Name: detecting protein subcellular localization by green fluorescence protein tagging and 4',6-diamidino-2-phenylindole staining in caenorhabditis elegans
Uploaded: 2018-07-30T13:00:00.000+00:00
Duration: 9 min 36 s
Description: Watch this Scientific Journal Video about Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans at JoVE.

이 연구에 사용 된 선 충 C. 긴장 건강-사무실의 연구 인프라 프로그램 (P40 OD010440)의 국가 학회에 의해 지원 되는 꼬마 유전학 센터에서 얻은 했다. 이 작품은 NIH에 의해 지원 되었다: 1R03AG048578-01 6 월 회담, 6 월 회담, 및 PSC CUNY CCRG 1501-CUNY 66184 00 44와 67248-00 45 6 월 회담을. 우리는 그녀의 실험실 공간을 공유 하는 친절 하 게 대 한 캐시 야만인-던 감사 합니다. 모든 형광 이미지는 퀸 즈 대학 핵심 시설에서 수집 되었다. 원고에 대 한 그의 의견에 감사 윌리엄 J. 쌀 하 고.

1입니다. 솔루션
<ol><li>
NGM 플레이트
	<ol>
<li>2l 삼각 플라스 크에 NaCl의 3 세대, 한 천의 17 g, 펩의 2.5 g와 dH2오 커버 알루미늄 호 일로 플라스 크의 입의 975 mL를 추가 합니다. 압력솥 50 분에 대 한 플라스 크 20-30 분 동안 그것을 멋진.</li>
		<li>멸 균된 솔루션 추가: 1 mL의 1 M CaCl2, 에탄올에 5 mg/mL 콜레스테롤의 1 mL, 1 mL의 1 M MgSO4, 및 1 M KPO4 (pH 6.0) 버퍼의 25 mL. 그들을 잘 혼합 솔루션을 소용돌이 친다.</li>
		<li>액체 디스펜서를 사용 하 여, 60 mm 페 트리 접시에 NGM 솔루션 분배. 한 천의 격판덮개 2/3 전체 채우기. 나중에 사용할 4 ° C에서 밀폐 용기에 그들을 저장 합니다.</li>
	</ol>
</li>
	<li>
1 M KPO4 버퍼 (pH 6.0)
	<ol>
<li>KH2포4 10.83 g와 dH2O, K2HPO4 의 3.56 g 녹 이십시오 다음 100 ml 전체 볼륨을 조정 합니다. 압력솥 15 분에 대 한 솔루션입니다.</li>
	</ol>
</li>
	<li>
M9
	<ol>
<li>KH2포4, 나2HPO 6 g의 3 세대 분해4및 5 g dH2O, NaCl의 1 mL의 1 M MgSO4, 추가 하 고 1 L. 총 볼륨 조정</li>
	</ol>
</li>
	<li>
10 µ g/mL DAPI
	<ol>
<li>DAPI dH2오의 999 µ L에 10 mg/mL의 1 µ L을 추가</li>
	</ol>
</li>
	<li>
95% 알콜 (v/v)
	<ol>
<li>100% 알코올의 95 mL를 측정 하 고 증류수 100ml에 볼륨 조절 추가.</li>
	</ol>
</li>
	<li>
30% 아세톤 (v/v)
	<ol>
<li>DH2O 30 mL의 아세톤을 추가 하 고 100 ml 전체 볼륨을 조정 합니다.</li>
	</ol>
</li>
</ol>2. 열 스트레스
<ol><li>Extrachromosomal 배열 라인과 대상 유전자9,10의 변환 구문 생성 합니다. 또는,에서 꼬마 유전학 센터 (CGC), C. 선 충 연구를 위한 공공 자원 인 또는 이전에 게시 연구 실험실에서 같은 라인을 얻을.</li>
