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>
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The authors declare that they have no competing financial interests.

往其他亚细胞蛋白质一样，线粒体有针对性的蛋白质具有自己的原发性或继发性结构中的定位信号，即引导他们以精心的蛋白质易位和折叠机21,22的帮助下，细胞器。线粒体靶向从独占线粒体驻地蛋白质如COX8获得的序列（MTS）可以被添加到任何基因序列的N末端 ​​至特异性蛋白靶向进入线粒体11,23,24。这里，其CyclinB1和CDK1基因克隆到含有载体COX8 MTS和表达时，重组CyclinB1的和CDK1被本地化为线粒体。这种方法的优点是，它使基因表达的修饰在特定亚细胞隔室，在此情况下线粒体，在不改变整体的基因表达。有了这个战略，线粒体特定核激酶的功能，CDK1被取消termined。同样，通过加入MTS来显性负CDK1，击倒的CDK1的特定线粒体功能达到了，这使线粒体CDK1的特定目标的识别，并启用的线粒体缺失CDK1功能的功能后果的分析。 CDK1的表达，而不在这两个线粒体和细胞核CD​​K1表达增强多边贸易体制的标签结果，因此，复杂的CDK1比线粒体行动的后果进一步调查。 然而，正如我们已经在通过加入MTS的标签把一些激酶进入线粒体经历了几次失败的尝试这一方法可能不适合所有基因产物。某些细胞系可以是转染相当抗性，并发现最佳的协议可能是耗时。即使当细胞是健康的，转染是成功的，荧光标记的蛋白质的工作表现出一些问题SUCH作为聚合，不正确的定位，非功能性融合体，和弱的信号。 线粒体和mitoplasts的隔离的主要限制包括从其它细胞或亚线粒体区室的低收率和可能的污染。有人建议，贴壁细胞不通过常规的化学或机械的方法有效地破裂，因此，使得难以从培养的粘附细胞获得高数量的线粒体。在这里，我们利用MCF10A细胞贴壁培养。得到约30 - 50微克线粒体蛋白质，3的起始量- 5×分别利用10 7个细胞。同质化的步骤是线粒体制剂的最终产量的另一个临界点。根据所使用的细胞系，中风和/或均化的时间的数量可以变化。为MCF10A细胞，我们观察到，通过玻璃/玻璃匀浆均质化10分钟是必需的，而小鼠胚胎fibroblaSTS（MEF）只需要3 - 5均化分钟。因为过度均化可能造成对线粒体膜的损伤，并触发线粒体成分的释放，对于每种细胞系的标准条件应根据经验来确定。使用玻璃 - 玻璃匀浆的增加相比，玻璃/特氟龙匀浆线粒体制剂的产率。细胞的起始量以及细胞的冷冻/解冻可能改变必需破开细胞笔划的数量。最后，蛋白质变性和聚集可能由于均化过程中样品的局部加热发生。它是，因此，必须预先冷却的组织研磨机和在此过程中保持在冰上的样品。 在这篇文章中提出的另一种方法是使用的EdU标记，以实时监测细胞周期。的EdU是一种改性的胸苷类似物，其荧光是一个明亮，光稳定的Alexa Fluor染料标记。的EdU是艾菲ciently掺入新合成的DNA。这个方法是利用增殖细胞与核苷类似物，溴脱氧尿苷（BrdU）标记的传统标签的一个更好的选择。活性的DNA合成过程中的BrdU掺入DNA中。的BrdU标记的DNA的定量通过比较苛刻的方法，如高温或高酸性暴露了BrdU的抗体结合的BrdU分子要求DNA变性。用于DNA变性严酷治疗可能影响样品的质量和费时。用EdU，洗涤剂透通常是足够的的EdU检测试剂来访问该DNA。而不需要使用苛刻的化学品或热进行DNA连接，所述的EdU方法是更容易使用，更准确和一致。除了提供的EdU的优势，一直存在关于使用埃杜研究扩散的担忧。的EdU表明超过72小时的治疗轻微的抗增殖活性，这可以减少到negl当脉冲的EdU保持igible水平在1小时25。 用EdU标记采用的一个变形例是在固定的时间和方法。该试剂盒建议一个15分钟的固定与所提供的定影液。然而，用70％乙醇的额外的固定是利用在该实验。有两个原因使用乙醇:1）跟踪随时间变化的细胞周期进程，进行时间点实验，其中收集细胞，每2小时。将细胞在 4℃保持在70％的乙醇，直到所有的实验时间点的已经完成。实际上，细胞可以保持在70％乙醇较长（数周至数月），如果需要的话。 2）用于实验的细胞稳定地与GFP标记CDK1和/或RFP标记CyclinB1的转染。若要从GFP / RFP荧光分开的EdU和PI信号，乙醇固定是利用变性GFP和RFP的蛋白质，因此在执行这些EDU的前解其荧光ðPI染色。变性的GFP / RFP蛋白是基本上完全无荧光，这大概是因为在色团不再从淬火26,27的保护。 以确定CDK1的线粒体靶，2D-DIGE法，这是优于传统的2D凝胶在许多方面使用。在2D-DIGE，不同的荧光染料， 例如 :中，Cy 3，第5和2中，用于标记的样品，其允许在一个凝胶上高达3个样品，减少的变异，而不需要运行重复像在标准的2D凝胶。在2D-DIGE使用的荧光染料具有相比，在100毫微克/点，因此，这需要在2D-DIGE凝胶与高点的分辨率和运行蛋白量较小三苯甲烷类染料的0.2纳克/现货灵敏度非常高出版质量凝胶扫描。自动软件被用于检测，量化和限定差异表达的蛋白质。由于高点分辨率，差分样品中蛋白质表达CAn为使用软件辅助现货量化比较准确;低至10％的差异可以通过2D-DIGE进行检测，容易使翻译后修饰的可视化。利用软件辅助在凝胶分析也可以更快地获得数据。然而，由于设备，如荧光扫描仪，需要进行图像采集，使用这种方法产生额外的费用。 2D-DIGE的其它限制包括疏水蛋白的差表示，以及与极端等电点和很大的分子量28,29蛋白质。使用替代的技术，如免疫细胞化学或免疫印迹以2D-DIGE获得的结果进一步验证须确认新发现。

