적 결정 및 중요 한 신호 분자의 전 좌 허용. 세포 분별 법의 다른 방법이 있다, 하는 동안 여기에 설명 된 하나 쉽고 간단한 방법입니다. 이러한 방법은 더 명료이 펩 티이 드 및 다른 치료제의 행동의 메커니즘을 사용할 수 있습니다.

여기에 제시 된 절차를 사용 하 여, 우리 1 μ M와 100 μ M HEK293와 SH SY5Y 셀 치료 S14G-humanin, 강력한 humanin 아날로그21, 각각, 완료 미디어 (그림 1 및 그림 1 지정 된 기간에 대 한 B). 우리는 다음 세202총 단백질 추출 물에서 /Tyr204 에서 ERK1/2의 총 및 phosphorylated 형태 검사. S14G...

Watch this Scientific Journal Video about Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment at JoVE.com

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

이 프로토콜에는 미토 콘 드리 아에서 파생 된 펩 티 드와 세포 신호 캐스케이드 인 단백질의 지 방화를 평가 하는 방법을 설명 합니다.

미토 콘 드리 아에서 파생 된 펩 티 드 치료 시 ERK 활성화에 대 한 subcellular 분류

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the ...

subcellular-fractionation-for-erk-activation-upon-mitochondrial

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JoVE Journal

Biology

7.8K 조회수.  University of Southern California. 이 프로토콜에는 미토 콘 드리 아에서 파생 된 펩 티 드와 세포 신호 캐스케이드 인 단백질의 지 방화를 평가 하는 방법을 설명 합니다.

비디오: Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

방사성 표지 된 ATP로 단백질 키나아제 활성 측정

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

연구

생화학

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

인간 배아 줄기 세포에서 Mitochondrial 복잡한 활동에 대한 탐색

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

생물학

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

활성 ERK의 공간 및 시간 분석<em&gt; C. 엘레</em&gt; 생식선

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

발생학

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

고속 원심 분리의 부재에서 U937 세포의 세포 분별 법

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

미토콘드리아 - 관련 ER의 멤브레인 (MAMs)와 Glycosphingolipid 강화 Microdomains (보석) : 마우스 뇌에서 절연

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

신경과학

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

신진 효모 사카로미세스 세레비시아에 제출 단백질 지역화 평가

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing. 
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral F&#246;rster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

Live FRET 현미경 검사 및 면역형광을 통해 초기 제브라피시 누난 증후군 모델에서 ERK 활동 조절을 평가하기 위한 효율적인 프로토콜

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

신장 세포 Overexpressing 단백질 키나제 C Isozymes에 Mitochondrial 기능 및 세포 생존 능력의 평가

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

갭 접합부 시험에 대한 기능 분석; 인듐 - 주석 산화물에 자기편 세포의 전기 천공 법 (electroporation)

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

미토 콘 드리 아에서 파생 된 펩 티 드 치료 시 ERK 활성화에 대 한 subcellular 분류

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