	<li>Extrachromosomal 라인을 3800 Rad γ 방사선9벌레를 노출 하 여 게놈에 통합할. 그런 다음 선택 안정적으로 표현한 라인 롤러 등 GFP 마커 단백질을 표현 하는 동물.</li>
	<li>방사선 중에 생성 된 돌연변이 제거 하려면 outcross 라인 적어도 2 x. 이 유전자 변형 라인을 사용 하 여 스트레스 단백질 식 패턴 변화 감지.</li>
	<li>1.1 단계에서 설명한 대로 NGM 웜 접시를 준비 합니다. OP50 복제를 행진 하 고 하룻밤 액체 LB 국물에 박테리아 문화. 벌레를 위한 음식 근원으로 박테리아 잔디를 성장 씨앗 NGM 접시 액체 OP50와 문화 고 37 ° C 배양 기에서 번호판을 품 어 하룻밤. 사용 하기 전에 적어도 30 분 동안 실내 온도에 격판덮개를 식혀.</li>
	<li>열 스트레스 분석 결과 이전에 적어도 2 세대에 대 한 기아 없이 20 ° C에서 유전자 변형 라인을 성장.</li>
	<li>새로운 벌레 플레이트, 그리고 하자 그들에 대 한 3-5 h에 대 한 알을 8-10 젊은 벗 성인 (자 궁 가득 계란)을 선택 하 고 모든 성인 판에서 제거.</li>
	<li>벌레를 동기화 하려면 계란 해치 고 약 48 h에 대 한 애벌레 애벌레 (L4)의 4 단계로 성장 하자. 그들의 외 음부 구조 (반달 구조) L4 단계 (그림 1, 화살표)11를 식별 하기 위해 검사 합니다.</li>
	<li>열 스트레스를 달성 하기 위해 2-5 h 35 ° C 배양 기에서 L4 애벌레와 벌레 접시를 놓습니다. 정확 하 게 온도 측정 하는 인큐베이터에 온도계를 넣습니다.</li>
</ol>3. DAPI 얼룩
<ol><li>
에탄올 고정
	<ol>
<li>유리 슬라이드 중앙에 M9 버퍼의 10 µ L를 추가 합니다.</li>
		<li>단계 2.8에서에서 열 충격 벌레를 선택 하 고 유리 슬라이드에 M9 버퍼에 넣어.<ol>
<li>또는, M9와 접시를 씻어, 실내 온도에서 1 분 동안 1000 x g 에서 원심 고는 상쾌한 삭제 합니다. 유리 슬라이드에 벌레를 추가 합니다.</li>
		</ol>
</li>
		<li>부드러운 휴지를 사용 하 여 모든 초과 액체를 배출 하. 조심 해 현미경이 단계.</li>
		<li>벌레에 95% 알코올의 10 µ L을 추가 하 고 그들이 건조 한 공기. 해 현미경 과정을 시청 하 고 너무 건조 얻을 하는 동물을 두지 마세요. 에탄올은 동물에서 증발 하 고, 후에 바로 다음 단계로 진행 합니다. 참고: 에탄올 매우 빠르게 증발.</li>
		<li>두 번 반복 단계 3.1.4 벌레 고정 되도록. 참고: 그것은 어두운 하 고 왜곡을 보고 동물에 대 한 정상입니다.</li>
		<li>DAPI의 10 µ L 추가 (10 µ g/mL) 벌레를. 즉시는 coverslip로 그들을 커버 하 고 투명 한 매니큐어 젤 슬라이드 인감.</li>
		<li>형광 현미경에서 약 10 분 후 동물을 보기.</li>
	</ol>
</li>
	<li>
아세톤 고정 (대체 방법)
	<ol>
<li>단계 2.8 m 9에서에서 열 충격 격판덮개 떨어져 세척 하 고 실내 온도에서 1 분 동안 1000 x g 에서 원심은 상쾌한 삭제. 씻어 dH2O의 1 mL와 펠 릿, 펠 릿, 수집 하 고 삭제는 상쾌한.</li>
		<li>1000 x g 실 온에서 1 분에 15 분 원심 분리기에 대 한 30% 아세톤의 400 µ L을 추가 하 고 삭제는 상쾌한. DH2O의 500 µ L를 사용 하 여 씻어 동물 2 x. 폐기는 상쾌한.</li>