在哺乳动物中，细胞周期进展取决于由细胞周期蛋白和周期蛋白依赖性激酶（的Cdks）1控制的高度有序的事件。通过其胞质，细胞核，和中心体定位，其CyclinB1 / CDK1能够在有丝分裂的不同的事件，如核膜破裂和中心体分离2同步。其CyclinB1 / CDK1保护有丝分裂细胞抗凋亡3，促进线粒体分裂，线粒体平均分配给新成立的子细胞4的关键一步。 在增殖哺乳动物细胞中，线粒体ATP经由氧化磷酸化（OXPHOS）机械（电子传递链），它是由5多亚基复合物生成;复杂的I - 复杂V（CI-CV）。烟酰胺腺嘌呤二核苷酸（NADH）:泛醌氧化还原酶或复合物I（CI）是理解的五个配合物5的最大和最小。在复杂的C的45亚基，其中14 onsists形成催化核心。一旦组装好，复杂的假定的L形结构有一个臂突出到基体和嵌入在内膜6,7-另一个臂。在CI亚基突变是多种线粒体紊乱8的原 ​​因。在OXPHOS一支高效CI不仅需要整体线粒体呼吸9，而且对成功的细胞周期进程10。 Unravelling在健康和疾病这种膜结合酶复合物的功能所依据的机制可以启用新的诊断程序和先进的治疗策略的开发。在最近的研究中，我们已经发现，中CyclinB1 / CDK1复杂易位到在（峡2），G2 /（有丝分裂）M期线粒体和磷酸CI亚基以提高线粒体的能量生产，潜在地抵消细胞中的细胞的增加的能源需求周期11。在这里，我们笑wcase实验程序，并且可以被用来研究否则核/质激酶的线粒体易位策略，其线粒体底物，以及使用其CyclinB1 / CDK1，例如他们的线粒体定位的功能的后果。 这一发现在需要的时候提示特定线粒体表达这种复杂​​的击倒的研究中CyclinB1 / CDK1复杂易位到线粒体。以实现蛋白质的特异性线粒体表达，人们可以在感兴趣的蛋白的N-末端添加一个线粒体靶向序列（MTS）。线粒体靶向序列使线粒体蛋白到他们正常居住12线粒体的排序。我们使用来自人的细胞色素C氧化酶亚基8A（COX8）的前体衍生的87碱基线粒体靶向序列，并克隆到绿色荧光蛋白（GFP）-tagged CyclinB1的或红色荧光蛋白（RFP）-tagged含质粒的帧CDK1。该方法使我们能够针对CyclinB1的和CDK1进入线粒体，具体地改变这些蛋白质的线粒体表达而不影响它们的核池。通过荧光标记这些蛋白质，我们能够监视他们的实时定位。同样，我们推出了MTS到含有RFP标记的显性负CDK1的质粒，使我们能够明确打掉CDK1的线粒体表达和功能。它具有双本地化一样CDK1激酶的线粒体和核函数来区分是必不可少的。工程MTS到这些双功能激酶的N端提供了一个很好的策略，很容易被采用和有效的。 因为CDK1是细胞周期激酶，它是基本的，以确定细胞周期进展时CDK1被本地化为线粒体。为了实现这一目标，我们已利用的新方法具d，来监测细胞中的DNA含量。传统的方法包括使用的BrdU（溴脱氧尿苷），一种合成的胸苷类似物，它在细胞周期的S期合并到新合成的DNA代替胸苷。然后正在积极复制它们的DNA的细胞可以使用抗BrdU抗体进行检测。这种方法的一个缺点是，它需要的DNA变性通过苛刻的方法，如酸或热处理，这可能导致在结果中13,14之间的不一致性，以提供所述的BrdU抗体的访问。另外，我们采用了类似的方法，以监测不同的胸苷类似物，埃杜的积极分裂的细胞。检测的EdU不需要苛刻的DNA变性，中性清洁剂处理使检测试剂来访问这些EDU在新合成的DNA。所述的EdU方法已被证明是更可靠的，一致的，并与高通量分析15的潜力。 最后，T○确定CDK1的线粒体基质中，我们使用了一种名为2D-DIGE蛋白质组学的工具，这是经典的双向电泳的高级版本。二维电泳根据在第二第一维和分子量等电点分离蛋白。由于翻译后修饰，如磷酸化影响的蛋白质的等电点和分子量，2D凝胶可检测不同样品中的蛋白质的磷酸化状态之间的差异。蛋白质的大小（面积和强度）掩护变化与蛋白质的表达水平，允许多个样品间的定量比较。使用这种方法，我们能够区分磷酸化蛋白质在野生型与突变线粒体靶向CDK1表达细胞。特定蛋白质斑点，在野生型显示，但在线粒体靶向突变CDK1制备失踪分离并通过质谱法鉴定。 在传统的2D凝胶，三苯甲烷染料被用来可视化凝胶上的蛋白质。 2D-DIGE使用荧光蛋白标签与蛋白质的电泳迁移率的影响最小。不同的蛋白质样品可以用不同的荧光染料，混合在一起，并通过相同的凝胶上分离，从而允许在单一凝胶16多个样品的共电泳进行标记。这最大限度地减少了凝胶到凝胶变体，它是在基于凝胶的蛋白质组学研究的一个关键问题。

