This work was supported in part by NIH grants (1R01CA137494, R01CA132115, R01CA086072 to R.G.P.), the Kimmel Cancer Center NIH Cancer Center Core grant P30CA056036 (R.G.P.), a grant from the Breast Cancer Research Foundation, generous grants from the Dr. Ralph and Marian C. Falk Medical Research Trust (R.G.P.) and a grant from the Pennsylvania Department of Health (R.G.P.). In part this work was supported by an American Italian Cancer Foundation postdoctoral fellowship (G.D.) and Bioimaging Shared Resource of the Sidney Kimmel Cancer Center (NCI 5 P30 CA-56036).The Department specifically disclaims responsibility for an analysis, interpretations or conclusions. There are no conflicts of interest associated with this manuscript.

모두 발현 벡터 EYFP - 파킨과 pDsRed2 - 미토와 섬유 아세포 1. Electroporation에 <ol><li> 100 ㎎ / ㎖ DMEM (둘 베코 변형 이글 배지), 10 % 소 태아 혈청, 2 밀리몰 / ℓ L- 글루타민, 100 U / ㎖의 페니실린으로 보충 된 배지를 이용하여 10cm 조직 배양 플레이트에 불멸화 마우스 배아 섬유 아세포 성장 및 37 ℃에서 5 % CO 2를 함유하는 가습 분위기 스트렙토. <ol><li> 80 %의 세포 생장에서 HPO 4 KH 2.4 g을 멸균 흡인 완전한 DMEM 배지를 버리고 멸균 1X 인산 완충 용액 10 ㎖ (PBS) (NaCl을 80 g, KCl을 2.0 g, 나트륨이 14.4 g을 추가 7.4) : 2 PO 4 1 L은 H 2 O, PH 증류. </li><li> 멸균 흡입하여 PBS를 버리고 트립신 - EDTA 0.25 % 1 ㎖를 추가합니다. (- 3 분 2) 세포가 분리 될 때까지 37 ℃에서 플레이트를 인큐베이션. _, 완전한 DMEM 배지 4 ML을 추가 세포를 재현 탁하고 10을 꺼내(6) 상기 혈구 계산을위한 세포 현탁액 L. 참고 : 16 코너 사각형의 한 세트의 셀의 개수는 셀 × 104 / ㎖의 수에 해당되도록 혈구가 설계된다. </li></ol></li><li> 일렉트로 과정 24 시간 이전에 10cm의 조직 배양 접시 위에 시드 1 × 106 세포. </li><li> 멸균 흡입에 의해 완전한 DMEM 매체를 취소하고 멸균 PBS 10 ㎖를 추가합니다. 멸균 흡입하여 PBS를 버리고 트립신 - EDTA 0.25 % 1 ㎖를 추가합니다. (- 3 분 2) 세포가 분리 될 때까지 37 ℃에서 플레이트를 인큐베이션. 완전 DMEM 배지 4 ㎖에 첨가하고 15 ㎖의 튜브에 세포를 재현 탁. </li><li> 냉장 원심 분리기 (4 °에 C)를 사용하여 5 분 동안 250 XG에서 세포를 스핀 다운. 멸균 흡인에 의해 뜨는을 취소하고 멸균 PBS 1 ml의 펠렛을 재현 탁. 4 ° C에서 5 분 동안 250 XG에서 세포를 스핀 다운. </li><li> 멸균 흡입하여 상층 액을 버리고, 100 μL O를 추가전기에 대한 F 솔루션 믹스 세포 펠렛에 (82 기업 솔루션 (1)의 전기 솔루션 V + 18 μL의 μL) 부드럽게 피펫 팅하여 펠렛을 재현 탁 (자세한 내용은 사용 설명서를 참조하십시오). 세포 현탁액에서 EYFP - 파킨 (여기 / 방출 527분의 514) 및 1 μg의 pDsRed2 - 미토 (여기 / 방출 620분의 565)의 2 μg의 추가. </li><li> 일회용 파스퇴르를 사용하여 멸균 큐벳에 대한 해결책을 (모두 도구 키트와 함께 제공)로 이동하고, 미리 설정된 프로그램 NIH / 3T3 U-030 (단일 펄스 전압을 200 V, 용량 960 μF, 펄스 시간이 20 밀리 초를 사용 electroporate , 펄스 수 : 1). </li><li> 37 ℃에서 5 % CO 2를 함유하는 가습 분위기 중에서 즉시 일렉트로 후 신선한 미리 예열 완전 DMEM 배지 500 ㎕를 추가하고, 6cm 조직 배양 플레이트 라이브 영상 급의 전지를 시드 및 부화 24 시간. </li></ol> 2. 