这项工作得到了埃里森/阿法尔博士后研究员在老龄化研究项目 SJK, 和格伦基金会奖和 NIH 授予 PC (1P01AG034906, 1R01GM 090311, 1R01ES 020812)。所有的作者都出现在影片中。

1. 对细胞的多肽处理 <ol> <li> 板 200万 SH-SY5Y 或 HEK293 细胞 (2 x 10 6 ) 放入10厘米的盘中并长出2天 </li> <li> (可选) 第二天, 用无血清的 Dulbecco 和 #39 清洗细胞; s 修改鹰培养基 (DMEM) 培养基一旦需要在血清自由介质中进行, 可以在一夜之间用血清自由 DMEM. </li>
	<li> 在3天, 溶解 S14G-humanin 肽在0.2 和 #181; m 过滤, 蒸馏水, 并重组为1mM 库存解决方案. 
	注: 多肽应溶于适当的溶剂, 这可以由肽的序列特征 ( 例如 , 总电荷) 来确定, 如果多肽是商用的, 可以由制造商提供。例如, S14G-humanin, SHLP2, 和 SHLP6, 和妙语溶解在水中。在适当的体积 ( 例如 , 50 和 #956; L) 中分多肽, 以避免冻结和解冻循环。一旦解冻, 不要再使用它们. </li>
	<li> 分在50毫升锥形管中5ml 含血清或无血清培养基, 并添加室温1毫米的溶液, 使其进入工作浓度 ( 如 , 1 和 #181; m, 10 和 #181; m). </li>
	<li> 从步骤1.4 中用6毫升肽溶液替换介质. 并在适当的时间内孵育细胞. 
	注意: 根据激活 ERK 的条件选择孵育时间. </li>
</ol> 2。胞和核的亚细胞分馏 <ol> <li> 用10毫升的 ice-cold pbs 清洗两次电池, 在5毫升的 ice-cold pbs 中刮掉板上的细胞, 将细胞悬浮液转化为15毫升的锥形管. </li>
	<li> 离心 500 x g 的细胞5分钟在4和 #176; C. </li>
	<li> 在200和 #181 中吸出 PBS 并 re-suspend 颗粒; ice-cold, 分馏缓冲 (10 毫米 HEPES pH = 7.6, 3 毫米氯化镁 2 , 10 毫米氯化钾, 5% (v/v) 甘油, 1% 非离子表面活性剂 (c 13 h 22 o (c 2 h 4 o) n ), 蛋白酶/磷酸酶抑制剂). 
	注意: 为了保持蛋白质的磷酸化状态, 磷酸酶抑制剂和蛋白酶抑制剂应新鲜添加, 样品应保持在冰上的时刻。应避免重复冻结和解冻样品循环。将样本分为少量 ( 例如 50 和 #956; g), 然后将其冻结在-80 和 #176; C. </li>
	<li> 在冰上孵育悬浮颗粒15分钟, 然后在 250 x g 离心5分钟, 在4和 #176; C. </li>
	<li> 收集上清液为细胞质分数, 并保持颗粒为核分数. </li>
	<li> 离心上清液去除细胞碎片和其他污染物在 1.8万 x g 为10分钟在4和 #176; C 然后转移上清成一个新的离心管。这是胞的分数. </li>
	<li> 重200和 #956 中的颗粒; L ice-cold 洗涤缓冲器 (10 毫米 HEPES pH = 7.6, 1.5 毫米氯化镁 2 , 420 毫米氯化钠, 25% (v/v) 甘油, 0.2 毫米 EDTA, 蛋白酶/磷酸酶抑制剂) 和离心机在 250 x g 为 5 min 在4和 #176; C </li>
	<li> 在100和 #956 中除去上清和重颗粒; L ice-cold, 核萃取缓冲 (20 毫米 HEPES pH = 7.6, 1.5 毫米氯化镁 2 , 420 毫米氯化钠, 25% (v/v) 甘油, 0.2 毫米 EDTA, 蛋白酶/磷酸酶抑制剂) 和几种10次 (5 s on, 十年代关闭, 30% 安培)在冰上. </li>
	<li> 1.8万 x g 离心10分钟在4和 #176; C. </li>