		<li>DAPI의 200 µ L 추가 (10 µ g/mL), 또는 15 분 동안 관에서 총 벌레 볼륨의 금액 x 4.</li>
		<li>1 분 동안 1000 x g 에서 centrifuge 고는 상쾌한 삭제. 2 펠 릿을 세척 dH2오 500 µ L x 벌레 유리 슬라이드에, coverslips와 함께 그들을 커버, 투명 매니큐어 젤, 슬라이드 봉인 놓고 형광 현미경 동물을 관찰.</li>
	</ol>
</li>
</ol>4. 현미경 이미징
<ol><li>UV 광원 및 형광 현미경 켭니다. 현미경에 연결 된 컴퓨터를 켭니다.</li>
	<li>형광 현미경에 슬라이드를 로드 합니다. 낮은 전력 목표 렌즈 (10 배)를 사용 하 여 벌레, 찾아서, 400 X, 확대 힘 증가 표본에 집중.</li>
	<li>현미경으로 제공 하는 소프트웨어를 엽니다. 메뉴;에서 "인수" 클릭 "카메라"에 클릭 하 고 현미경;에 연결 된 카메라를 선택 소프트웨어와 통신 하기 위해 선택 된 카메라 수 있습니다. 참고: 다른 현미경 작업에서 달라질 수 있습니다.</li>
	<li>같은 "수집", "다차원 수집"에 클릭 합니다. 새로운 "다차원 수집" 탐색 창이 열립니다. "다중 채널" 버튼을 클릭 하 고 모든 사용할 수 있는 채널이 표시 됩니다.</li>
	<li>파랑, 녹색, 및 표본 관찰 하 DIC (미분 간섭 명암)를 선택 합니다. "다차원 수집" 탐색 창에서 두고는 "블루-DAPI", "그린-GFP", 및 "회색-DIC", "Nomarski" 채널 "레드-rhodamine" 및 "백색 밝은 분야", 등의 다른 채널을 마우스 오른쪽 단추로 그들을 닫습니다.</li>
	<li>
첫째, 각 채널에 대 한 노출 시간 측정:
	<ol>
<li>"블루-DAPI" 채널을 선택 하 고 다음 자동된 노출 시간 DAPI 채널에서 라이브 이미지 "측정"에 클릭 왼쪽 클릭 하십시오. 라이브 이미지와 함께 새로운 창이 나타납니다.</li>
		<li>라이브 이미지 창의 왼쪽에 원하는 대로 이미지를 만드는 데 필요한 경우 수동으로 노출 시간을 조정 합니다.</li>
		<li>마지막으로, "확인 클릭" 라이브 이미지 창에서. 이 DAPI 채널에서 노출 시간을 설정합니다.</li>
	</ol>
</li>
	<li>다른 채널, "그린-GFP" 및 "회색-DIC", "Nomarski", 그 채널에 대 한 노출 시간을 설정 하려면 4.6 단계를 반복 합니다.</li>
	<li>"다차원 수집" 탐색 창에서 "시작" 설정으로 특정 노출 시간 순서 (위 참조) 3 다른 채널에서 3 이미지를 자동으로 수집을 클릭 합니다.</li>
	<li>파일을 저장 하려면 주 메뉴에, "파일" 다음 "다른 이름으로 저장"을 클릭 합니다. 파일 이름 및 폴더 이름을 입력 합니다. 나중에 참조할 수에 대 한 상세한 이미지 정보를 유지 하려면 기본 파일 형식으로 파일을 저장 합니다.</li>
	<li>
다른 형식으로 파일을 내보내려면:
	<ol>
<li>"파일"에 고 "내보내기"를 클릭 합니다. 이것은 수출 설정 창을 열 것 이다.</li>
		<li>파일 이름 및 폴더를 설정 합니다. 데이터 구성 "프로젝트 폴더 만들기"를 선택 합니다. 소프트웨어는 또한 다른 4 옵션을 지정 하려면 사용자 수 있습니다: "생성 병합 이미지", "이미지에 대 한 색상을 사용", "사용 하 여 채널 이름" 및 "결합 된 이미지에 만".</li>
		<li>"TIF", "BMP", "PSD", 또는 "JPG" 및 다른 형식 예: 드롭-다운 메뉴에서 원하는 파일 형식을 선택 합니다. 다음 클릭 하십시오 "시작" 내보낼.</li>
	</ol>
</li>
</ol>