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

中CyclinB1和CDK1的亚线粒体定位 碳酸钠提取用于确定一种蛋白质是否位于线粒体内或外表面，即外膜上。一旦蛋白被示出为在线粒体内本地化，亚线粒体定位的进一步确定可以通过用蛋白酶消化mitoplasting组合制成。来指定其CyclinB1或CDK1的副线粒体定位，mitoplasts通过稀释在低渗缓冲液线粒体与200毫渗透蔗糖浓度降低至25mM的隔离。外膜开始于蔗糖的150mM的破裂，而内膜保持完好直至在（ 图1A）的蔗糖的25mM的终浓度。在与mitoplasting组合，蛋白酶保护测定可使用tryps进行在消化之后外膜破裂暴露的蛋白质。这将导致在间空间的蛋白质消化。如果感兴趣的蛋白是从胰蛋白酶消化的保护，这表明该蛋白质的线粒体基质定位。在这个代表性的人物，线粒体基质蛋白Hsp60的，和膜间隙蛋白质Timm13被用作子线粒体定位标记。与Hsp60的但不像Timm13，CyclinB1的和CDK1类似被保护，胰酶消化，表明其线粒体基质中的定位（ 图1B）。 MTS-和GFP标记中CyclinB1和CDK1蛋白表达线粒体 MTS是在帧中的CyclinB1的或CDK1基因的N末端，其在它们的C-末端具有GFP或RFP的标签克隆。所得的重组蛋白是线粒体定位的GFP-或RFP标记赛扬clinB1或CDK1。产生并在此研究中使用的构建体的列表显示在图中。使用这些构建体，CyclinB1的和/或CDK1在线粒体的过表达来实现，由分离的线粒体级分的蛋白质印迹这里所示（ 图2）。 其CyclinB1 / CDK1的潜在目标线粒体通过2D-DIGE测定 CDK1属于丝氨酸/苏氨酸（S / T）激酶家族催化磷酸从ATP转移至脯氨酸（P）的取向S或T残基。 ，在CDK1（D146N）的146位替换天冬酰胺（N）天冬氨酸（D）残留的点突变产生显性负（DN）CDK1突变19。研究线粒体CDK1的功能，通过构建的质粒产生的线粒体靶向CDK1-DN蛋白（pERFP-N1 MTS-CDK1-dn）中含有29个氨基酸，经度与吸烟有关的RFP标记的DN-CDK1人类细胞色素C氧化的VIII亚基的G线粒体定位序列（MTS）。制备线粒体靶向ERFP蛋白pERFP-N1 MTS被用作空载体对照。在G2线粒体磷酸化蛋白/既构建体转染M细胞被与pH值4-10胶条的二维凝胶分析概要分析。用空载体转染（ 图3，上图）相比，一组线粒体磷蛋白的显然是不存在或在CDK1-DN转染（ 图3，下图）下降。检测到的点的质谱分析测定由CDK1在线粒体磷酸的蛋白的身份。 细胞周期和测定期长度的用EdU脉冲追踪分析 为了研究细胞周期磨片的进展Ñ ​​线粒体中CyclinB1 / CDK1水平提高，使用胸苷类似物的脉冲追踪标记实验，乙炔基脱氧尿苷（EDU）进行标记经历DNA合成的20个细胞的群。此方法允许细胞周期通过跟踪的EdU阳性人群时，细胞通过S和G2 / M期的进展和G1期积累捕获在一个22小时的窗口的可视化。结果表明标记的S期细胞的进展通过G2 / M期并出现在G1期尽可能快4小时在细胞中表达野生型线粒体中CyclinB1 / CDK1相比，6小时在用矢量控制或突变CyclinB1的/转染的细胞CDK1（ 图4A），这表明线粒体中CyclinB1的该增强/ CDK1加速的细胞周期进程。 <img alt="图1" src="/files/ftp_upload/53417/53417fig1.jpg" /> 图1. 线粒体细胞周期蛋白1 / CDK1本土化矩阵。（AB）中CyclinB1和CDK1的亚线粒体定位由mitoplasting和蛋白酶保护法检测，数字已经从王某等人在 2014年11修改。总（T），沉淀（P）和上清液（S）组分进行用所示抗体免疫印迹分析。 TIMM13（一个空间间蛋白）和HSP60（基质蛋白）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53417/53417fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53417/53417fig2.jpg" /> （质粒表示图2 线粒体CDK1构建体的表达。