This work was supported by NIH grants CA133402, CA152313 and Department of Energy Office of Science DE-SC0001271. We thank the University of California Davis Flow Cytometry Shared Resource Laboratory with funding from the NCI P30 CA0933730, and NIH NCRR C06-RR12088, S10 RR12964 and S10 RR 026825 grants and with technical assistance from Ms. Bridget McLaughlin and Mr. Jonathan Van Dyke for their help with the flow cytometry experiments.

<ol>
	<li>Weinert, T., Hartwell, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+G2+delay+by+the+rad9+gene+of+Saccharomyces+cerevisiae.">Control of G2 delay by the rad9 gene of Saccharomyces cerevisiae.</a> J Cell Sci Suppl. 12, 145-148 (1989).</li><li>Takizawa, C. G., Morgan, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+mitosis+by+changes+in+the+subcellular+location+of+cyclin-B1-Cdk1+and+Cdc25C.">Control of mitosis by changes in the subcellular location of cyclin-B1-Cdk1 and Cdc25C.</a> Curr Opin Cell Biol. 12 (6), 658-665 (2000).</li><li>Allan, L. A., Clarke, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+caspase-9+by+CDK1/cyclin+B1+protects+mitotic+cells+against+apoptosis.">Phosphorylation of caspase-9 by CDK1/cyclin B1 protects mitotic cells against apoptosis.</a> Mol Cell. 26 (2), 301-310 (2007).</li><li>Cerveny, K. L., Tamura, Y., Zhang, Z., Jensen, R. E., Sesaki, H. <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+mitochondrial+fusion+and+division.">Regulation of mitochondrial fusion and division.</a> Trends Cell Biol. 17 (11), 563-569 (2007).</li><li>Brandt, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+converting+NADH:quinone+oxidoreductase+(complex+I).">Energy converting NADH:quinone oxidoreductase (complex I).</a> Annu Rev Biochem. 75, 69-92 (2006).</li><li>Hofhaus, G., Weiss, H., Leonard, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopic+analysis+of+the+peripheral+and+membrane+parts+of+mitochondrial+NADH+dehydrogenase+(complex+I).">Electron microscopic analysis of the peripheral and membrane parts of mitochondrial NADH dehydrogenase (complex I).</a> J Mol Biol. 221 (3), 1027-1043 (1991).</li><li>Weiss, H., Friedrich, T., Hofhaus, G., Preis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+respiratory-chain+NADH+dehydrogenase+(complex+I)+of+mitochondria.">The respiratory-chain NADH dehydrogenase (complex I) of mitochondria.</a> Eur J Biochem. 197 (3), 563-576 (1991).</li><li>Janssen, R. J., Nijtmans, L. G., van den Heuvel, L. P., Smeitink, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+complex+I:+structure,+function+and+pathology.">Mitochondrial complex I: structure, function and pathology.</a> J Inherit Metab Dis. 29 (4), 499-515 (2006).</li><li>Roessler, M. 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=Direct+assignment+of+EPR+spectra+to+structurally+defined+iron-sulfur+clusters+in+complex+I+by+double+electron-electron+resonance.">Direct assignment of EPR spectra to structurally defined iron-sulfur clusters in complex I by double electron-electron resonance.</a> Proc Natl Acad Sci U S A. 107 (5), 1930-1935 (2010).</li><li>Owusu-Ansah, E., Yavari, A., Mandal, S., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+mitochondrial+retrograde+signals+control+the+G1-S+cell+cycle+checkpoint.">Distinct mitochondrial retrograde signals control the G1-S cell cycle checkpoint.</a> Nat Genet. 40 (3), 356-361 (2008).</li><li>Wang, Z., 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=Cyclin+B1/Cdk1+coordinates+mitochondrial+respiration+for+cell-cycle+G2/M+progression.">Cyclin B1/Cdk1 coordinates mitochondrial respiration for cell-cycle G2/M progression.</a> Dev Cell. 29 (2), 217-232 (2014).</li><li>Omura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria-targeting+sequence,+a+multi-role+sorting+sequence+recognized+at+all+steps+of+protein+import+into+mitochondria.">Mitochondria-targeting sequence, a multi-role sorting sequence recognized at all steps of protein import into mitochondria.</a> J Biochem. 123 (6), 1010-1016 (1998).</li><li>Konishi, T., Takeyasu, A., Natsume, T., Furusawa, Y., Hieda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+heavy+ion+tracks+by+labeling+3'-OH+termini+of+induced+DNA+strand+breaks.">Visualization of heavy ion tracks by labeling 3'-OH termini of induced DNA strand breaks.</a> J Radiat Res. 52 (4), 433-440 (2011).</li><li>Leif, R. C., Stein, J. H., Zucker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+short+history+of+the+initial+application+of+anti-5-BrdU+to+the+detection+and+measurement+of+S+phase.">A short history of the initial application of anti-5-BrdU to the detection and measurement of S phase.</a> Cytometry A. 58 (1), 45-52 (2004).