시간 경과 비디오 현미경 <ol><l난&gt; 사용하기 전에 37 ° C에서 현미경의 챔버의 온도를 설정합니다. </li><li> 이때, 실험 프로토콜을 수행 할 특정 라이브 영상 매체를 준비한다. 다음과 같이 라이브 영상 매체를 준비; 혼합 DMEM 페놀없이 10 % 소 태아 혈청, 2 밀리몰 / ℓ L- 글루타민, 100U / ml 페니실린 및 100 ㎎ / ㎖ 스트렙토 마이신으로 보충. 수욕에서 37 ° C에서 라이브 영상 매체를 미리 가온. </li><li> 멸균 흡인 일렉트릭 세포의 배지를 버리고 P1000 피펫을 사용하여, 미리 예열 라이브 영상 배지 1 ㎖을 추가하고, 37 ℃에서 30 분 동안 플레이트를 배양한다. </li><li> 37 ℃의 온도에서 안정한 이미 현미경의 챔버 내의 플레이트 잠복기, 유입 5 % CO 2 중. </li><li> 주요 움직임이나 진동을 방지 현미경의 챔버로 일렉트릭 세포와 접시를 놓습니다. </li><li> 소프트웨어 인터페이스를 사용하여, 두 fluore를 검출하기 위해서 현미경을 설정할공동 형질 벡터에 의해 코딩되는 융합 단백질에서에서 scence 신호 EYFP-파킨 및 (여진 범위 495 nm의 발광 범위 녹색 520-550 나노 미터, 510)를 pDsRed2-미토 (여진 최대 558 nm의 발광 최대 583 nm의 빨간). <ol><li> 시간 경과 비디오 현미경 소프트웨어를 엽니 다. 상단 메뉴에서 pDsRed2 - 미토에 대한 EYFP - 파킨과 로다 민에 대한 FITC를 선택합니다. 상단 메뉴에서 배율 (20 배)을 선택합니다. </li></ol></li><li> 소프트웨어 인터페이스를 사용하여, 두 공동 형질 발현 벡터를 단일 셀의 모양과 위치를 등록한다. 10 셀의 최소마다 실험 조건 (실험 그룹)에 대해 수집 될 때까지이 단계를 반복합니다. <ol><li> 메뉴 &quot;앱&quot;을 선택하고 멀티 차원 취득을 클릭합니다. 다중 차원 취득 창에서 이러한 인수의 수, 각 수집 및 기록 된 셀의 위치 사이의 시간 간격과 필요한 매개 변수를 선택합니다. 획득 &quot;을 클릭합니다&quot;인수 과정을 시작합니다. </li></ol></li><li> EYFP - 파킨과 15 분의 총 간격 이미지마다 5 분을 수집 DsRed2 - 미토 형광 신호 모두의 기저 인수를 시작합니다. </li><li> 최종 작업 농도보다 두 배 높은 FCCP의 농도로 예열 라이브 영상 매체를 준비 (0.1-10 μM, 세포 유형에 따라 다름). </li><li> 인수 과정을 중단하고 P1000 피펫을 사용하여 현미경의 챔버 내부의 플레이트에 FCCP에 미리 예열 라이브 영상 매체의 부드럽게 1ml를 추가합니다. </li><li> 이전에 기록 된 위치를 이용하여 3 시간의 총 시간 동안, 한 바와 같이, 획득 프로세스를 재시작. 를 분석하고 수집이 끝날 때 mitophagic 과정의 비디오를 만들기 위해 모든 획득 된 영상을 저장합니다. </li></ol> 3. 분석 <ol><li> 개인용 컴퓨터의 &quot;티파니&quot;형식의 모든 획득 된 영상을 수집 </li><li> 그래픽 소프트와 이미지를 엽니 다도자기와 시간 간격을 정의는 (사진 매 5 분을 취득) 미토콘드리아 막에 파킨의 채용을 보여주는 첫 번째 이미지에 FCCP와 미토콘드리아 유도 - 탈분극에서 발생했습니다. 실험 그룹의 각 셀에 대해 이러한 분석을 반복합니다. </li><li> 실험 그룹 이름의 스프레드 시트에 열의 첫 번째 셀을 레이블. 각 실험군의 경우, 10 셀의 최소의 파킨 모집을위한 시간 간격을 측정한다. 파킨 모집하기위한 계산 시간 간격의 평균을 각 실험군의 표준 편차를 정의하는 (평균 ± SD를, N = 10). <ol><li> 열 하나의 계산 된 시간 간격으로 구성합니다. 모든 열은 N = 실험군의 10 측정이 포함되어 있습니다. </li><li> 수식 작성기 메뉴에서 기능 &quot;평균&quot;을 선택합니다. 열의 데이터를 선택합니다. 수식 작성기 메뉴에서 기능 &quot;표준 편차&quot;를 선택합니다. 실리열의 데이터 캐럿. </li></ol></li><li> 통계적 방법을 사용하여, 실험군 간의 mitophagy의 동적 상당한 차이를 정의하기 위해 적절한 통계인가 실험군과 비교. (예를 들어, 두 그룹 = 스튜던트 t- 검정 세 이상의 그룹 = ANOVA 플러스 사후 시험) </li></ol>