	<li> 将上清液转移到新的离心管。这是核子分数. </li>
	<li> 使用一个免疫分析方法量化蛋白质量 </li> </ol>
 3. 粗的线粒体分数 <ol> <li> 用10毫升的 ice-cold pbs 洗涤每 10 cm 盘中的细胞, 加入5毫升 ice-cold pbs, 并使用细胞刮板分离细胞. 
	注: 使用三10厘米的细胞, 以获得良好的产量线粒体为西方印迹. </li>
	<li> 将电池悬浮液转换为15毫升锥形管, 并将所有从三10厘米的菜肴组合成一个15毫升的锥形管. </li>
	<li> 离心 600 x g 的细胞10分钟在4和 #176; C. </li>
	<li> 吸 PBS 和 re-suspend 1 毫升的 ice-cold 线粒体隔离缓冲 (10 毫米三拖把, 1 毫米 EGTA/三, 200 毫米蔗糖, 调整 pH = 7.4). </li>
	<li> 质25冲程2毫升均质机与聚四氟乙烯涂层杵在冰上的细胞. 
	注意: 这一步是关键的保持线粒体的完整性和最大的收益率的线粒体分数。应针对每个单元格类型优化笔画数。在开始操作前冷匀浆机. </li>
	<li> 将匀浆转移到离心管, 并将其离心于 600 x g, 用于10分钟的4和 #176; C 以除去细胞核和完整的细胞. </li>
	<li> 收集上清液, 转移到一个新的离心管, 并离心 7000 x g 为10分钟, 在4和 #176; C. 
	注: 颗粒松散, 用小心收集上清, 并尽量不扰乱颗粒. </li>
	<li> 去除上清液, re-suspend 200 和 #956 的颗粒; ice-cold 线粒体隔离缓冲, 并将溶液转移到新的离心管。离心管在 7000 x g 为10分钟在4和 #176; C. </li>
	<li> 重复步骤3.8 以清洗颗粒。在这一步中, 没有必要将上清液转移到新的离心管上. </li>
	<li> 从洗涤过的颗粒中去除上清液, 并 re-suspend 50 和 #956 的含有线粒体的颗粒; 帕缓冲. </li>
	<li> 在冰上孵育悬浮液10分钟. </li>
	<li> 离心悬浮在 1.6万 x g 为15分钟在4和 #176; C. </li>
	<li> 将上清液转移到新的离心管。这是线粒体的分数. </li>
	<li> 使用定量检测方法量化蛋白质量. </li>
</ol> 4。磷特异蛋白的西部印迹 <ol> <li> 执行 SDS-聚丙烯酰胺凝胶电泳 (8-16% 预制凝胶) 并将蛋白质转移到 PVDF 膜上 4 。
	<li> 在室温下孵育 5% BSA 的 TBST (0.1% 聚 20), 以阻止背景不特定的结合点. 
	注意: 对于磷酸化蛋白检测, 用 BSA 不加牛奶阻止细胞膜。牛奶含有酪蛋白, 一个丰富的磷, 导致高非特异性信号. </li>
	<li> 在一夜之间用主抗体 (anti-phospho-ERK1/2) 在4和 #176 孵育膜. </li>
	<li> 第二天, 在室温下用 TBST (0.1% 聚 20) 冲洗膜三次, 为5分钟. </li>
	<li> 室温下, 用二次抗体 (兔 HRP) 孵育膜1小时. </li>
	<li> 在室温下用 TBST (0.1% 聚 20) 清洗膜三次, 以5分钟. </li>
	<li> 用 ECL 溶液孵育膜, 并在图像分析仪中对膜进行图像处理. </li>
	<li> 在室温下剥去15分钟的剥离缓冲膜, 用 TBST 三次清洗5分钟. </li>
	<li> 使用 anti-total ERK1/2 抗体重复步骤4.2 到 4.7. </li>
	<li> 检查每个分数的纯度, 运行 SDS 页和执行 immunoblots 使用抗体识别每个舱室的标记 ( 如 , Lamin B1 核, GAPDH 胞, TOM20 线粒体). </li>
</ol>