</li><li>Li, K., Lee, L. A., Lu, X., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorogenic+'click'+reaction+for+labeling+and+detection+of+DNA+in+proliferating+cells.">Fluorogenic 'click' reaction for labeling and detection of DNA in proliferating cells.</a> Biotechniques. 49 (1), 525-527 (2010).</li><li>Kondo, T., 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=Application+of+sensitive+fluorescent+dyes+in+linkage+of+laser+microdissection+and+two-dimensional+gel+electrophoresis+as+a+cancer+proteomic+study+tool.">Application of sensitive fluorescent dyes in linkage of laser microdissection and two-dimensional gel electrophoresis as a cancer proteomic study tool.</a> Proteomics. 3 (9), 1758-1766 (2003).</li><li>Gundry, R. L., 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=Chapter+10,+Preparation+of+proteins+and+peptides+for+mass+spectrometry+analysis+in+a+bottom-up+proteomics+workflow.">Chapter 10, Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow.</a> Curr Protoc Mol Biol. , Unit 10.25 (2009).</li><li>Davies, D., Macey, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11,+Cell+Sorting+by+Flow+Cytometry.">Chapter 11, Cell Sorting by Flow Cytometry.</a> Flow Cytometry. , 257-276 (2007).</li><li>van den Heuvel, S., Harlow, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+cyclin-dependent+kinases+in+cell+cycle+control.">Distinct roles for cyclin-dependent kinases in cell cycle control.</a> Science. 262 (5142), 2050-2054 (1993).</li><li>Terry, N. H. A., White, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+after+bromodeoxyuridine+labeling+to+measure+S+and+G2+M+phase+durations+plus+doubling+times+in+vitro+and+in+vivo.">Flow cytometry after bromodeoxyuridine labeling to measure S and G2+M phase durations plus doubling times in vitro and in vivo.</a> Nat. Protcols. 1 (2), 859-869 (2006).</li><li>Becker, L., 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=Preprotein+translocase+of+the+outer+mitochondrial+membrane:+reconstituted+Tom40+forms+a+characteristic+TOM+pore.">Preprotein translocase of the outer mitochondrial membrane: reconstituted Tom40 forms a characteristic TOM pore.</a> J Mol Biol. 353 (5), 1011-1020 (2005).</li><li>Karniely, S., Pines, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+translation--dual+destination:+mechanisms+of+dual+protein+targeting+in+eukaryotes.">Single translation--dual destination: mechanisms of dual protein targeting in eukaryotes.</a> EMBO Rep. 6 (5), 420-425 (2005).</li><li>Candas, 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=CyclinB1/Cdk1+phosphorylates+mitochondrial+antioxidant+MnSOD+in+cell+adaptive+response+to+radiation+stress.">CyclinB1/Cdk1 phosphorylates mitochondrial antioxidant MnSOD in cell adaptive response to radiation stress.</a> J Mol Cell Biol. 5 (3), 166-175 (2013).</li><li>Nantajit, 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=Cyclin+B1/Cdk1+phosphorylation+of+mitochondrial+p53+induces+anti-apoptotic+response.">Cyclin B1/Cdk1 phosphorylation of mitochondrial p53 induces anti-apoptotic response.</a> PLoS One. 5 (8), e12341 (2010).</li><li>Cappella, P., Gasparri, F., Pulici, M., Moll, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+based+on+click+chemistry,+which+overcomes+limitations+of+cell+cycle+analysis+by+classical+determination+of+BrdU+incorporation,+allowing+multiplex+antibody+staining.">A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining.</a> Cytometry A. 73 (7), 626-636 (2008).</li><li>Niwa, H., 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=Chemical+nature+of+the+light+emitter+of+the+Aequorea+green+fluorescent+protein.">Chemical nature of the light emitter of the Aequorea green fluorescent protein.</a> Proc Natl Acad Sci U S A. 93 (24), 13617-13622 (1996).</li><li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Annu Rev Biochem. 67, 509-544 (1998).</li><li>Marouga, R., David, S., Hawkins, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+DIGE+system:+2D+fluorescence+difference+gel+analysis+technology.">The development of the DIGE system: 2D fluorescence difference gel analysis technology.</a> Anal Bioanal Chem. 382 (3), 669-678 (2005).</li><li>Timms, J. F., Cramer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difference+gel+electrophoresis.">Difference gel electrophoresis.</a> Proteomics. 8 (23-24), 4886-4897 (2008).</li><li>Crane, J., Mittar, D., Soni, D., McIntyre, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Cycle+Analysis+Using+the+BD+BrdU+FITC+Assay+on+the+BD+FACSVerse+System.">Cell Cycle Analysis Using the BD BrdU FITC Assay on the BD FACSVerse System.</a> BD Biosciences Appl Notes. , (2011).</li></ol>