<ol>
	<li>Essers, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FOXO+transcription+factor+activation+by+oxidative+stress+mediated+by+the+small+GTPase+Ral+and+JNK.">FOXO transcription factor activation by oxidative stress mediated by the small GTPase Ral and JNK.</a> The EMBO Journal. 23 (24), 4802-4812 (2004).</li><li>Oh, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JNK+regulates+lifespan+in+Caenorhabditis+elegans+by+modulating+nuclear+translocation+of+forkhead+transcription+factor/DAF-16.">JNK regulates lifespan in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription factor/DAF-16.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (12), 4494-4499 (2005).</li><li>Gross, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+cleaved+BID+targets+mitochondria+and+is+required+for+cytochrome+c+release,+while+BCL-XL+prevents+this+release+but+not+tumor+necrosis+factor-R1/Fas+death.">Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death.</a> The Journal of Biological Chemistry. 274 (2), 1156-1163 (1999).</li><li>Doerflinger, M., Glab, J. A., Puthalakath, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BH3-only+proteins:+a+20-year+stock-take.">BH3-only proteins: a 20-year stock-take.</a> The FEBS Journal. 282 (6), 1006-1016 (2015).</li><li>Mayorova, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+containing+aragonite+crystals+mediate+responses+to+gravity+in+Trichoplax+adhaerens+(Placozoa),+an+animal+lacking+neurons+and+synapses.">Cells containing aragonite crystals mediate responses to gravity in Trichoplax adhaerens (Placozoa), an animal lacking neurons and synapses.</a> PLoS One. 13 (1), e0190905 (2018).</li><li>Terron-Camero, L. C., Molina-Moya, E., Sanz-Fernandez, M., Sandalio, L. M., Romero-Puertas, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+reactive+oxygen+and+nitrogen+species+(ROS/RNS)+during+hypersensitive+cell+death.">Detection of reactive oxygen and nitrogen species (ROS/RNS) during hypersensitive cell death.</a> Methods in Molecular Biology. , 97-105 (2018).</li><li>Pedrazzini, E., Vitale, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+biosynthesis+and+maturation+in+the+ER.">Protein biosynthesis and maturation in the ER.</a> Methods in Molecular Biology. , 179-189 (2018).</li><li>Liang, J., Shaulov, Y., Savage-Dunn, C., Boissinot, S., Hoque, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride+intracellular+channel+proteins+respond+to+heat+stress+in+Caenorhabditis+elegans.">Chloride intracellular channel proteins respond to heat stress in Caenorhabditis elegans.</a> PLoS One. 12 (9), e0184308 (2017).</li><li>Evans, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+and+microinjection.">Transformation and microinjection.</a> Wormbook. , (2006).</li><li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transformation.">DNA transformation.</a> Methods in Cell Biology. 48, 451-482 (1995).</li><li>Yochem, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nomarski+images+for+learning+the+anatomy,+with+tips+for+mosaic+analysis.">Nomarski images for learning the anatomy, with tips for mosaic analysis.</a> Wormbook. , (2006).</li><li>Dulhunty, A., Gage, P., Curtis, S., Chelvanayagam, G., Board, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glutathione+transferase+structural+family+includes+a+nuclear+chloride+channel+and+a+ryanodine+receptor+calcium+release+channel+modulator.">The glutathione transferase structural family includes a nuclear chloride channel and a ryanodine receptor calcium release channel modulator.</a> The Journal of Biological Chemistry. 276 (5), 3319-3323 (2001).</li><li>Suh, K. S., Malik, M., Shukla, A., Yuspa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIC4,+skin+homeostasis+and+cutaneous+cancer:+surprising+connections.">CLIC4, skin homeostasis and cutaneous cancer: surprising connections.</a> Molecular Carcinogenesis. 46 (8), 599-604 (2007).</li><li>Yao, Q., Qu, X., Yang, Q., Wei, M., Kong, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIC4+mediates+TGF-beta1-induced+fibroblast-to-myofibroblast+transdifferentiation+in+ovarian+cancer.">CLIC4 mediates TGF-beta1-induced fibroblast-to-myofibroblast transdifferentiation in ovarian cancer.</a> Oncology Reports. 22 (3), 541-548 (2009).</li><li>Ronnov-Jessen, L., Villadsen, R., Edwards, J. C., Petersen, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+a+chloride+intracellular+channel+gene,+CLIC4,+in+transforming+growth+factor-beta1-mediated+conversion+of+fibroblasts+to+myofibroblasts.">Differential expression of a chloride intracellular channel gene, CLIC4, in transforming growth factor-beta1-mediated conversion of fibroblasts to myofibroblasts.