<ol>
	<li>Yen, K., Lee, C., Mehta, H., Cohen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+role+of+the+mitochondrial-derived+peptide+humanin+in+stress+resistance.">The emerging role of the mitochondrial-derived peptide humanin in stress resistance.</a> J. Mol. Endocrinol. 50 (1), 11-19 (2013).</li><li>Muzumdar, R. H., Huffman, D. 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=Humanin:+a+novel+central+regulator+of+peripheral+insulin+action.">Humanin: a novel central regulator of peripheral insulin action.</a> PloS one. 4 (7), 6334 (2009).</li><li>Lee, C., Yen, K., Cohen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanin:+a+harbinger+of+mitochondrial-derived+peptides.">Humanin: a harbinger of mitochondrial-derived peptides.</a> Trends Endocrinol. Metab. 24 (5), 222-228 (2013).</li><li>Kim, S. J., Guerrero, N., 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-derived+peptide+humanin+activates+the+ERK1/2,+AKT,+and+STAT3+signaling+pathways+and+has+age-dependent+signaling+differences+in+the+hippocampus.">The mitochondrial-derived peptide humanin activates the ERK1/2, AKT, and STAT3 signaling pathways and has age-dependent signaling differences in the hippocampus.</a> Oncotarget. 7 (30), 46899-46912 (2016).</li><li>Ying, G., Iribarren, P., 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=Humanin,+a+newly+identified+neuroprotective+factor,+uses+the+G+protein-coupled+formylpeptide+receptor-like-1+as+a+functional+receptor.">Humanin, a newly identified neuroprotective factor, uses the G protein-coupled formylpeptide receptor-like-1 as a functional receptor.</a> J. Immunol. 172 (11), 7078-7085 (2004).</li><li>Zhang, W., Liu, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAPK+signal+pathways+in+the+regulation+of+cell+proliferation+in+mammalian+cells.">MAPK signal pathways in the regulation of cell proliferation in mammalian cells.</a> Cell Res. 12 (1), 9-18 (2002).</li><li>Huang, C., Jacobson, K., Schaller, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAP+kinases+and+cell+migration.">MAP kinases and cell migration.</a> J. Cell Sci. 117, 4619-4628 (2004).</li><li>Cagnol, S., Chambard, J. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERK+and+cell+death:+mechanisms+of+ERK-induced+cell+death--apoptosis,+autophagy+and+senescence.">ERK and cell death: mechanisms of ERK-induced cell death--apoptosis, autophagy and senescence.</a> FEBS J. 277 (1), 2-21 (2010).</li><li>Raman, M., Chen, W., Cobb, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+and+properties+of+MAPKs.">Differential regulation and properties of MAPKs.</a> Oncogene. 26 (22), 3100-3112 (2007).</li><li>Morrison, D. K., Davis, 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=Regulation+of+MAP+kinase+signaling+modules+by+scaffold+proteins+in+mammals.">Regulation of MAP kinase signaling modules by scaffold proteins in mammals.</a> Annu. Rev. Cell Dev. 19 (1), 91-118 (2003).</li><li>Wainstein, E., Seger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+subcellular+localization+of+ERK:+mechanisms+of+translocation+and+role+in+various+organelles.">The dynamic subcellular localization of ERK: mechanisms of translocation and role in various organelles.</a> Curr. Opin. Cell Biol. 39, 15-20 (2016).</li><li>Zehorai, E., Yao, Z., Plotnikov, A., Seger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+subcellular+localization+of+MEK+and+ERK--a+novel+nuclear+translocation+signal+(NTS)+paves+a+way+to+the+nucleus.">The subcellular localization of MEK and ERK--a novel nuclear translocation signal (NTS) paves a way to the nucleus.