The authors declare that they have no competing financial interests.

다른 세포 내 소기관으로 향하는 단백질 마찬가지로 미토콘드리아 표적 단백질은 정교한 단백질 전좌 접지기 (21, 22)의 도움 소기관에 직접 일차 또는 이차 구조 내의 표적 신호를 갖는다. 이러한 COX8 같은 독점적으로 미토콘드리아 상주 단백질에서 얻은 시퀀스 (MTS)을 대상으로 미토콘드리아는 미토콘드리아 11,23,24에 특정 단백질을 대상으로하는 유전자 서열의 N 말단에 추가 할 ?...

포유 동물에서 세포주기의 진행은 cyclins과 cyclin-dependent kinases (Cdks) 1에 의해 제어 높은 순서 이벤트에 따라 달라집니다. 그 세포질, 핵 및 centrosomal 지역화를 통해 CyclinB1 /는 Cdk1는 핵 봉투 고장 및 중심체 분리 2와 유사 분열에서 다양한 이벤트를 동기화 할 수 있습니다. CyclinB1 /는 Cdk1는 세포 사멸 (3)에 대해 유사 분열 세포를 보호하고 미토콘드리아 분열, 새로 형성된 딸 세포 4 미토콘드리아의 평등 한 분배를위한 중요한 단계를 촉진한다. 포유류 세포 증식에​​서 미토콘드리아 ATP 5 멀티 소단위 복합체로 구성되어 산화 적 인산화 (OXPHOS) 기계 (전자 전달계)를 통해 생성된다; 복잡한 I - 복잡한 V (CI-CV). 니코틴 아미드 아데닌 디 뉴클레오티드 (NADH) : 유비 퀴논 산화 환원 효소 복잡한 I (CI)는 다섯 단지 (5)의 이해 최대 규모 이상이다. 복잡한 C45 서브 유닛의 14 onsists는 촉매 코어를 형성한다. 일단 복합 한 팔이 매트릭스 내부의 막에 매립 -6,7- 다른 아암에 돌출하여 L 자형 구조를 가정 조립. CI 단위체의 돌연변이는 미토콘드리아 장애 8의 다양한 원인이다. OXPHOS에서 기능적으로 효율적 CI는 전체 미토콘드리아 호흡 9, 또한 성공적인 세포주기 진행 10뿐만 아니라 필요합니다. 건강과 질병이 막 결합 효소 복합체의 기능을 기본 메커니즘을 해결했습니다 것은 소설 진단 절차 및 고급 치료 전략의 개발을 가능하게 할 수있다. 최근 연구에서는 CyclinB1 /는 Cdk1 착체 셀 중 셀 증가 에너지 요구를 상쇄하기 위해 GAP (2) G2 / (유사 분열) M 단계에서 미토콘드리아에 전위 시키며 잠재적으로, 미토콘드리아의 에너지 생산을 향상 CI 소단위 인산화 것을 발견 사이클 11. 여기에서 우리는 쇼실험 절차와 달리 핵 / 세포질 키나제의 미토콘드리아 전위을 연구하는데 사용될 수있는 전략들은 미토콘드리아 기질뿐만 아니라 예를 들어 CyclinB1 /는 Cdk1을 사용하여 미토콘드리아 지역화 기능적 결과 wcase. 필요한 미토콘드리아 고유의 과발현이 단지의 최저의 연구를 묻는 메시지가 나타나면 CyclinB1 /는 Cdk1 단지가 미토콘드리아로으로 전위 것을 발견. 단백질의 미토콘드리아 - 특이 적 발현을 달성하기 위하여, 하나는 관심있는 단백질의 N 말단에 미토콘드리아 타겟팅 서열 (MTS)을 추가 할 수있다. 시퀀스를 대상으로 미토콘드리아는 일반적으로 12있는 미토콘드리아에 미토콘드리아 단백질의 정렬을 할 수 있습니다. 