从线粒体CyclinB1的针对性和/或野生型或显性负突变体转染CDK1细胞中分离出的线粒体分数的印迹<html>在底部11。的pEGFP-N1-MTS和pERFP-N1-MTS载体是为MTS-CyclinB1的和MTS-CDK1分别）空载体对照。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53417/53417fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53417/53417fig3.jpg" /> 图3. CDK1的潜在线粒体基质。从线粒体有针对性的空载体（pERFP-N1-MTS，上图）或突变CDK1（pERFP-N1-MTS-CDK1-DN转G2 / M-见顶细胞中提取的线粒体蛋白质，下图）分别用Cy5标记（绿色），由2-D凝胶上分离和磷酸化蛋白染色用磷染料（红色）。这个数字已经从王某等人。2014年11修改。"&gt;请点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/53417/53417fig4.jpg" /> 图4. 线粒体CDK1提高G2 / M过渡和整体周期进程。 用EdU脉冲追踪标记细胞周期分析。的EdU标记的细胞的散点图直方图绘制为DNA含量（X轴）和埃杜（Y轴）。在每个面板的下图显示的EdU的平均荧光强度标记核。分别表示在小时时间点脉冲的EdU 11日之后。对于所有时间点，门显示以下种群得出:G0 / G1期，S和G2 / M期。 6，8和10小时的时间点，埃杜标记G1 *，S / G2 *，和G2 / M *种群被示出。这个数字已经从王某等人修改，2014年11 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53417/53417fig4large.jpg" target="_blank">请点击</a>此处查看该图的放大版本。 <table fo:keep-together.within-page="1"><tbody><tr><td colspan="6">使用浓度蔗糖</td></tr><tr height="43"><td>没有胰蛋白酶</td><td> 25毫米</td><td> 50毫米</td><td> 100毫米</td><td> 150毫米</td><td> 200毫米</td></tr><tr height="43"><td> +胰蛋白酶</td><td> 25毫米</td><td> 50毫米</td><td> 100毫米</td><td> 150毫米</td><td> 200毫米</td></tr></tbody></table> 表1.低渗蔗糖缓冲器用于步骤4.2 <table fo:keep-together.within-page="1"><tbody><tr><td>步骤1 </td><td> 30 V </td><td> 12小时</td><td>步骤和保持</td></tr><tr><td>第2步</td><td> 300 V </td><td> 0.5小时</td><td>步骤和保持</td></tr><tr><td height="21" style="height:21px;">第3步</td><td> 1,000V， </td><td> 0.5小时</td><td>梯度</td></tr><tr><td heigHT ="21"的风格="高度:21px;"&gt;第4步</td><td>至5,000V </td><td> 1.33小时</td><td>梯度</td></tr><tr><td height="21" style="height:21px;">第5步</td><td>至5,000V </td><td> 20000 V小时</td><td>步骤和保持</td></tr></tbody></table> 表2.等电协议用 ​​于步骤6.4.5