</a> The American Journal of Pathology. 161 (2), 471-480 (2002).</li><li>He, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CLIC4+in+the+host+innate+responses+to+bacterial+lipopolysaccharide.">Role of CLIC4 in the host innate responses to bacterial lipopolysaccharide.</a> European Journal of Immunology. 41 (5), 1221-1230 (2011).</li><li>Zhong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+CLIC4+enhances+autophagy+and+triggers+mitochondrial+and+ER+stress-induced+apoptosis+in+human+glioma+U251+cells+under+starvation.">Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation.</a> PLoS One. 7 (6), e39378 (2012).</li><li>Liang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+schnurri+homolog+sma-9+mediates+stage-+and+cell+type-specific+responses+to+DBL-1+BMP-related+signaling.">The Caenorhabditis elegans schnurri homolog sma-9 mediates stage- and cell type-specific responses to DBL-1 BMP-related signaling.</a> Development. 130 (26), 6453-6464 (2003).</li><li>Leopold, L. E., Heestand, B. N., Seong, S., Shtessel, L., Ahmed, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+pairing+during+meiosis+triggers+multigenerational+transgene+silencing+in+Caenorhabditis+elegans.">Lack of pairing during meiosis triggers multigenerational transgene silencing in Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (20), E2667-E2676 (2015).</li><li>Schultz, R. D., Bennett, E. E., Ellis, E. A., Gumienny, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+extracellular+matrix+organization+by+BMP+signaling+in+Caenorhabditis+elegans.">Regulation of extracellular matrix organization by BMP signaling in Caenorhabditis elegans.</a> PLoS One. 9 (7), e101929 (2014).</li><li>Matus, D. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+cell+fate+requires+G1+cell-cycle+arrest+and+histone+deacetylase-mediated+changes+in+gene+expression.">Invasive cell fate requires G1 cell-cycle arrest and histone deacetylase-mediated changes in gene expression.</a> Developmental Cell. 35 (2), 162-174 (2015).</li><li>Reddy, K. C., Villeneuve, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+HIM-17+links+chromatin+modification+and+competence+for+initiation+of+meiotic+recombination.">C. elegans HIM-17 links chromatin modification and competence for initiation of meiotic recombination.</a> Cell. 118 (4), 439-452 (2004).</li><li>Ames, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-cell-autonomous+role+of+BEC-1/BECN1/Beclin1+in+coordinating+cell-cycle+progression+and+stem+cell+proliferation+during+germline+development.">A non-cell-autonomous role of BEC-1/BECN1/Beclin1 in coordinating cell-cycle progression and stem cell proliferation during germline development.</a> Current Biology. 27 (6), 905-913 (2017).</li><li>Pekar, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+the+environment,+DAF-7/TGFbeta+signaling+and+LAG-2/DSL+ligand+expression+in+the+germline+stem+cell+niche.">Linking the environment, DAF-7/TGFbeta signaling and LAG-2/DSL ligand expression in the germline stem cell niche.</a> Development. 144 (16), 2896-2906 (2017).</li><li>Schvarzstein, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+helicase+HIM-6/BLM+both+promotes+MutSgamma-dependent+crossovers+and+antagonizes+MutSgamma-independent+interhomolog+associations+during+caenorhabditis+elegans+meiosis.">DNA helicase HIM-6/BLM both promotes MutSgamma-dependent crossovers and antagonizes MutSgamma-independent interhomolog associations during caenorhabditis elegans meiosis.</a> Genetics. 198 (1), 193-207 (2014).</li><li>Flemming, A. J., Shen, Z. Z., Cunha, A., Emmons, S. W., Leroi, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+polyploidization+and+cellular+proliferation+drive+body+size+evolution+in+nematodes.">Somatic polyploidization and cellular proliferation drive body size evolution in nematodes.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (10), 5285-5290 (2000).</li><li>Nagamatsu, Y., Ohshima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+the+control+of+body+size+by+a+G-kinase+and+a+downstream+TGFbeta+signal+pathway+in+Caenorhabditis+elegans.">Mechanisms for the control of body size by a G-kinase and a downstream TGFbeta signal pathway in Caenorhabditis elegans.</a> Genes to Cells. 9 (1), 39-47 (2004).</li></ol>