우리는 CyclinB1 또는 적색 형광을 -tagged 인간 시토크롬 C 산화 효소 서브 유닛 8A (COX8)의 전구체에서 파생 된 87베이스 미토콘드리아 대상 시퀀스를 사용하여 녹색 형광 단백질 (GFP)로 그것을 복제 한단백질 (RFP)는는 Cdk1 프레임에 플라스미드를 포함 -tagged. 이 방법은 특히 핵 풀에 영향을주지 않고 이들 단백질의 미토콘드리아 표현을 변경, 우리가 미토콘드리아에 CyclinB1과는 Cdk1를 대상으로 할 수있었습니다. 형광이 단백질에 태그를 추가함으로써, 우리는 실시간으로 자신의 위치 파악을 모니터링 할 수 있었다. 마찬가지로, 우리는 우리가 구체적으로는 Cdk1의 미토콘드리아 표현과 기능을 노크 할 수 RFP 태그가 지배적 인 부정적인는 Cdk1 포함하는 플라스미드로 MTS를 도입했습니다. 는 Cdk1 같은 듀얼 현지화를 키나제의 미토콘드리아 및 핵 기능을 구별하는 것이 필수적이다. 이러한 이중 기능성 키나제의 N 말단에 MTS 공학이 사용될 쉽고 효과적인 다양한 전략을 제공한다. 는 Cdk1은 세포주기 키나제이므로,는 Cdk1은 미토콘드리아에 국소 때 세포주기 진행을 결정하는 기본이다. 이를 위해, 우리는 새로운 운전 방식을 이용했다D는 세포의 DNA 함량을 모니터한다. 전통적인 방법은 티미 딘으로 대체하는 세포주기의 S 단계에서 새로 합성 된 DNA 내로 통합 BrdU (브로 모데 옥시 우리 딘), 티미 딘의 합성 유사체를 포함하여. 이어서 적극적으로 DNA를 복제 세포는 항 BrdU의 항체를 이용하여 검출 될 수있다. 이 방법의 하나의 단점은, 결과 (13, 14) 사이의 불일치가 발생할 수 산 또는 열처리 등 열악한 방법에 의해 BrdU의 항체에 대한 액세스를 제공하는 DNA의 변성이 필요하다는 것이다. 대안 적으로, 우리는 다른 티미 딘 유사체, 듀와 적극적 분할 셀을 모니터링하는 유사한 방법을 이용했다. 중성 세제를 처리 새로 합성 된 DNA에 EDU에 액세스 할 수있는 검출 시약을 가능으로 에듀 검출 거친 DNA 변성을 필요로하지 않습니다. 듀 방법보다 신뢰성 일관된 높은 처리량 분석 15 가능성이 입증되었다. 마지막으로, tO는 Cdk1의 미토콘드리아 기질을 결정, 우리는 고전 이차원 전기 영동의 향상된 버전 2D-DIGE라는 프로테오믹스 도구를 사용했다. 두 차원 전기 영동은 두 번째의 첫 번째 차원과 분자량에서의 등전점에 따라 단백질을 분리한다. 이러한 인산화와 같은 번역 후 변형은 단백질의 등전점과 분자량을 결정하므로, 2D 겔 상이한 샘플 내 단백질의 인산화 상태의 차이를 검출 할 수있다. 단백질의 크기 (면적 강도)는 다수의 샘플들 사이의 양적 비교를 허용하는 단백질의 발현 수준의 변화 스폿. 이 방법을 사용하여, 우리는 돌연변이 미토콘드리아 타겟팅는 Cdk1 발현 세포에 비해 야생형에서 인산화 단백질을 구별 할 수 있었다. 야생형에 보여하지만 미토콘드리아 타겟 돌연변이는 Cdk1 준비에 누락 된 특정 단백질 관광 명소 격리하고,질량 분석을 통해 확인 하였다. 전통적인 2D 젤에서 트리 페닐 메탄 염료 겔 단백질을 가시화하기 위해 사용된다. 2D-DIGE 단백질 전기 영동 이동성에 최소한의 효과 형광 단백질 라벨을 사용합니다. 다른 단백질 시료는 다른 형광 염료를 함께 혼합 한 겔 (16)상의 복수의 샘플 공동 전기 있도록, 동일한 겔에 의해 분리로 표지 될 수있다. 이 겔 기반 프로테오믹스 연구에서 중요한 문제이다 겔을 겔 변형을 최소화한다.

