Name: yeast luminometric and xenopus oocyte electrophysiological examinations of the molecular mechanosensitivity of trpv4
Uploaded: 2013-12-31T13:15:00
Description: Watch this Scientific Journal Video about Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4 at JoVE.com

我们要感谢安德烈Kremsreiter优秀的技术援助。麦迪逊 - 我们实验室的工作是由美国国立卫生研究院GM096088和威斯康星大学的拉斯信托基金的支持。

1。酵母发光法方法使用酿酒酵母的菌株BYYT。这是BY4741（ 马塔，ura3D0，his3D1，leu2D0，lys2D0，yvc1 :: HIS3，tok1 :: kanMX4）的yvc1-tok1衍生 。转化细胞与列伊可选水母发光蛋白表达质粒pEVP1/Aeq及市建局可选鼠TRPV4表达质粒p416GPDV4，如Batiza 等 26 Loukin 等 24所描述的质粒建设和改造27使用标准方法。酵母菌株和质粒可根...

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正如引言所述，通常用于研究TRPV4功能的方法有时会导致不一致和争议。两套此处描述的方法提供了一些优点，它可以补充现有的方法。虽然我们只描述上TRPV4的研究中，该方法可以被扩展以研究其他离子通道为好。 1。酵母发光法方法出芽酵母具有天然的Ca 2 +内流响应是受影响的脂质组合物30的突变敏低渗休克。这个信号可以被擦除与外部...

跨国激进党（瞬时受体电位）包括服务于感觉功能1,2阳离子通道七个亚科。在哺乳动物中，TRPV，跨国激进党的香草亚科，有六个品种。 TRPV4（类型4）3,4响应于热，某些化学品，渗透膨胀，或剪切应力。该TRPV4基因反复分离候选基因和/或表达克隆5-8。后一种方法遵循了基因产物的反应，低渗透压。 TRPV4表达于几乎所有的器官和功能的开发，生理学，或许多不同的细胞类型3,4的病理。 引人注目的是&gt; 50人常染色体显性TRPV4基因突变，引起周围神经病变和/或骨骼发育不良（在骨骼发育异常）9-11。骨骼发育不良的范围从轻微brachyomia 3型（3型侏儒症），spondylometaphyseal发育不良科兹洛夫斯基类型，以塞弗重新发育不良，有的引起新生儿或胚胎死亡。虽然机制的所有举止似乎不可能，没有解释的多样性，复杂性，多变性，而这些疾病4偶尔重叠。 像其他TRP通道1，TRPV4是一个四聚体。在大鼠或人TRPV4，每个子单元包括871个残基。其核心内容是六跨膜螺旋一（S1〜S6），这是有可能安排在类似电压门控K +通道的方式。还有，S1至S4形成外围域和S5和S6形成渗透毛孔领域。之间S5和TRPV4的S6是一个短孔螺旋后跟序列LDLFKLTIGMGDL，其中四个想必会聚以形成阳离子的过滤器。 470个残基的N-末端胞质段包含6个锚蛋白重复序列​​，已知结合的蛋白质或小分子配体。的C-末端152个残基的胞质片段包括其中结合OT其它可能位点的钙调蛋白结合序列她元素3。 TRPV4是，基本上不包括阴离子1阳离子通道。而其生理功能是转导的刺激与Ca 2 +内流，这也是可渗透的其它阳离子与埃森曼IV渗透性序列，在p利于钙的二价：P 娜 〜7 12。单通道电导在整流〜90 PS向外〜40 PS向内6,13,14。异源表达的电流（下图）可以通过低渗膨胀，剪切应力，或温暖15被激活。它也是由多不饱和脂肪酸16,17和合成phorbal酯4αPDD18激活。目前，最有效的激动剂是GSK1016790A 19和拮抗剂是GSK2193874，显效10 -9〜10 -8 M 20，这两个发现通过高通量的，小分子画面。 TRPV4研究的两个关键领域仍扑朔迷离：（1）即使TRPV4主要是研究其mechanosensitivity，其分子基础是有争议的。一个模型描述了低渗透压不知何故激活磷脂酶A2（PLA2），以产生不饱和脂肪酸（PUFA）花生四烯酸（AA），这是转换由一个环氧化物酶，以环氧二十碳三烯酸（EET）和EET的结合激活TRPV4 16 17。然而，TRPV4本身已被证明能直接响应膜拉伸14（下图），提供了一个简单的解释。 （2）TRPV4突变病理学是扑朔迷离。在该基础上，人们需要知道的疾病是否是由于损失，减少，或信道活动的增加。即使在这里，文学是很不明朗。虽然多个骨骼发育异常等位基因据报道，有较高的活动，（功能获得的，GOF）4,21，数据报道，有活动减少（功能丧失的，LOF）10,22。 14个等位基因的系统研究发现它们全部GOF突变（下）23。有些是外场所的说法似乎矛盾的TRPV4-/表型-基因敲除小鼠，这是可行的或肥沃，只有轻微的缺陷，尽管TRPV4功能完全丧失。 这些争议的部分有方法的起源。实验室使用不同的方法，或一种方法的变体，并使用不同的判断标准。最常见的是，TRPV4短暂表达在培养的哺乳动物细胞（HEK，CHO，HeLa细胞）和内部的[Ca 2 +]的上升时的激动剂或低渗性刺激被注册到的Ca 2 +敏感的荧光染料Fura-2的。在此荧光测定法的过度依赖已被诟病1。在不同人群中的表达水平，分布在其中，以及在有效的染料浓度，需要加以控制和记录。更可靠的是直接的电生理检查。即使是这样，由于commonlŸ练，也并非没有问题。因为在单个培养的细胞中的表达水平是难以控制的，全细胞电流有大的变化。此外，由于电流很小，可靠的统计数据将不得不依赖于大样本的大小，往往不实用。膜片钳考试却很少被执行。一些这样的唱片展团活动爆发，使统计评价具有挑战性16,17。 为了更好地理解分子mechanosensitivity，我们已经开发了两个额外的系统来检查TRPV4。 （1）要隔离TRPV4远离其一贯的哺乳动物的合作伙伴，我们在芽殖酵母24表示大鼠TRPV4。 TRPV4在这遥远的进化方面的功能表现表明，它仍然可以应对渗透力不其一贯的合作伙伴。因为酵母是没有多不饱和脂肪酸，如AA或EET，其基因组有没有磷脂酶A2或表氧化酶基因，这种EXPRE裂变也表明，对于TRPV4感知力它们不是必需的。有TRPV4在分子生物学上最适合真核生物还允许高效的正向或反向遗传操作25。 （2）为深入生物物理TRPV4的分析，我们在非洲爪蟾卵母细胞表达的TRPV4。不同的培养细胞，其产生子nA到NA（10 -10〜10 -9 A）的电流，卵母细胞表达在μA的（10 -6〜10 -4 A）的电流。在噪音大得多信号可以更好地量化和更自信的比较。 TRPV4，所以表达，也可以检测作为使用膜片钳单个分子。单卵母细胞可重复修补采样，再次使定量更可靠。这些研究表明，TRPV4通道本身可直接通过膜拉伸力14被激活。分析还表明，14代表骨骼发育不良等位基因是功能获得的所有突变。另外，DEGR此构的Ca 2 +渗漏的ee值平行的骨骼疾病23的严重性。 因为他们的新颖性和实用性，这两种方法的详细程序在这里集会，允许重复在未来的TRPV4或类似途径的研究。

<table border="0" cellpadding="0" cellspacing="0" width="595">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vapor Pressure Osmometer</td>
			<td style="width:93px;">Wescor</td>
			<td style="width:119px;">Wescor 5500</td>
			<td style="width:167px;">Logan, UT, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sirius Single Tube Luminometer </td>
			<td style="width:93px;">Titertek-Berhold</td>
			<td style="width:119px;">Sirius </td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microplate luminometer</td>
			<td style="width:93px;">Titertek-Berthold </td>
			<td style="width:119px;">Mithras LB940</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Luminometry software</td>
			<td style="width:93px;">Titertek-Berthold</td>
			<td style="width:119px;">MikroWin2000</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:216px;">Patch clamp and software</td>
			<td style="width:93px;">Axon Instrument</td>
			<td style="width:119px;">HS2A headstage, VG-2Ax100 virtual grounded bath clamp, GeneClamp 500 amplifier, digidata 1440 digitizer, pClamp10 software </td>
			<td>Union City, CA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">manometer</td>
			<td style="width:93px;">Bio-Tec</td>
			<td style="width:119px;"> </td>
			<td>Winooski, VT, USA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pipet puller</td>
			<td style="width:93px;">Sutter Instrument Co</td>
			<td style="width:119px;">Model P-97</td>
			<td>Novata, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microinjector</td>
			<td style="width:93px;">Drummond Scientific</td>
			<td style="width:119px;">Nanoinject II</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Material:</td>
			<td style="width:93px;"> </td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">luciferin coelenterazine</td>
			<td style="width:93px;">Invitrogen</td>
			<td style="width:119px;"> </td>
			<td>Carlsbad, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">T7 RNA polymerase reaction</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;">mMessage mMachine</td>
			<td>Austin, TX, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gentamicin</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ruthenium red </td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micropipets </td>
			<td style="width:93px;">Drummond</td>
			<td style="width:119px;">100 microliter micropipets</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">high-fidelity PfuUtra polymerase </td>
			<td style="width:93px;">Stratagene</td>
			<td style="width:119px;"> </td>
			<td>La Jolla, CA, USA</td>
		</tr>
	</tbody>
</table>

yeast luminometric and xenopus oocyte electrophysiological examinations of the molecular mechanosensitivity of trpv4

TRPV4（瞬时受体电位，香草素家族，类型4）是广泛表达于脊椎动物的组织和由几个刺激，包括通过机械力活化。某些TRPV4基因突变导致遗传性复杂骨或神经元病症的人。野生型或突变型TRPV4转基因通常表达在培养的哺乳动物细胞并通过Fura-2的荧光和由电极检测。在mechanosensitivity与疾病的分子基础的机制方面，目前的文献是混乱和争议。为了补充现有的方法中，我们描述了另外两种方法来检查TRPV4的分子特性。 （1）大鼠TRPV4和水母发光蛋白基因转化为芽殖酵母。转化人口的低osmtic冲击产生一个发光计量信号，由于水母发光蛋白与钙离子结合，通过TRPV4通道释放。在这里，TRPV4从其一贯哺乳动物伴侣蛋白分离并揭示了其自身的mechanosensitivity。 （2）TRPV4的的cRNA注入爪蟾卵母细胞。后培养一段合适的宏观TRPV4电流检测用双电极电压钳。在去除惰性渗透剂从卵母细胞浴目前的上涨是指示mechanosensitivity的。从卵母细胞中的微安（10 -6〜10 -4 A）的电流是比从培养的细胞中的subnano到毫微安（10 -10〜10 -9 A）的电流大得多，产生更清晰的量化和更自信评估。微观电流反映个别通道蛋白质的活性也膜片钳下直接注册，在对小区或切模式。同卵母细胞提供了多个补丁的样品，允许更好的数据复制。适用于补丁吸力可以激活TRPV4直接评估mechanosensitivity。这些方法也应该是在其他类型的TRP通道的研究是有用的。

在一个典型的发光法的实验中，一系列400-1,200毫渗量低渗震荡回落是通过添加200微升不同的低渗透压冲击的解决方案，以20微升的文化在1400毫渗量来实现。转化了空p416GPDV4质粒或1，其中包含TRPV4基因有突变的离子过滤器（ 图1）24的酵母细胞：在实验的RLU进行比较，该两个阴性对照的TRPV4活性是显而易见的。 在一个典型的双电极电压钳实验中，卵母?...

Watch this Scientific Journal Video about Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4 at JoVE.com

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

以补充常用的方法来研究TRPV4的两个方法被描述：其mechanosensitivity可以通过在低渗挑战的转基因酵母的Ca 2 +-水母发光蛋白发光法进行研究。它也可以用在TRPV4-RNA检测注入非洲爪蟾卵母细胞的全细胞双电极电压钳或膜片钳在对小区或切模式。

酵母发光计量和<em&gt;爪蟾</emTRPV4的分子Mechanosensitivity的&gt;卵母细胞电生理检查

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

Research

JoVE Journal

Biology

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

一个现场的固定化酵母细胞可视化的快速技术

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

科研

生物学

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

补丁钳爪蟾卵母细胞中表达的离子通道

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Visualizing RNA Localization in Xenopus Oocytes

Visualizing RNA Localization in Xenopus Oocytes

可视化的RNA中的本地化爪蟾卵母细胞

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

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

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

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

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

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

小鼠卵母细胞显微注射，成熟和倍性评估

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Competitive Genomic Screens of Barcoded Yeast Libraries

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Chromatographic Purification of Highly Active Yeast Ribosomes

高活性酵母核糖体的色谱分离纯化

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

绿色怪物承载多基因缺失的酵母菌株的产生过程

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

该<em&gt;爪蟾</em&gt;卵母细胞切割开凡士林隙电压钳技术与荧光

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Surface Spreading and Immunostaining of Yeast Chromosomes

表面扩散和酵母染色体免疫染色

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...