</a> Mol. Cell. Endocrinol. 314 (2), 213-220 (2010).</li><li>Kolch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinating+ERK/MAPK+signalling+through+scaffolds+and+inhibitors.">Coordinating ERK/MAPK signalling through scaffolds and inhibitors.</a> Nat. Rev. Mol. Cell Biol. 6 (11), 827-837 (2005).</li><li>Horbinski, C., Chu, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinase+signaling+cascades+in+the+mitochondrion:+a+matter+of+life+or+death.">Kinase signaling cascades in the mitochondrion: a matter of life or death.</a> Free Radic. Biol. Med. 38 (1), 2-11 (2005).</li><li>Aebersold, D. M., Shaul, Y. 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=Extracellular+signal-regulated+kinase+1c+(ERK1c),+a+novel+42-kilodalton+ERK,+demonstrates+unique+modes+of+regulation,+localization.">Extracellular signal-regulated kinase 1c (ERK1c), a novel 42-kilodalton ERK, demonstrates unique modes of regulation, localization.</a> Mol Cell Biol. 24 (22), 10000-10015 (2004).</li><li>Zhu, J. -. H., Guo, F., Shelburne, J., Watkins, S., Chu, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+phosphorylated+ERK/MAP+kinases+to+mitochondria+and+autophagosomes+in+Lewy+body+diseases.">Localization of phosphorylated ERK/MAP kinases to mitochondria and autophagosomes in Lewy body diseases.</a> Brain Pathol. 13 (4), 473-481 (2003).</li><li>Cobb, L. J., Lee, 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=Naturally+occurring+mitochondrial-derived+peptides+are+age-dependent+regulators+of+apoptosis,+insulin+sensitivity,+and+inflammatory+markers.">Naturally occurring mitochondrial-derived peptides are age-dependent regulators of apoptosis, insulin sensitivity, and inflammatory markers.</a> Aging. 8 (4), 796-809 (2016).</li><li>Ihle, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Stat+family+in+cytokine+signaling.">The Stat family in cytokine signaling.</a> Curr. Opin. Cell Biol. 13 (2), 211-217 (2001).</li><li>Xu, Y. S., Liang, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT3+Undergoes+Acetylation-dependent+Mitochondrial+Translocation+to+Regulate+Pyruvate+Metabolism.">STAT3 Undergoes Acetylation-dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism.</a> Sci. Rep. 6 (1), 39517 (2016).</li><li>You, L., 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=The+role+of+STAT3+in+autophagy.">The role of STAT3 in autophagy.</a> Autophagy. 11 (5), 729-739 (2015).</li><li>Hashimoto, Y., Niikura, 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=Detailed+characterization+of+neuroprotection+by+a+rescue+factor+humanin+against+various+Alzheimer's+disease-relevant+insults.">Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer's disease-relevant insults.</a> The J Neurosci. 21 (23), 9235-9245 (2001).</li><li>Ying, G., Iribarren, P., 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=Humanin,+a+newly+identified+neuroprotective+factor,+uses+the+G+protein-coupled+formylpeptide+receptor-like-1+as+a+functional+receptor.">Humanin, a newly identified neuroprotective factor, uses the G protein-coupled formylpeptide receptor-like-1 as a functional receptor.</a> J. Immunol. 172 (11), 7078-7085 (2004).</li><li>Hashimoto, Y., Kurita, 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=Humanin+inhibits+apoptosis+in+pituitary+tumor+cells+through+several+signaling+pathways+including+NF-&#954;B+activation.">Humanin inhibits apoptosis in pituitary tumor cells through several signaling pathways including NF-&#954;B activation.</a> Mol. Biol. Cell. 20 (12), 2864-2873 (2009).</li></ol>