<table border="0" cellpadding="0" cellspacing="0" width="765">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:273px;">32P ATP&#160;</td>
			<td style="width:207px;">PerkinElmer</td>
			<td style="width:119px;">BLU002001MC&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse secondary antibody</td>
			<td>Invitrogen&#160;</td>
			<td>A-11003</td>
			<td>Alexa-546 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-rabbit secondary antibody</td>
			<td>Invitrogen&#160;</td>
			<td>A11029</td>
			<td>Alexa-488 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATP</td>
			<td>Research Organics</td>
			<td>1166A</td>
			<td>For in vitro kinase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cdk1 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cdk1 kinase buffer</td>
			<td>New England Biolabs</td>
			<td>P6020S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Click-iT EdU Alexa Fluor 488 Imaging Kit</td>
			<td>Life Technologies</td>
			<td>C10337</td>
			<td>For cell cycle analysis with EdU labeling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">COX IV antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4844S</td>
			<td>For mitochondrial immunostaining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyclin B1 antibody</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-752</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CyclinB1/Cdk1 enzyme complex</td>
			<td>New England Biolabs</td>
			<td>P6020S</td>
			<td>Avoid freeze/thaw</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CyDye DIGE Fluor Labeling Kit</td>
			<td>GE Healthcare Life Sciences</td>
			<td>25-8009-83</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DIGE Gel and DIGE Buffer Kit</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28-9480-26 AA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethylformamide&#160;</td>
			<td>Sigma Aldrich</td>
			<td>319937</td>
			<td>DMF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol</td>
			<td>Bio-Rad</td>
			<td>161-0611</td>
			<td>DTT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dNTP</td>
			<td>EMD Millipore</td>
			<td>71004</td>
			<td>For site-directed mutagenesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dpn I enzyme</td>
			<td>Stratagene</td>
			<td>200519-53</td>
			<td>For site-directed mutagenesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dry Strip cover fluid</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17-1335-01</td>
			<td>Used as mineral oil</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>J.T. Baker</td>
			<td>4040-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Acros Organics</td>
			<td>409910250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf Vacufuge Concentrator</td>
			<td>Fisher Scientific</td>
			<td>07-748-13</td>
			<td>Used as vacuum centrifuge concentrator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoromount G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>Anti-fade mounting solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fortessa Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>649908</td>
			<td>For cell cycle analysis with EdU labeling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Histone H1</td>
			<td>Calbiochem</td>
			<td>382150</td>
			<td>For in vitro kinase assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>For purifying DNA fragments from agarose gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Immobiline DryStrip Gels</td>
			<td>GE Healthcare Life Sciences</td>
			<td>18-1016-61</td>
			<td>IEF (isoelectric focusing) strips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immobilized Glutathione</td>
			<td>Thermo Scientific</td>
			<td>15160</td>
			<td>Glutathione-agarose beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iodoacetamide</td>
			<td>Sigma Aldrich</td>
			<td>I1149</td>
			<td>IAA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IPGphor 3 Isoelectric Focusing Unit</td>
			<td>GE Healthcare Life Sciences</td>
			<td>11-0033-64</td>
			<td>IPGphor strip holders</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropyl-b-D-thio-galactopyranoside&#160;</td>
			<td>RPI Corp</td>
			<td>156000-5.0</td>
			<td>IPTG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leupeptin</td>
			<td>Sigma Aldrich</td>
			<td>L9783</td>
			<td>For cell lysis buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668027</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysine</td>
			<td>Sigma Aldrich</td>
			<td>L5501</td>
			<td>For CyDye labeling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysozyme</td>
			<td>EMD Chemicals</td>
			<td>5960</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mitoctracker Red/Green</td>
			<td>Invitrogen&#160;</td>
			<td>M7512/M7514</td>
			<td>Mitochondrial fluorescent dyes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MOPS</td>
			<td>EMD Chemicals</td>
			<td>6310</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pEGFP-N1</td>
			<td>Clonetech</td>
			<td>6085-1</td>
			<td>GFP-expressing vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pfu</td>
			<td>Stratagene</td>
			<td>600-255-52</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pGEX-5X-1&#160;</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28-9545-53</td>
			<td>GST-expressing vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenylmethylsulfonyl fluoride</td>
			<td>Shelton Scientific</td>
			<td>IB01090</td>
			<td>PMSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline</td>
			<td>Life Technologies</td>
			<td>14040</td>
			<td>PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectra/Por 4 dialysis tubing</td>
			<td>Spectrum Labs</td>
			<td>132700</td>
			<td>as porous membrane tubing for dialysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pro-Q Diamond Phosphoprotein Gel Stain</td>
			<td>Life Technologies</td>
			<td>P-33300</td>
			<td>For staining phosphoproteins on 2D gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase inhibitor cocktail</td>
			<td>Calbiochem</td>
			<td>539134</td>
			<td>For cell lysis buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QuikChange site-directed mutagenesis kit</td>
			<td>Stratagene</td>
			<td>200519-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td>MiniPrep Plasmid Isolation Kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RO-3306</td>
			<td>Alexis Biochemicals</td>
			<td>270-463-M001</td>
			<td>Cdk1 inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotenone</td>
			<td>MP Biomedicals</td>
			<td>150154</td>
			<td>Complex I inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium carbonate</td>
			<td>Fisher Scientific</td>
			<td>S93359</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride</td>
			<td>EMD Chemicals</td>
			<td>SX0420-5</td>
			<td>For cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium orthovanadate</td>
			<td>MP Biomedicals</td>
			<td>159664</td>
			<td>For cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium pyrophosphate decahydrate</td>
			<td>Alfa Aesar</td>
			<td>33385</td>
			<td>For cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium &#946;-glycerophosphate</td>
			<td>Alfa Aesar</td>
			<td>L03425</td>
			<td>For cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SpectraMax M2e&#160;</td>
			<td>Molecular Devices</td>
			<td>M2E</td>
			<td>Microplate reader</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Fisher Scientific</td>
			<td>57-50-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Grinder pestle</td>
			<td>Kimble Chase</td>
			<td>885301-0007</td>
			<td>For mitochondria isolation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Grinder tube</td>
			<td>Kimble Chase</td>
			<td>885303-0007</td>
			<td>For mitochondria isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichloroacetic acid solution</td>
			<td>Sigma Aldrich</td>
			<td>T0699</td>
			<td>TCA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris</td>
			<td>MP Biomedicals</td>
			<td>103133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton-x-100</td>
			<td>Teknova</td>
			<td>T1105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin</td>
			<td>Calbiochem</td>
			<td>650211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Typhoon Imager</td>
			<td style="width:207px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">28-9558-09</td>
			<td>Laser gel scanner fro 2D-DIGE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ubiquinone</td>
			<td>Sigma Aldrich</td>
			<td>C7956</td>
			<td></td>
		</tr>
	</tbody>
</table>