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	<li>Youle, R. J., Narendra, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+mitophagy.">Mechanisms of mitophagy.</a> Nat Rev Mol Cell Biol. 12, 9-14 (2011).</li><li>Zhi, X., Zhong, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+cancer.">Autophagy in cancer.</a> F1000Prime Rep. 7, 18 (2015).</li><li>Ding, W. X., Yin, X. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy:+mechanisms,+pathophysiological+roles,+and+analysis.">Mitophagy: mechanisms, pathophysiological roles, and analysis.</a> Biol Chem. 393, 547-564 (2012).</li><li>Lemasters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+mitochondrial+autophagy,+or+mitophagy,+as+a+targeted+defense+against+oxidative+stress,+mitochondrial+dysfunction,+and+aging.">Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging.</a> Rejuvenation Res. 8, 3-5 (2005).</li><li>Kissova, I., Deffieu, M., Manon, S., Camougrand, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uth1p+is+involved+in+the+autophagic+degradation+of+mitochondria.">Uth1p is involved in the autophagic degradation of mitochondria.</a> J Biol Chem. 279, 39068-39074 (2004).</li><li>Kubli, D. A., Gustafsson, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+mitophagy:+the+yin+and+yang+of+cell+death+control.">Mitochondria and mitophagy: the yin and yang of cell death control.</a> Circ Res. 111, 1208-1221 (2012).</li><li>Redmann, M., Dodson, M., Boyer-Guittaut, M., Darley-Usmar, V., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+mechanisms+and+role+in+human+diseases.">Mitophagy mechanisms and role in human diseases.</a> Int J Biochem Cell Biol. 53, 127-133 (2014).</li><li>Di Sante, 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=Loss+of+sirt1+promotes+prostatic+intraepithelial+neoplasia,+reduces+mitophagy,+and+delays+park2+translocation+to+mitochondria.">Loss of sirt1 promotes prostatic intraepithelial neoplasia, reduces mitophagy, and delays park2 translocation to mitochondria.</a> Am J Pathol. 185, 266-279 (2015).</li><li>Tanaka, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkin-mediated+selective+mitochondrial+autophagy,+mitophagy:+Parkin+purges+damaged+organelles+from+the+vital+mitochondrial+network.">Parkin-mediated selective mitochondrial autophagy, mitophagy: Parkin purges damaged organelles from the vital mitochondrial network.</a> FEBS Lett. 584, 1386-1392 (2010).</li><li>Shiba-Fukushima, 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=PINK1-mediated+phosphorylation+of+the+Parkin+ubiquitin-like+domain+primes+mitochondrial+translocation+of+Parkin+and+regulates+mitophagy.">PINK1-mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocation of Parkin and regulates mitophagy.</a> Sci Rep. 2, 1002 (2012).</li><li>Trinh, J., Farrer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+genetics+of+Parkinson+disease.">Advances in the genetics of Parkinson disease.</a> Nat Rev Neurol. 9, 445-454 (2013).</li><li>Murphy, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+mitochondria+produce+reactive+oxygen+species.">How mitochondria produce reactive oxygen species.</a> Biochem J. 417, 1-13 (2009).</li><li>Heytler, P. G., Prichard, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+class+of+uncoupling+agents--carbonyl+cyanide+phenylhydrazones.">A new class of uncoupling agents--carbonyl cyanide phenylhydrazones.</a> Biochem Biophys Res Commun. 7, 272-275 (1962).</li><li>Karan, G., Yang, Z., Zhang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+wild+type+and+mutant+ELOVL4+in+cell+culture:+subcellular+localization+and+cell+viability.">Expression of wild type and mutant ELOVL4 in cell culture: subcellular localization and cell viability.</a> Mol Vis. 10, 248-253 (2004).</li><li>Jin, S. M., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINK1-+and+Parkin-mediated+mitophagy+at+a+glance.">PINK1- and Parkin-mediated mitophagy at a glance.</a> J Cell Sci. 125, 795-799 (2012).</li><li>Kerr, M. C., 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=Visualisation+of+macropinosome+maturation+by+the+recruitment+of+sorting+nexins.">Visualisation of macropinosome maturation by the recruitment of sorting nexins.</a> J Cell Sci. 119, 3967-3980 (2006).</li><li>Edin, F., 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=3-D+gel+culture+and+time-lapse+video+microscopy+of+the+human+vestibular+nerve.">3-D gel culture and time-lapse video microscopy of the human vestibular nerve.</a> Acta Otolaryngol. 134, 1211-1218 (2014).