저자는 선언할 수 없다.

이 기사는 핵을 세포질 로부터 단백질 subcellular 변경 사항을 확인 하는 신속 하 고 효율적인 방법을 제시. 핵 구조 DAPI 얼룩 (그림 3)에 의해 확인 하는 동안 단백질 식 GFP 융해에 의하여 표시 했다. Immunostaining C. 선 충 단백질 도전 이기 때문에, 대부분 선 충 C. 단백질 subcellular 지 방화 마커 단백질 GFP, 나사로, mCherry, 등 그들을 태그 특징으로하는18</su...

단백질 subcellular 지 방화의 변화는 열 스트레스, 기아, 산화 스트레스, apoptosis, 단백질 인 산화 등 내부 또는 외부 신호에 응답에서 일반적인 메커니즘입니다. 예를 들어 열 스트레스는 FOXO 회원 DAF 16 핵 전 좌1,2, 및3,4신호 수신 죽음 미토 콘 드리 아에 입찰 translocates 프로 apoptotic BCL-2 단백질을 유도 합니다. 다양 한 기술을 이러한 변화를 감지할 수 있습니다. 서 부 럽 고 화학적 분리 subcellular 구조 (예, 미토 콘 드리 아 또는 핵)의 조합을 잘3목표 달성할 수 있었다. 그러나, 그것은 관심사의 단백질에 대하여 특정 항 체를 요구 한다. 따라서, 잘 설립 된 항 체는 성공에 열쇠 된다. 다른 접근은 다른 subcellular 구조 또는 세포 녹색 형광 단백질 (GFP), 빨간 형광 단백질 (RFP), 노란색 형광 단백질 (YFP), mCherry, 등 다양 한 마커 라벨 한편 라벨의 단백질을 다른 표시와 관심입니다. 그런 다음 대상5,6지역화 confocal 현미경 그들을 관찰 합니다. 방사성 동위 원소는 대상 단백질 레이블 및 다음 그들의 subcellular 지 방화7감지 대안 선택 이다. 그러나,이 메서드는 적절 한 교육 및 방사성 폐기물의 처리를 해야합니다. 특정 항 체의 부족, 적절 한 마커의 부재 또는 confocal 현미경 등의 scarceness 상황 대체 접근 고려 될 필요가 있다. 단백질의 핵 전 좌를 식별 하려면 그것만 표시와 함께 대상 단백질을 분류 하 고 얼룩 핵 화학 시 약 4', 6-diamidino-2-phenylindole (DAPI)이만 일반 형광 현미경을 필요로 하기 때문에 함께 매력적입니다.
Immunolabeling C. 선 충 항 체와 달걀 껍질 또는 동물을 둘러싼 collagenous 표 피에의 낮은 침투성으로 인해 도전 이다. 한편, 선 충 C. 단백질 그들의 척추 orthologs에서 크게 분기 이기 때문에, 몇몇 상업 회사 특정 제품 C. 선 충 을 제공 합니다. 자신에 대 한 C. 선 충 항 체를 생성 하는 작은 실험실 어렵습니다. 종종 지역 사회에 있는 연구원은 단백질 지역화 또는 유전자 발현을 보여 주기 위해 태그 단백질 마커로 사용 합니다. 이 문서의 예를 들어 EXL 1::GFP를 사용 하 여 열 스트레스8에서 단백질 핵 전 좌를 추적. 동물 게놈에는 통합된 변환 exl 1::gfp 는 안정적으로 gfp 융해 유전자를 표현 하는 데 사용 됩니다. 연구 exl-1 소장, 체 벽 근육, 및 다른 subcellular 구조8에 표현 했다. 이 프로토콜에서는 벌레 애벌레 4 (L4) 단계로 동기화, 열 스트레스 실험, 및 행위 DAPI 얼룩, 에탄올 고정, 아세톤 고정 및 일반 형광 현미경 이미징 수행 하는 방법을 보여 줍니다.

