我们感谢博士。埃雷兹埃利亚胡，丹尼尔·梅拉梅德，Ophry松树和多伦拉帕波特建立这个协议的过程中帮助和意见。这项工作是由ISF（批准号1193至1109年）的资助。

1。线粒体提纯权衡细胞沉淀。大约0.6克是用100ml的细胞获得。 <ol><li>增长100-150毫升酵母细胞的OD 600 = 1-1.5 30 °C于一个非发酵生长介质中，例如半乳糖基生长培养基中，以富集线粒体。 </li><li>离心细胞在3000×g离心5分钟，在室温下，弃上清。 </li><li>用双蒸水，离心再次洗涤沉淀。弃上清。 </li><li>每0.5 G细胞的悬浮颗粒1毫升缓冲液的DTT。它治疗的细胞与一种还原剂，以细胞壁内打破二硫键，从而提高水解是重要的。 </li><li>于室温孵育10分钟，在30 °下缓慢摇动。与此同时，重达6毫克酶解酶每克细胞，并暂停其在的Zymolyase缓冲区。 </li><li>离心细胞在3000×g离心5分钟一吨室温，并弃去上清液。 </li><li>每0.15克细胞重悬沉淀于1毫升的Zymolyase缓冲。不要旋涡。 </li><li>测量的细胞在990微升的水将10μl外径600。 </li><li>加的Zymolyase（步骤1.5），以使细胞水解的葡萄糖聚合物的β-1，3 - 葡聚糖的联系，并产生原生质球。从该步骤中，球状体应保持在等渗溶液中，以避免溶解。 </li><li>轻轻摇动孵育细胞15分钟，在30℃（对于酶解酶的最佳活性）。为了验证细胞壁和原生质发生水解，混合10微升的细胞与990微升的水。球状体，预计以裂解由于渗透压差，并且OD 600应至少10倍小于在步骤1.9 ​​中确定的值。如果没有，继续孵育酶解酶的另一个15分钟。 </li><li>离心细胞在3000×g离心5分钟，在室温下进行。仔细存款保险计划卡上清液，作为沉淀可能是不稳定的。 </li><li>同的Zymolyase缓冲液洗涤原生质球，弃上清。 </li><li>重悬原生质球用100毫升恢复介质。转移到球状体的锥瓶中，并孵育1小时，在30 °下振荡。此恢复步骤是必要的，因为在治疗的Zymolyase翻译被逮捕和mRNA的定位被破坏（ 图1B）。 </li><li>添加0.1毫克/毫升CHX和转移球状体到预冷的50ml锥形管中。 CHX除了重要的是要冻结的mRNA与核糖体协会。因此，核糖体依赖的线粒体基因的关联保持不变。 </li><li>离心原生质球在3000×g离心5分钟，在4℃，并弃去上清液。 </li><li>用冷甘露醇缓冲液洗两次。 </li><li>重悬原生质球用4毫升冷甘露醇缓冲液，并将其转移到15毫升容量一个Dounce匀浆配备紧身杵。轻轻挣脱原生质球15招裂解液转移至13 ml管。 </li><li>离心裂解物在1,500×g离心6分钟，在4℃下以沉淀细胞核和完整的细胞。 </li><li>将上清液小心转移到新管中。抛开将1ml样品（25％）为普通肝素样品（“合计”样品）。将50毫升该样品到一个新的管中，加入4倍LSB免疫印迹分析为15毫升。沉淀RNA的“总计”样品的其余部分（见协议3，RNA沉淀）。 </li><li>离心的上清液在10,000×g离心10分钟，在4 °C至颗粒的线粒体。 </li><li>上清液（〜3ml）中转移到一个新的管，并保持它在冰上。这是在“胞质”分数。将50毫升该样品到一个新的管中，加入4倍LSB免疫印迹分析为15毫升。沉淀的RNA从“细胞质”样品的其余部分（见普罗特OCOL 3，RNA沉淀）。 </li><li>用3毫升缓冲液甘露醇和离心再以10000×g在4℃下洗涤沉淀10分钟</li><li>重悬沉淀用3毫升甘露醇缓冲。此示例包含线粒体，而被称为“线粒体”部分。将50毫升该样品到一个新的管中，加入4倍LSB免疫印迹分析为15毫升。 </li></ol> 2。 RNA提取<ol><li>加至8M的盐酸胍盐酸每个样品1卷和2体积的100％乙醇中。振荡后，至少2小时，在-20 °C。 </li><li>离心样品以10,000 xg离心20分钟，在4 °C。弃上清液。要小心，因为小球可能是不稳定的。 </li><li>用70％乙醇洗涤沉淀。 </li><li>重悬沉淀用400毫升的不含RNA酶的水和样品转移到新的Eppendorf管中。 </li><li>通过加入0.1体积的3M钠ACETAT再次沉淀RNAËpH值5.2和两册100％乙醇。振荡后，至少2小时，在-20 °C。 </li><li>离心样品以20,000 xg离心20分钟，在4℃下弃上清，洗用70％乙醇。 </li><li>空气干燥沉淀，重悬用无RNA酶的水。商店的RNA样品在-80 °C。样品可用于Northern分析19或微阵列分析18（参见方案3）。 </li></ol> 3。准备RNA的微阵列分析<ol><li>从步骤2.2 650毫升无RNase水重悬mRNA的表达。 </li><li>以除去任何残留的DNA或蛋白质，添加酸性酚等体积（650毫升）：氯仿（5:1，pH值4.7）和涡流大力。以最大速度离心2分钟。 </li><li>转移500毫升上层相（水相）的至新管。避免服用相间，因为它包含的DNA。另外要注意的服用酚，它可以抑制逆转录反应。&lt;/ LI&gt; <li> RNA样品中含有显著量肝素，这是逆转录酶的强效抑制剂。因此，对于RT-PCR或微阵列标记需要被删除。通过氯化锂沉淀去除肝素：添加氯化锂，以2M的最终浓度，并培育样品过夜，在-20 °C。 </li><li>解冻的样品在4 ℃，离心机以20,000×g离心20分钟，在4℃下</li><li>小心弃去上清液，并用80％乙醇洗涤沉淀。离心机再次按照步骤3.5中所述。 </li><li>弃去上清液，空气干燥，重悬沉淀在150毫升的不含RNA酶的水。 </li><li>以除去任何残余的LiCl，再沉淀用0.1体积的3M醋酸钠pH值为5.3和3倍体积的100％乙醇。孵育-20 °C至少2小时。 </li><li>离心机的最高速度为20分钟，在4 °C。用80％的乙醇和空气干燥仔细清洗透明颗粒。 </li><li>重悬沉淀用25ml不含RNA酶的水，并保持样品在-80 °C。 </li><li>按照步骤18,20中描述的RNA标记和杂交。 </li></ol>