</li><li>Ramsden, A. E., Mota, L. J., Munter, S., Shorte, S. L., Holden, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SPI-2+type+III+secretion+system+restricts+motility+of+Salmonella-containing+vacuoles.">The SPI-2 type III secretion system restricts motility of Salmonella-containing vacuoles.</a> Cell Microbiol. 9, 2517-2529 (2007).</li><li>Meseguer, 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=Embryo+incubation+and+selection+in+a+time-lapse+monitoring+system+improves+pregnancy+outcome+compared+with+a+standard+incubator:+a+retrospective+cohort+study.">Embryo incubation and selection in a time-lapse monitoring system improves pregnancy outcome compared with a standard incubator: a retrospective cohort study.</a> Fertil Steril. 98, 1481-1489 (2012).</li><li>Campbell, 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=Modelling+a+risk+classification+of+aneuploidy+in+human+embryos+using+non-invasive+morphokinetics.">Modelling a risk classification of aneuploidy in human embryos using non-invasive morphokinetics.</a> Reprod Biomed Online. 26, 477-485 (2013).</li><li>Bingol, B., 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+mitochondrial+deubiquitinase+USP30+opposes+parkin-mediated+mitophagy.">The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy.</a> Nature. 510, 370-375 (2014).</li><li>Carroll, R. G., Hollville, E., Martin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkin+sensitizes+toward+apoptosis+induced+by+mitochondrial+depolarization+through+promoting+degradation+of+Mcl-1.">Parkin sensitizes toward apoptosis induced by mitochondrial depolarization through promoting degradation of Mcl-1.</a> Cell Rep. 9, 1538-1553 (2014).</li><li>Batlevi, Y., La Spada, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+autophagy+in+neural+function,+neurodegenerative+disease,+neuron+cell+death,+and+aging.">Mitochondrial autophagy in neural function, neurodegenerative disease, neuron cell death, and aging.</a> Neurobiol Dis. 43, 46-51 (2011).</li><li>Frank, 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=Mitophagy+is+triggered+by+mild+oxidative+stress+in+a+mitochondrial+fission+dependent+manner.">Mitophagy is triggered by mild oxidative stress in a mitochondrial fission dependent manner.</a> Biochim Biophys Acta. 1823, 2297-2310 (2012).</li><li>Neumann, E., Schaefer-Ridder, M., Wang, Y., Hofschneider, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+mouse+lyoma+cells+by+electroporation+in+high+electric+fields.">Gene transfer into mouse lyoma cells by electroporation in high electric fields.</a> EMBO J. 1, 841-845 (1982).</li><li>Sugar, I. P., Neumann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+model+for+electric+field-induced+membrane+pores.+Electroporation.">Stochastic model for electric field-induced membrane pores. Electroporation.</a> Biophys Chem. 19, 211-225 (1984).</li><li>Lemasters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variants+of+mitochondrial+autophagy:+Types+1+and+2+mitophagy+and+micromitophagy+(Type+3).">Variants of mitochondrial autophagy: Types 1 and 2 mitophagy and micromitophagy (Type 3).</a> Redox Biol. 2, 749-754 (2014).</li><li>Aggarwal, B. B., Quintanilha, A. T., Cammack, R., Packer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damage+to+mitochondrial+electron+transport+and+energy+coupling+by+visible+light.">Damage to mitochondrial electron transport and energy coupling by visible light.</a> Biochim Biophys Acta. 502, 367-382 (1978).</li><li>Alexandratou, E., Yova, D., Handris, P., Kletsas, D., Loukas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fibroblast+alterations+induced+by+low+power+laser+irradiation+at+the+single+cell+level+using+confocal+microscopy.">Human fibroblast alterations induced by low power laser irradiation at the single cell level using confocal microscopy.</a> Photochem Photobiol Sci. 1, 547-552 (2002).</li><li>Kim, I., Lemasters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+selectively+degrades+individual+damaged+mitochondria+after+photoirradiation.">Mitophagy selectively degrades individual damaged mitochondria after photoirradiation.</a> Antioxid Redox Signal. 14, 1919-1928 (2011).</li><li>Coutu, D. L., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+cellular+processes+by+long-term+live+imaging--historic+problems+and+current+solutions.">Probing cellular processes by long-term live imaging--historic problems and current solutions.</a> J Cell Sci. 126, 3805-3815 (2013).</li></ol>