<table><tbody><tr><td>DAPI</td><td>Biotium</td><td>40043</td><td>both Hoechst 33258 and Hoechst 33342 also works well</td></tr><tr><td>OP50</td><td>CGC</td><td>OP50</td><td>https://cgc.umn.edu/</td></tr><tr><td>Caenorhabditis Genetics Center (CGC)</td><td></td><td></td><td>https://cgc.umn.edu/</td></tr><tr><td>Kim Wipe</td><td>Kimberly-Clark Corporation</td><td></td><td>soft tissue</td></tr><tr><td>Zeiss&amp;nbsp; AX10</td><td>Zeiss</td><td></td><td>fluorescence microscope</td></tr><tr><td>Axiovision Rel 4.8</td><td>Zeiss</td><td></td><td>microscope software</td></tr><tr><td>AxioCam MR Rev3</td><td>Zeiss</td><td></td><td>digital camera</td></tr><tr><td>Incubator&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Micro centrifuge</td><td></td><td></td><td></td></tr><tr><td>Transparent nail polish gel</td><td></td><td></td><td></td></tr><tr><td>60 mm petri dishes</td><td></td><td></td><td></td></tr><tr><td>Glass slides</td><td></td><td></td><td></td></tr><tr><td>Glass coverslips</td><td></td><td></td><td></td></tr><tr><td>1-20 &amp;micro;L pipettor</td><td></td><td></td><td></td></tr><tr><td>20-200 &amp;micro;L pipettor</td><td></td><td></td><td></td></tr><tr><td>200-1000 &amp;micro;L pipettor</td><td></td><td></td><td></td></tr><tr><td>1-200 &amp;micro;L pipet tips</td><td></td><td></td><td></td></tr><tr><td>200-1000 &amp;micro;L pipet tips</td><td></td><td></td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>Platinum wire worm pick</td><td></td><td></td><td></td></tr></tbody></table>

detecting protein subcellular localization by green fluorescence protein tagging and 4',6-diamidino-2-phenylindole staining in caenorhabditis elegans

이 프로토콜, 녹색 형광 단백질 (GFP) 융합 단백질 및 4', 6-diamidino-2-phenylindole (DAPI) 단백질 subcellular 지 방화 변화;을 추적 하는 데는 얼룩 특히, 아래는 열 핵 전 스트레스 조건. 단백질 반응 대응에 외부 및 내부 신호. 공통 메커니즘의 subcellular 지 방화를 변경 하는. 이 문서는 항 체, 방사성 라벨 또는 confocal 현미경 요구 하지 않는 단백질 지 방화를 추적 하는 프로토콜을 설명 합니다. 이 문서에서는, GFP 포유류 CLIC4를 포함 하 여 염화 세포내 채널 단백질 (CLICs) 가족의 C. 선 충, 구성원에에서 대상 단백질 EXL-1 태그 데 사용 됩니다. (모터와 전체 유전자 시퀀스) 통합된 변환 exl 1::gfp 유전자 변형 라인 변환 및 γ 방사선에 의해 창조 되 고 안정적으로 유전자와 gfp를 표현 한다. 최근 연구는 열 스트레스, 아니라 산화 스트레스, 따라 EXL 1::GFP 핵에서 축적 했다. 핵 구조와는 DAPI GFP 신호 겹치는 신호 스트레스 EXL 1 subcellular 지 방화 변경 확인 합니다. 이 프로토콜 선물 DAPI 얼룩에 대 한 두 가지 다른 고정 방법: 고정은 에탄올과 아세톤 고정. 이 문서에 제공 된 DAPI 얼룩 프로토콜 신속 하 고 효율적 이며 GFP 신호 및 단백질 subcellular 지 방화 변경. 이 메서드는만 Nomarski, FITC 필터와 DAPI 필터 형광 현미경을 요구 한다. 그것은 작은 실험실 설정, 학부 학생 연구, 고 등 학생 연구 및 생명 공학 교실에 적합 합니다.

염화 물 채널 세포내 단백질 (클릭)는 높은 종12에 걸쳐 보존 다기능 단백질. 많은 연구가 보여줍니다 CLICs 세포, autophagy, apoptosis, 발암, 신생, 긴장과는 포유류 시스템13,,1415,16 대 식 세포 타고 난 면역 반응 조절 ,17. C. 선 충에...