Pinchas Cohen 是 CohBar 的股东和顾问, Inc. 是 CohBar 的顾问, Inc。

在这里, 我们证明了 humanin 肽介导的 ERK1/2 活化发生在两种不同的细胞类型, 并根据条件 (例如, 肽的剂量, 时间点和细胞, 活化 ERK1/2 的亚单位的本地化可以是不同的。类型)。它已经表明, humanin 信号通过两个不同的受体22,23, 这可能解释两个细胞线之间的信号的差异, 以及对不同剂量的 humanin 的要求。这方面的生理意义还没有得到充分的研究, 但它可能建议一种可能的机制, 组织依赖反应的。
磷酸化 ERK1/2 在 ERK1/2 信号中起着至关重要的作用, 而 ERK1/2 化状态则表现出动态变化。这是很重要的, 以优化的条件, 以寻找时, ERK1/2 是磷酸化在特定条件/刺激。为了达到这个目的, 尝试不同的刺激条件 (例如, 剂量和培养基), 并进行时间课程, 以找到什么时候最高水平的磷酸化是实现。大多数的研究只集中在 ERK1/2 磷酸化的总细胞裂解。然而, 结果可能提供有限的功能信息, 因为 ERK1/2 可以与不同的目标和调节不同的下游叶栅在特定的亚细胞舱室。因此, 研究磷酸化 ERK1/2 的亚细胞定位能更好地了解 ERK1/2 活化的作用。
ERK1/2 活化后亚细胞分馏可作为进一步研究的来源 (如, co-immunoprecipitation), 以确定新的下游目标, 因为亚细胞分馏可以丰富的下游目标 ERK1/2。这种方法可以应用于其他的信号分子, 被移位到不同的亚细胞室后的刺激条件优化。
尽管在过去的几十年中, 已经提出了各种分离线粒体的方法, 但也有来自其他亚细胞隔离的部分污染。本文提出的粗线粒体分离方法也含有少量的细胞质污染物。由于污染的数量很小, 我们认为它仍然是一种有效的方法, 以检测 ERK1/2 的定位到线粒体激活时。由于肽在溶液中不一定是稳定的, 所以有时很难保持实验间的一致性。确保多肽溶液的小等分, 以避免冻融效果, 提高肽活性的一致性。