The authors have nothing to disclose.

저속 현미경은 장시간 휴대 역학의 관​​찰 시간에 하나의 관측으로부터 생균 촬상 확장 기술로 정의 될 수있다. 그것은 관찰자 실시간 단일 형광 표지 된 단백질을 확인하고 단일 생균 내부 동적를 수행 할 수 있기 때문에이 방법은 단순 또는 공 초점 현미경 생균과 구별된다. 사실, 공 초점 현미경 쉽게 형광 항체를 이용하여 면역 표지 된 단백질을 식별하지만, 상기 셀 및 그 실제 환경에서의 ?...

Macro autophagy is an intracellular process that involves the catabolic degradation of both damaged and dysfunctional cellular components, such as organelles and proteins for the purpose of either recycling or energy production. To initiate this metabolic process, the cell engulfs the damaged cellular components into a double-membrane structure, known as an autophagosome, which fuses with a lysosome and its content is degraded and recycled 1,2. There are two major types of autophagy, the non-selective and selective. The non-selective autophagy process occurs when the cell is under nutrient deprivation conditions and needs to scavenge for both essential nutrients and energy. However, selective autophagy occurs to mediate the removal of both dysfunctional/damaged organelles and proteins that otherwise could be toxic. One of the most studied selective autophagy process is the removal of mitochondria, termed mitophagy 1,3-5.
Mitochondria are the central organelles for cell metabolism and the primary source of adenosine triphosphate (ATP) via oxidative phosphorylation through the electron transport chain, fatty acid oxidation, and tricarboxylic acid (TCA) cycle. Moreover, mitochondria regulate reactive oxygen species (ROS) production and release proteins that participate in cell death pathways 6-8.
PTEN-induced putative kinase 1 (PINK1) and Parkin RBR E3 ubiquitin ligase (Parkin) are the key proteins implicated in the mitophagy process. Parkin can protect against cell death by keeping the cell healthy through mitochondrial quality control9. Upon the loss of mitochondrial membrane potential, cytosolic Parkin is recruited to the mitochondria by PINK1. This recruitment triggers the sequential events of mitophagy 10. There is a broad range of evidence that mitophagy is a fundamental mitochondria quality control process and abnormalities in this process drive disease 7. For instance, autosomal recessive Parkinson's disease has been associated with mutations in the genes that encode for Parkin and PINK1 (PARK2 and PINK1, respectively) 11. The quality control of mitochondrial health is essential for the removal of mitochondria that contribute to the accumulation of ROS12. Excessive presence of intracellular ROS can lead to damage of both nuclear and mitochondrial DNA (DNA and mt DNA, respectively).