Watch this Scientific Journal Video about Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans at JoVE.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

이 프로토콜 열 스트레스 단백질 핵 전 좌는 녹색 형광 단백질 (GFP) 퓨전 단백질 마커로 4', 6-diamidino-2-phenylindole (DAPI)를 사용 하 여 추적 하는 방법을 보여 줍니다 얼룩. 프로토콜을 얼룩이 DAPI 빨리 고 GFP 단백질 subcellular 지 방화 신호 유지.

녹색 형광 단백질 태그와 4', 6-Diamidino-2-phenylindole 꼬마 선 충 에 얼룩이 단백질 Subcellular 지 방화를 감지

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

detecting-protein-subcellular-localization-green-fluorescence-protein

Research

JoVE Journal

Biology

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

9.5K 조회수.  Borough of Manhattan Community College/CUNY. 이 프로토콜 열 스트레스 단백질 핵 전 좌는 녹색 형광 단백질 (GFP) 퓨전 단백질 마커로 4', 6-diamidino-2-phenylindole (DAPI)를 사용 하 여 추적 하는 방법을 보여 줍니다 얼룩. 프로토콜을 얼룩이 DAPI 빨리 고 GFP 단백질 subcellular 지 방화 신호 유지.

비디오: Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

C. 선 충 장 한 모델으로 루멘 Morphogenesis 세포를 단일 세포 수준에서 Vivo에서 편광 막 속: 항 체 얼룩이 지기, RNAi 손실의 기능 분석 및 이미징 라벨

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

연구

발생학

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

세포내 루멘 Morphogenesis 그리고 Vivo에서 편광 막 속 단일 셀에 대 한 모델로 선 충 C. Excretory 운하: GFP 융해, RNAi 상호 작용 화면 및 이미징 라벨

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

신경 세포에서 세포내 수송 분석 꼬마 선 충 의 동원 정지

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.의 생체 Neuronal 칼슘 이미징에서 elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

신경과학

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using "Freeze-Cracking"

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

항체 염색법에<em&gt; C. 엘레</em&gt; &quot;동결 크래킹 사용&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

생물학

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

RNA 형광 제자리 혼성화(FISH)를 통해 Caenorhabditis elegans 장에서 미생물 집락화 및 감염을 시각화합니다.

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

면역학 및 감염병학

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

방법은 근육의 세포 내 구획을 평가하기 위해<em&gt; C. 엘레</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

생체 내에서 처리되는 신경 칼슘의 세포 내 이미징

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

예쁜꼬마선충의 세포구조와 세포세포형태를 연구하기 위한 정량적 접근법

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally important for cell function and cell integrity of neurons. Drosophila photoreceptor cells have become a model for studying membrane protein trafficking. Besides rhodopsin, which upon illumination becomes internalized from the photoreceptor membrane and is degraded, the transient receptor potential-like (TRPL) ion channel in Drosophila exhibits a light-dependent translocation between the rhabdomeral photoreceptor membrane (where it is located in the dark) and the photoreceptor cell body (to which it is transported upon illumination). This intracellular transport of TRPL can be studied in a simple and non-invasive way by expressing eGFP-tagged TRPL in photoreceptor cells. The eGFP fluorescence can then be observed either in the deep pseudopupil or by water immersion microscopy. These methods allow detection of fluorescence in the intact eye and are therefore useful for high-throughput assays and genetic screens for Drosophila mutants defective in TRPL translocation. Here, the preparation of flies, the microscopic techniques, as well as quantification methods used to study this light-triggered translocation of TRPL are explained in detail. These methods can be applied also for trafficking studies on other Drosophila photoreceptor proteins, for example, rhodopsin. In addition, by using eGFP-tagged rhabdomeral proteins, these methods can be used to assess the degeneration of photoreceptor cells.

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Here, non-invasive methods are described for localization of photoreceptor membrane proteins and assessment of retinal degeneration in the Drosophila compound eye using eGFP fluorescence.

eGFP 태그 단백질을 사용하여 초파리 광수용체 세포에서 막 단백질 밀매를 연구하기

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally ...

녹색 형광 단백질 태그와 4', 6-Diamidino-2-phenylindole 꼬마 선 충 에 얼룩이 단백질 Subcellular 지 방화를 감지

요약

더 많은 비디오 탐색

이 비디오의 챕터