新兴研究表明, 线粒体衍生肽 (mdp) 在细胞和新陈代谢中起重要作用1,2,3。了解 mdp 存在下的信号转导通路, 可以让我们洞察 mdp 调节各种功能的机制。第一个确定的 MDP, humanin, 已证明增加细胞外信号调节激酶 1/2 (ERK1/2) 磷酸化通过其受体结合4,5。然而, ERK1/2 活化的下游效应仍 (。
ERK1/2 级联在各种细胞过程中起着重要的中介作用, 包括增殖、细胞迁移、细胞代谢、存活和凋亡6、7、8。为了协调所有这些不同的细胞过程, ERK1/2 的活性和亚型定位受到磷酸和支架蛋白的严格调控9,10。除了修饰修改, ERK1/2 的动态穿梭还调节其信号功能、活动和特异性11,12。ERK1/2 主要在胞13中进行本地化。一组锚和支架蛋白有助于保留 ERK1/2 在骨架元素, 在细胞器表面, 或在细胞质中扩散13。在刺激下, ERK1/2 磷酸化并与它的锚固蛋白分离, 允许 ERK1/2 转移到其他亚细胞室, 包括细胞核、线粒体、高尔基体和溶酶体14,15,16。
虽然 humanin 是已知的激活 ERK1/2 信号通路, ERK1/2 的激活只观察到总细胞裂解。如前所述, 由于 ERK1/2 亚细胞定位在其下游效应中起着至关重要的作用, 对亚细胞定位和磷酸化 ERK1/2 的总水平的分析是必要的, 以提供全面的理解humanin 诱导的 ERK1/2 活化和下游靶点的活化。
为了了解活化 ERK1/2 的靶细胞器, 进行了亚细胞分离, 其次为磷酸化 ERK1/2。这种方法可以很容易地实现, 因为它利用标准的实验室设备和试剂。分离的亚细胞室纯度高, 能使结果得到直截了当的解释。免疫的 ERK1/2 可能产生类似的结果。然而, 某些亚细胞隔间相对来说很难形象化, 需要特殊的固定和性方法。ERK1/2 的水平在亚细胞间不同, 这种变化可能导致假阳性和假阴性信号时, 看整个单元裂解。因此, 一个免疫使用孤立的亚细胞舱室让我们更好地了解 ERK1/2 的本地化。
该方法的通用性, 使该协议的修改, 以调查其他兴奋剂的影响, 包括其他 mdp 或其他信号分子的易位, 如 STAT3。最近发现的小 humanin 样肽 (SHLPs) 是编码从 16S rRNA 地区的 humanin 编码, 他们有相似的, 但独特的属性相比,17。例如, SHLP2 和 SHLP3 激活 ERK1/2 后8小时, 虽然 humanin 激活 ERK1/2 在5分钟内. 研究 ERK1/2 的亚细胞定位对不同肽的反应将使我们更好地了解这些肽的生物学。新的证据表明, 信号分子的亚细胞定位在其下游效应中起着至关重要的作用。例如, STAT3 传统上是已知的主要是在胞的休眠细胞, 然后它 translocates 到细胞核激活基因表达, 以响应细胞因子18。STAT3 也 translocates 线粒体和调节 TCA 循环和 ATP 生产19。关于自噬调控, 不同的亚细胞定位 STAT3 调节自噬的各种方式20。例如, 核 STAT3 转录调节自噬相关基因, 并充当自噬调制器。胞质 STAT3 组成通过与自噬信号分子相互作用抑制吞噬。线粒体 STAT3 抑制和防止 mitophagy 的氧化应激诱导自噬。因此, 这种亚细胞隔离方法对于理解其他信号分子以及 ERK1/2 的作用是至关重要的。

<table>
	<tbody>
		<tr>
			<td>p44/42 MAPK (ERK1/2)</td>
			<td>Cell signaling</td>
			<td>9102</td>
			<td>Dilution 1:1,000</td>
		</tr>
		<tr>
			<td>phospho-p44/42 MAPK (ERK1/2)(Thr202/Tyr204)</td>
			<td>Cell signaling</td>
			<td>4370</td>
			<td>Dilution 1:1,000</td>
		</tr>
		<tr>
			<td>Lamin B1</td>
			<td>Cell signaling</td>
			<td>12586</td>
			<td>Nuclear Marker, Dilution 1:1,000</td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>Cell signaling</td>
			<td>5174</td>
			<td>Cytoplasmic marker, Dilution 1:2,000</td>
		</tr>
		<tr>
			<td>Tom20</td>
			<td>Santa cruz</td>
			<td>SC-17764</td>
			<td>Mitochondria marker, Dilution 1:2,000</td>
		</tr>
		<tr>
			<td>anti-Rabbit-HRP conjugated</td>
			<td>Cell signaling</td>
			<td>7074</td>
			<td>Dilution 1:30,000</td>
		</tr>
		<tr>
			<td>RIPA Lysis and Extraction Buffer</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>89900</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Culture Dish</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>12556002</td>
			<td></td>
		</tr>
		<tr>
			<td>HNG peptide</td>
			<td>Genescript</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25mm sylinge filter</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>09-719A</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>3609</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP152-1</td>
			<td></td>
		</tr>
		<tr>
			<td>HCL</td>
			<td>Sigma</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Lonza</td>
			<td>17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper</td>
			<td>FALCON</td>
			<td>353085</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt&#8482; Protease and Phosphatase Inhibitor Cocktail (100X)</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>78440</td>
			<td></td>
		</tr>
		<tr>
			<td>Thomas Pestle Tissue Grinder Assemblies with Smooth Pestles</td>
			<td>Thomas Scientific</td>
			<td>3432S90</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>8-16% Mini-PROTEAN TGX Precast Protein Gels</td>
			<td>BIO RAD</td>
			<td>4561104</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Module</td>
			<td>BIO RAD</td>
			<td>1658030</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo Transfer System</td>
			<td>BIO RAD</td>
			<td>1704150</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo RTA Mini PVDF Transfer Kit</td>
			<td>BIO RAD</td>
			<td>1704272</td>
			<td></td>
		</tr>
		<tr>
			<td>Clarity Western ECL Blotting Substrates</td>
			<td>BIO RAD</td>
			<td>1705060</td>
			<td></td>
		</tr>
		<tr>
			<td>Restore Western blot stripping buffer</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>21059</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator, Medel: FB120</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>695320-07-12</td>
			<td></td>
		</tr>
	</tbody>
</table>

subcellular fractionation for erk activation upon mitochondrial-derived peptide treatment

线粒体衍生肽 (mdp) 是一种新的肽类, 是由小开放阅读框架编码的其他已知基因的线粒体基因组。mdp 具有多种生物效应, 如保护神经细胞不受细胞凋亡, 改善代谢标志物, 以及保护细胞免受化疗。Humanin 是第一个被发现的 MDP, 是 MDP 家族中最受研究的肽。humanin 的膜受体和下游信号通路都经过了细致的表征。更多的 mdp, 如妙语和 SHLP1-6, 最近发现和信号机制尚未阐明。在这里, 我们描述了一种基于细胞培养的方法来确定这些肽的功能。特别是, 细胞分馏技术结合与西方印迹允许定量确定的激活和易位的重要信号分子。虽然还有其他的细胞分离方法, 这里描述的是一个简单明了的方法。这些方法可用于进一步阐明这些肽和其他治疗剂的作用机理。

使用这里介绍的过程, 我们对待 HEK293 和 SH-SY5Y 细胞与1μ m 和100μ m S14G-humanin, 一个强有力的 humanin 模拟21分别, 在完全媒介为被表明的时间期间 (图 1a和图 1 B). 然后, 我们检查了 ERK1/2 在202/Tyr204从总蛋白提取物的总和磷酸化形式。S14G-humanin 治疗显示, 在其监管的202/Tyr204站点上, ERK1/2 磷酸化的增加。ERK1/2 的亚细胞定位在其信号传递功能中起着重要作用, 特别是在获得其特异性方面。为了了解 S14G-humanin-mediated ERK1/2 活化的靶向性, 我们分析了 S14G-humanin 治疗后 ERK1/2 总和磷酸化的亚细胞定位。在 HEK293 细胞中, 磷酸化 ERK 在细胞质和细胞核中均有所增加, 但在线粒体 (图 2A) 中没有。有趣的是, 磷酸化 ERK 在 SH-SY5Y 细胞的细胞质、细胞核和线粒体中下降 (图 2B)。这些结果表明, S14G-humanin-mediated ERK 磷酸化可能在不同的细胞类型和不同的条件下发挥不同的作用。因此, 需要进一步的研究, 以分析 S14G-humanin-mediated 磷酸化的 ERK1/2 在其他亚细胞室, 作为磷酸化 ERK1/2 translocates 到细胞核, 线粒体, 体/溶酶体, 和各种膜。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56496/56496fig1.jpg"> 
图 1:S14G-humanin 增加 HEK293 和 SH-SY5Y 细胞中 ERK1/2 的磷酸化.在 (A) HEK293 和 (B) SH-SY5Y 细胞中, ERK 激活的量化和有代表性的印迹。总细胞裂解以下 (A) 1 µM 或 (B) 100 µM S14G-humanin 治疗 DMEM 补充与 10% FBS 的指定时间段 immunoblotted 使用 anti-phospho-和总 ERK 1/2 (Thr202/Tyr204)。此图已从 Kim et al.中修改4 <a href="//ecsource.jove.com/files/ftp_upload/56496/56496fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56496/56496fig2.jpg"> 
图 2:磷酸化 ERK1/2 在 HEK293 和 SH-SY5Y 细胞中 S14G-humanin 治疗后, 被定位到胞、细胞核和其他亚单位.有代表性的西方印迹显示 ERK1/2 亚细胞定位 (n = 3)。(A) HEK293 细胞用1µM S14G-humanin 治疗。胞浆 (15 µg), 核 (15 µg 和50µg), 线粒体 (15 微克) 裂解 immunoblotted 使用磷和总 ERK 抗体。(B) 用100µM S14G-humanin 治疗 SY5Y 细胞。S14G-humanin 治疗后获得30分钟的线粒体分数。胞浆, 核和线粒体 (15 µg) 裂解 immunoblotted 使用磷和总 ERK 抗体。抗 GAPDH、抗 Lamin B、anti-Tom20 抗体分别用作胞浆、核、线粒体分数标记。此图已从 Kim et al.中修改4.<a href="//ecsource.jove.com/files/ftp_upload/56496/56496fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Table 1" src="/files/ftp_upload/56496/56496table1v2.jpg"> 
表 1:胞、核和线粒体分数的缓冲.

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

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

该协议描述了如何刺激细胞线粒体衍生肽, 并评估信号级联和定位的磷蛋白。

线粒体源肽治疗中 ERK 活化的亚细胞分离

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

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

Universidad San Sebastian

Research

JoVE Journal

Biology

7.8K 次观看.  University of Southern California. 该协议描述了如何刺激细胞线粒体衍生肽, 并评估信号级联和定位的磷蛋白。

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

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

制备及分馏爪蟾卵提取

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

科研

生物学

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

原位贴壁和非贴壁的哺乳动物细胞的亚细胞分馏

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

RhoC GTPase Activation Assay

RhoC GTPase has 91% homology to RhoA GTPase. Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed. However, RhoC GTPase is expressed in metastatic cancer cells at relatively low levels. Therefore, few RhoC-specific reagents have been developed. We have adapted a Rho activation assay to detect RhoC GTPase. This technique utilizes a GST-Rho binding domain fusion protein to pull out active RhoC GTPase. In addition, we can harvest total protein at the beginning of the assay to determine levels of total (GTP and GDP bound) RhoC GTPase. This allows for the determination of active versus total RhoC GTPase in the cell. Several commercial versions of this procedure have been developed however, the commercial kits are optimized for RhoA GTPase and typically do not work well for RhoC GTPase. Parts of the assay have been modified as well as development of a RhoC-specific antibody.

This protocol utilizes a pull down assay to determine the levels of active RhoC GTPase within cells.

RhoC的GTP酶激活法

RhoC GTPase has 91% homology to RhoA GTPase.  Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed.  ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

成像生物膜损伤和参与亚细胞修复过程

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

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 ...

Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes

The osteocyte, once thought to be a passive resident of the bone given the backstage function of sensing mechanical loading, is now brought to the spotlight and has been shown to have multiple major functions like actively modifying the extracellular matrix and forming an endocrine organ with the lacunocanalicular system that encloses it sending messages to distant sites. Owing to the methods that made it possible to test the osteocyte in vitro from isolating primary osteocytes to osteocyte-like cell lines, osteocytes are now experiencing a resounding interest and a surge of knowledge on structure and function. Many aspects of the osteocyte biology and interaction with other molecular components are yet to be discovered. In this protocol, we describe in detail the efficient isolation of primary osteocytes from dmp1-topaz neonatal mouse calvaria, which express the green fluorescent protein in osteocytes, through cell fractionation and subsequently acquiring cultures of primary osteocytes by FACS.

This protocol describes the dissection of neonatal dmp1-topaz mouse calvaria and isolation of osteocytes expressing the green fluorescent protein through cell digestion and fractionation, in addition to osteocyte preparation for fluorescence activated cell sorting (FACS).