Herein, we show a time-lapse video microscopy approach to follow the aggregation of Parkin after the induction of Parkin-mediated mitophagy in immortalized mouse embryonic fibroblasts via in vitro administration of carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP), an uncoupling agent. FCCP disrupts ATP synthesis by short circuiting protons across the outer mitochondria membrane and hence uncoupling oxidative phosphorylation from the electron transport chain 13. Triggering the depolarization of the mitochondrial membrane leads to the disruption of mitochondria and selective Parkin-dependent removal. Therefore, transfecting the cells of interest with an expression vector encoding Parkin fused with a fluorescent marker (enhanced yellow fluorescent protein, EYFP) can be used as a fluorescent tag to follow the recruitment of Parkin during the mitophagic process. In order to visualize the mitochondria, we co-transfected pDsRed2-Mito, which encodes red fluorescent protein (DsRed2) that contains a mitochondrial targeting sequence of cytochrome c oxidase subunit VIII (Mito). pDsRed2-Mito is designed for fluorescent labeling of mitochondria14. The time required for Parkin translocation into the mitochondrial membrane can be measured and gives an indirect measure of cellular health. For example, we can say that if a cell line knocked-out for a particular gene of interest shows either a faster or slower recruitment of Parkin after the induction of mitophagy by FCCP, that gene product would be a key player in order to keep the metabolic rates of the cell at the physiological status and prevent the development of diseases. Therefore, the time-lapse video microscopy provides a very powerful tool for both basic and clinical research applications in following the dynamic of labeled proteins during their molecular processes and understanding how these processes are affected during a pathological condition.

<table><tbody><tr><td>DMEM</td><td>Corning Life Science</td><td>10-013-CV</td><td>Pre-warm at 37 &amp;#176;C before use</td></tr><tr><td>Phenol-free DMEM</td><td>Corning Life Science</td><td>17-205-CV</td><td>Pre-warm at 37 &amp;#176;C before use</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F2442</td><td>Pre-warm at 37 &amp;#176;C before use</td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030</td><td>Pre-warm at 37 &amp;#176;C before use</td></tr><tr><td>Penicillin/streptomycin</td><td>Corning Life Science</td><td>30-002-CI</td><td>Pre-warm at 37 &amp;#176;C before use</td></tr><tr><td>EYFP-PARKIN expression vector</td><td>Addgene</td><td>23955</td><td></td></tr><tr><td>pDsRed2-Mito expression vector</td><td>Clontech</td><td>632421</td><td></td></tr><tr><td>Nucleofector 2B device</td><td>LONZA</td><td>AAD-1001S</td><td></td></tr><tr><td>Nucleofector for kit R NIH/3T3</td><td>LONZA</td><td>VCA-1001</td><td></td></tr><tr><td>ZEISS AXIOVERT 200M inverted microscope</td><td>CARL ZEISS</td><td></td><td></td></tr><tr><td>Carbonyl Cyanide 4-(trifluoromethoxy)-Phenylhydrazone (FCCP)</td><td>Sigma-Aldrich</td><td>C2920</td><td></td></tr><tr><td>MetaMorph</td><td>Molecular Devices</td><td>Experimental Builder</td><td></td></tr><tr><td>ImageJ</td><td>National Institute of Health</td><td>Experimental Builder</td><td></td></tr></tbody></table>

time-lapse video microscopy for assessment of eyfp-parkin aggregation as a marker for cellular mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