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作者宣称没有竞争的财务权益。

在成像技术的进步已经产生了高解析度的工具来研究mRNA的定位。今天，人们可以在毫秒时间尺度23-26测量甚至单个mRNA分子的移动。然而，传统的生化方法，如上面所描述的，也都是有用的资讯和是优选的情况。生化隔离允许的mRNA和蛋白质的大型剧目的净化，因此是最好的全基因组的研究。在单个隔离，可以得到足够的mRNA水平，以允许数以千计的基因的表征，无论是由DNA微阵列或RNA-SEQ <su...

真核细胞被组织在具有特定功能的不同的隔间。为了实现它的功能，每个隔间中包含了一套独特的蛋白质，是其活性所必需的。通过这些蛋白质接近他们的隔间一种可能的机制是通过本地化翻译1,2。在这个过程中，蛋白质是由核糖体和mRNA的是位于那里合成了其目的地。其中本地化翻译的可能优点是增加蛋白质定位的效率，减少需要的蛋白质分子伴侣，并使特定地点的调节机制。另外，局部的mRNA和核糖体可以是机器翻译的一个僻静的水库在细胞应激，当一般翻译被抑制的案件。 线粒体在最近几年成为研究的本地化翻译中心模型。大多数线粒体蛋白质编码在细胞核中，翻译在胞质溶胶中，并导入到细胞器。各行的证据表明，许多这些蛋白质是通过一个本地翻译过程中产生的。最初，电子显微镜和生物化学分馏研究发现线粒体3-5相关核糖体。这些研究的地方，然后通过特定的mRNA，被发现要导入只在共翻译方式6,7的工作证实在体内 。与线粒体的mRNA关联的全基因组的研究显示，mRNAs的一个显著馏分被定位于线粒体附近8-10。一些这些mRNA的进一步特征在于在体内荧光的方法，如鱼或MTAG 9,11。该协会的一个直接的解释是，这些mRNA作为本地化翻译的模板。 由这些mRNA接近线粒体的机制是未知的。非编码区域（最significantly 3的&#39;非编码区）被证明是参与mRNA联想到线粒体12。这些领域有可能成为一个结合位点的RNA结合蛋白介导的运输。研究表明酵母，蛋白质的PUM系列（Puf3）的成员支持与线粒体8,13 mRNA的关联。对于Puf3，这是基于对其他家庭成员函数的一个似是而非的作用，是抑制翻译而表达的是途中 14。因此，可能的mRNA在非翻译状态被输送，由与非编码区相互作用的RNA结合蛋白。此外，庞大的身躯的工作表明，当蛋白质被合成的交通工具发生。特别是，翻译抑制剂被证明影响mRNA的结社8,13。此外，翻译等功能的AUG，线粒体定位序列（MTS），或ORF地区被证明，以协助定位8,11,15。热休克蛋白70家族的蛋白质通道aperone和蛋白质受体在线粒体外膜也显示，支持mRNA的关联，进一步暗示编码蛋 ​​白的功能是mRNA定位16重要。这是一个模型，其中核糖体蛋白新兴用作靶向的mRNA-核糖体-蛋白质复合物的线粒体17识别元件是一致的。 附近线粒体本地化翻译，研究了各种方法，包括电子显微镜（可视化核糖体）3，鱼9，绿色的RNA（检测特定mRNA的）11，和生化分馏（检测RNA和核糖体）10,18。而前者的方法检测定位在体内 ，并且可以允许传输动力学的可视化，后者允许检测在单次实验中多个不同的mRNA。此外，用于生化分离的编码或非编码的域不需要是人tered，因此其具体的作用进行评估。生化分馏已成功使用多年，对许多不同的细胞区室的隔离。它的校长和局限性已经非常成熟，并且可以很容易地修改为不同目的的现有协议。必要的仪器在许多实验室的标准，因此它通常是选择的第一个方法用于研究细胞内的定位。我们描述，这对于mRNA的分离优化，而核糖体与线粒体相关的协议。因此，该协议是最佳的学习参与线粒体附近本地化翻译的因素。

<table border="0" cellpadding="0" cellspacing="0" width="969">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yeast Extract</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BactoPeptone</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Galactose</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">Do not autoclave Galactose</td>
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		<tr height="60">
			<td height="60" style="height:60px;">Growth medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">For mitochondrial enrichment, you should use any non- fermentable carbon source, such as Galactose-based growth medium sterilized 1% Yeast Extract, 2% BactoPeptone, 2% Galactose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1 M TrisHCl pH9.4</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30 mM TrisHCl pH7.6</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol (DTT)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;10 mM DTT Buffer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">0.1 M TrisHCl pH 9.4, 10 mM Dithiothreitol (DTT). Make fresh every time</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.2 M Sorbitol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">4 mM KH2PO4</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">16 mM K2HPO4</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.2&#956;m filter</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;">Zymolyase Buffer&#160;</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">1.2 M Sorbitol, 4 mM KH2PO4 , 16 mM K2HPO4. Filter this&#160; buffer (0.2&#956;m) and keep at room temperature for future use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Zymolyase 20T&#160;</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">20,000 U/gr</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recovery medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">Galactose-based growth medium supplemented with 1 M Sorbitol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1 mg/mL Cycloheximide (CHX)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.6 M Mannitol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 mM MgAc</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 mM KCl</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5 mg/mL Heparin</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1 mM phenylmethanesulfonylfluoride (PMSF)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Mannitol Buffer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">0.6 M Mannitol, 30 mM TrisHCl pH7.6, 5 mM MgAc, 100 mM KCl. Add freshly: 0.5 mg/mL Heparin, 0.1 mg/mL CHX and 1mM (PMSF). Filter this buffer and use it ice-cold</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">8M Guanidinium-HCl</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;100% and 70% Ethanol (EtOH)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;3 M Sodium Acetate pH5.2</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;10 M LiCl stock solution</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 mM Tris HCl pH 6.8</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Glycerol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromophenolblue</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">&#160;4xLSB</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">250 mM Tris HCl pH 6.8, 8% SDS, 40% Glycerol, 20% &#946;-mercaptoethanol and 0.02% Bromophenol blue</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dounce homogenizer of 15-ml capacity equipped with tight- fitting pestle</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
	</tbody>
</table>

isolation of mrnas associated with yeast mitochondria to study mechanisms of localized translation

大多数线粒体蛋白质编码的细胞核和需要被导入到细胞器。而蛋白质在接近线粒体合成的，可能会出现导入。支持这种可能性是从最近的研究，在其中有许多mRNA的编码线粒体蛋白质被证明是定位于线粒体附近而得。连同早前与外膜核糖体协会的示威活动，这些结果表明本地化的翻译过程。这种本地化的翻译可能会提高进口效率，提供了独特的调控位点，并尽量减少异位表达的情况下。多种方法已被用于表征介导的本地化翻译的因素和因素。在这些标准是亚细胞分级差速离心。这个协议在单个程序的mRNA，核糖​​体和蛋白质的分离的优点。然后这些可以表征通过各种分子和双辉化工方法。此外，转录组学和蛋白质组学的方法可以应用到所得到的材料，从而使基因组范围的见解。酵母的利用率作为模式生物对这些研究具有速度快，成本和简单性的优点。此外，先进的基因工具和可用的缺失株便于核实候选人的因素。

该协议允许从胞浆成分含有线粒体组分的分离。以测试其成功的最好方法是从不同的隔离措施进行分析，北部和西部的分析（ 图1）的样品。三个参数的隔离质量均来自这些分析。首先，无论在样品中的RNA或蛋白是完整的 - 这些将被检测为不同的频带中进行分析。降级事件将导致额外的，更短的波段或污点的外观。其次，从每一步从输入（ 即总）的信号进行比较的信号提供了?...

Watch this Scientific Journal Video about Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation at JoVE.com

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

编码线粒体蛋白质多的mRNA与线粒体外膜有关。我们描述了亚细胞分离过程，旨在酵母线粒体与它相关的基因和核糖体的隔离。这个协议可以被应用到不同的条件下生长的细胞，以揭示mRNA定位的机制，并靠近线粒体本地化的翻译。

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

isolation-mrnas-associated-with-yeast-mitochondria-to-study

Research

JoVE Journal

Biology

12.8K 次观看.  Technion - Israel Institute of Technology. 编码线粒体蛋白质多的mRNA与线粒体外膜有关。我们描述了亚细胞分离过程，旨在酵母线粒体与它相关的基因和核糖体的隔离。这个协议可以被应用到不同的条件下生长的细胞，以揭示mRNA定位的机制，并靠近线粒体本地化的翻译。 

视频: Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

从酵母细胞线粒体的纯化

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

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.

无细胞测定中使用<em&gt;非洲爪蟾</em&gt;胚胎提取研究核的大小调节机制。

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 ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

The Use of the Patch-Clamp Technique to Study the Thermogenic Capacity of Mitochondria

Mitochondrial thermogenesis (also known as mitochondrial uncoupling) is one of the most promising targets for increasing energy expenditure to combat metabolic syndrome. Thermogenic tissues such as brown and beige fats develop highly specialized mitochondria for heat production. Mitochondria of other tissues, which primarily produce ATP, also convert up to 25% of the total mitochondrial energy production into heat and can, therefore, have a considerable impact on the physiology of the whole body. Mitochondrial thermogenesis is not only essential for maintaining the body temperature, but also prevents diet-induced obesity and reduces the production of reactive oxygen species (ROS) to protect cells from oxidative damage. Since mitochondrial thermogenesis is a key regulator of cellular metabolism, a mechanistic understanding of this fundamental process will help in the development of therapeutic strategies to combat many pathologies associated with mitochondrial dysfunction. Importantly, the precise molecular mechanisms that control acute activation of thermogenesis in mitochondria are poorly defined. This lack of information is largely due to a dearth of methods for the direct measurement of uncoupling proteins. The recent development of patch-clamp methodology applied to mitochondria enabled, for the first time, the direct study of the phenomenon at the origin of mitochondrial thermogenesis, H+ leak through the IMM, and the first biophysical characterization of mitochondrial transporters responsible for it, the uncoupling protein 1 (UCP1), specific of brown and beige fats, and the ADP/ATP transporter (AAC) for all other tissues. This unique approach will provide new insights into the mechanisms that control H+ leak and mitochondrial thermogenesis and how they can be targeted to combat metabolic syndrome. This paper describes the patch-clamp methodology applied to mitochondria to study their thermogenic capacity by directly measuring H+ currents through the IMM.

This method article details the main steps in measuring H+ leak across the inner mitochondrial membrane with the patch-clamp technique, a new approach to study the thermogenic capacity of mitochondria.

使用膜片钳技术研究线粒体的产热能力

Mitochondrial thermogenesis (also known as mitochondrial uncoupling) is one of the most promising targets for increasing energy expenditure to combat ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

应用被动头部运动在啮齿动物头部产生定义的加速度

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

从小鼠骨骼肌中分离线粒体用于呼吸测定

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

相关的mRNA分离与酵母线粒体研究本地化翻译的机制