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.

实验方法来研究线粒体定位和核细胞周期激酶CDK1功能

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

experimental-approaches-to-study-mitochondrial-localization-function

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11.8K 次观看.  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. 

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

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

线粒体钙在分离线粒体和培养细胞中的流入分析

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

科研

生物化学

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

贝克酵母糖精中线粒体基因组表达产品的标记和可视化

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

mtDNA 合成和分布的高通量定量

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

作者聚焦:基于图像的高通量线粒体 DNA 合成和分布定量  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

基于图像的高通量线粒体DNA合成和分布定量

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

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

Methods to Assess Subcellular Compartments of Muscle in C. elegans

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

方法来评估肌肉的亚细胞中<em&gt;温度。线虫</em

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

生物学

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

用于分析神经科学中线粒体网络的 MATLAB 优化

Optimized Automated Analysis of Live Neuronal Mitochondria Homeostasis Modulation by Isoform-Specific Retinoic Acid Receptors

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in timelapse files, considerably simplifying and speeding up the process of image processing. Nonetheless, existing tools produce a large output volume, requiring individual manual attention, and basic experimental setups have an output of thousands of files, each requiring extensive and time-consuming handling. 
To address these issues, a routine optimization was developed, in both MATLAB code and live-script forms, allowing for swift file analysis and significantly reducing document reading and data processing. With a speed of 100 files/min, the optimization allows an overall rapid analysis. The optimization achieves the results output by averaging frame-specific data for individual mitochondria throughout time frames, analyzing data in a defined manner, consistent with those output from existing tools. Live confocal imaging was performed using the dye tetramethylrhodamine methyl ester, and the routine optimization was validated by treating neuronal cells with retinoic acid receptor (RAR) agonists, whose effects on neuronal mitochondria are established in the literature. The results were consistent with the literature and allowed further characterization of mitochondrial network behavior in response to isoform-specific RAR modulation. 
This new methodology allowed rapid and validated characterization of whole-neuron mitochondria network, but it also allows for differentiation between axon and cell body mitochondria, an essential feature to apply in the neuroscience field. Moreover, this protocol can be applied to experiments using fast-acting treatments, allowing the imaging of the same cells before and after treatments, transcending the field of neuroscience.

作者聚焦:研究神经元线粒体中视黄酸受体的优化自动化方法

The mitochondrial network is extremely complex, making it very challenging to analyze. A novel MATLAB tool analyzes live confocal imaged mitochondria in timelapse images but results in a large output volume requiring individual manual attention. To address this issue, a routine optimization was developed, allowing for speedy file analysis.

通过亚型特异性视黄酸受体对活神经元线粒体稳态调节进行优化的自动分析

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in ...

实验方法来研究线粒体定位和核细胞周期激酶CDK1功能

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