여기서, 우리는 시간 경과 비디오 현미경은 단일 세포에 형광 태그 단백질의 분자 이벤트를 수행하는 데 사용할 수있는 강력한 기술이다 ​​방법을 보여줍니다. 대표 결과는 또한이 기술은 높은 품질의 이미지의 취득을 허용하는 방법을 보여줍니다. 분자 프로세스의 이미지가 획득 될 때, 우리는 다른 방법을 분석 할 수있는 기회를 가질 수있다. 여기서는 선택적 미토콘...

Watch this Scientific Journal Video about Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy at JoVE.com

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

휴대 Mitophagy을위한 마커 EYFP - 파킨 집계의 평가를위한 시간 경과 비디오 현미경

Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

time-lapse-video-microscopy-for-assessment-eyfp-parkin-aggregation-as

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JoVE Journal

Biology

7.1K 조회수.  Thomas Jefferson University, SKCC. Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

비디오: Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

곰팡이 병원체 Phagocytosis의 라이브 셀 비디오 현미경

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

연구

면역학 및 감염병학

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

효모에서 모니터링 Mitophagy을위한 형광 현미경 분석 Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

생물학

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

단일 세포 형광 현미경 검사 법에 의해 Efferocytosis의 정량화

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17.
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8 in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.

Mitochondrial fusion was measured by tracking the equilibration of photoconverted matrix-targeted GFP across the mitochondrial network over time. Thus far, only one cell could be subjected to an hour long kinetic analysis at a time. We present a method that simultaneously measures multiple cells, thereby speeding up the data collection process.

병렬 공촛점 현미경을 사용하여 여러 세포의 운동 데이터를 취득 빠르고 높은 해상도, mtPA-GFP 기반 Mitochondrial 퓨전 분석

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

생체 외에서 그리고 Vivo에서 인간 세포, C. 선 충, 쥐에서 Mitophagy의 검출

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

의학

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

ImageJ를 사용 하 여 붙일 태그가 단백질의 편견된 분석 통해 초파리 에서 Neurodegeneration의 양적 세포 생물학

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

신경과학

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

초기 자식 작용 이벤트의 셀 이미징 라이브 : Omegasomes을 넘어

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

근접 결찰 분석분석서를 활용한 인간 췌장 베타 세포에서 내인성 미토파기 복합체의 시각화

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

형광 기자 흐름 Cytometry 사용 pH-종속 mt-Keima에 의해 Mitophagy의 중요 한 측정

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and further explore whether its mechanism is related to the impact of mitophagy. This study also aimed to demonstrate the therapeutic effect of safflower acid on oxidative stress in cardiomyocytes and explore whether its mechanism is related to the effect of mitophagy. Here, an H2O2-based oxidative stress model was constructed and assessed the degree of oxidative stress injury of cardiomyocytes by detecting the levels of lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH Px). Reactive oxygen species (ROS)-detecting fluorescent dye DCFH-DA, JC-1 dye, and TUNEL dye were employed to assess mitochondrial damage and apoptosis. Autophagic flux was measured by transfecting Ad-mCherry-GFP-LC3B adenovirus. Mitophagy-related proteins were then detected via western blotting and immunofluorescence. However, crocetin (0.1-10 &#181;M) could significantly improve cell viability and reduce apoptosis and oxidative stress damage caused by H2O2. In cells with excessive autophagic activation, crocetin could also reduce autophagy flow and the expression of mitophagy-related proteins PINK1 and Parkin, and reverse the transfer of Parkin to mitochondria. Crocetin could reduce H2O2-mediated oxidative stress damage and the apoptosis of H9c2 cells, and its mechanism was closely related to mitophagy.