experimental approaches to study mitochondrial localization and function of a nuclear cell cycle kinase, cdk1

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

CyclinB1과는 Cdk1의 하위 미토콘드리아 현지화 탄산나트륨 추출 단백질이 미토콘드리아의 내부 또는 외부 표면, 즉, 외막에 위치하는지 여부를 결정하는데 사용된다. 단백질이 미토콘드리아 안에 지역화 도시되면 서브 미토콘드리아 지역화 상기 판정 프로테아제 소화 결합 mitoplasting 통해 이루어질 수있다. CyclinB1 ?...

Watch this Scientific Journal Video about Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1 at JoVE.com

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

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

실험 접근법은 미토콘드리아 현지화 및 핵 세포주기 키나제,는 Cdk1의 기능을 연구하는

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

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11.8K 조회수.  University of California, Davis. Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest. 

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

Experimental Approaches to Tissue Engineering

조직 공학 실험 접근법

연구

생물학

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

머릿속 RNA 현지화에서 Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

zebrafish의 시각 기능을 연구하기 위해 optokinetic 응답을 사용하여

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

안달 Ratiometric하여 세포주기를 통해 키나제와 인산 가수 분해 효소 활동을 모니터링

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

포유 동물 세포의 세포주기의 위치 분석

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

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

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

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

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

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

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

3D 원자력기구의 변화를 공부하기 위해 3D 보존 된 계면 핵에 결합 Immunofluorescence와 DNA의 물고기

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

키나제 억제제에 대해 내성 돌연변이의 검사 및 검증을위한 방법

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

셀 무료 분석은 유사 분열의 끝에서 염색질 Decondensation을 공부하기

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

사용하여 셀 - 무료 분석<em&gt; Xenopus의의 laevis의</em&gt; 배아 핵 크기 규제의 메커니즘을 연구하기 위해 추출

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally ...