Name: bottom-up in vitro methods to assay the ultrastructural organization, membrane reshaping, and curvature sensitivity behavior of septins
Uploaded: 2022-08-17T13:10:00
Description: Watch this Scientific Journal Video about Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins at JoVE.com

我们感谢帕特里夏·巴塞罗和丹尼尔·莱维的有益建议和讨论。这项工作得益于国家研究机构（ANR-13-JSV8-0002-01）、ANR SEPTIMORF ANR-17-CE13-0014和“SEPTSCORT”项目ANR-20-CE11-0014-01的支持。B. Chauvin由巴黎高等医学院“ED564：法兰西岛的体质”和医学研究基金会资助。K.中泽得到了索邦大学（AAP新兴）的支持。G.H.科恩德林克得到了荷兰组织联合会（NWO/OCW）通过“BaSyC-构建合成细胞”的支持。引力授予（024.003.019）。我们感谢拉博细胞（n）量表（ANR-11-LABX0038）和巴黎科学与文学（ANR-10-IDEX-0001-02）。我们感谢法国国家研究基础设施法国生物成像（ANR10-INBS-04）成员居里研究所的细胞和组织成像（PICT-IBiSA）。

1. 使用巨型单层囊泡 （GUV） 测定膜重塑
注意：在本节中，生成GUV是为了模拟隔膜在细胞环境中可能由隔膜引起的膜变形。事实上，在细胞中，隔膜蛋白经常在具有微米曲率的位点发现。GUV的尺寸从几微米到几十微米不等，可能会变形。因此，它们适用于测定任何微米级隔膜蛋白引起的变形。荧光脂质以及荧光标记的隔膜蛋白（使用绿色荧光蛋白[GFP]）用于 通?...

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如上所述，已经使用了脂质混合物来增强脂质双层内的PI（4，5）P2 掺入，从而促进隔膜相互作用。事实上，我们已经在其他地方展示了25 ，萌芽的酵母隔膜以PI（4，5）P2特异性方式与囊泡相互作用。该脂质组成通过筛选多种组合物进行经验调整，现在被作者广泛使用。PI（4，5）P2 脂质必须小心处理。储备溶液必须小体积等分，以便特定小瓶不会打开两?...

败类是与脂质膜相互作用的细胞骨架细丝形成蛋白。败类在真核生物中无处不在，对许多细胞功能至关重要。它们已被确定为萌芽酵母和哺乳动物细胞分裂的主要调节因子1，2。它们参与膜重塑事件，纤毛发生3和精子发生4。在哺乳动物细胞中，隔膜蛋白还可以以Rho GTPas酶（BORG）依赖性方式的结合剂8与肌动蛋白和微管5，6，7相互作用。在各种组织（神经元9，纤毛3，精子10）中，隔膜蛋白已被鉴定为膜结合组分11的扩散屏障的调节因子。隔膜也被证明可以调节膜起泡和突出形成12。败类是多任务蛋白质，与各种流行疾病的出现有关13.他们的失调与癌症14和神经退行性疾病15的出现有关。
根据生物体的不同，几个隔膜亚基（秀丽隐杆线虫中的两个到人类的13个）聚集在一起形成复合物，其组织以组织依赖性方式变化16。基本的隔膜构建块聚集了两到四个亚基，以两个副本存在并以棒状回文方式自我组装。在萌芽酵母中，七分素是八聚体17，18。在原位，隔膜通常局限于具有微米曲率的部位;它们被发现在分裂收缩部位，纤毛和树突的底部，以及精子19，20的环空。在膜上，隔膜的作用似乎是双重的：它们与重塑脂质双层和维持膜完整性有关21。因此，研究隔膜上形成隔膜的长丝蛋白和/或亚基的生物物理性质对于理解它们的作用至关重要。为了在控制良好的环境中解剖隔膜蛋白的特定性质，自下而上的体外方法是合适的。到目前为止，只有少数小组在体外描述了隔膜蛋白20，22，23的生物物理性质。因此，与其他细胞骨架丝相比，目前关于隔膜蛋白在体外行为的知识仍然有限。
该协议描述了如何分析隔膜长丝的组织，膜重塑和曲率敏感性19。为此，使用了光学和电子显微镜方法（荧光显微镜，冷冻电子显微镜[冷冻电镜]和扫描电子显微镜[SEM]）的组合。使用荧光光学显微镜可视化微米级巨型单层囊泡（GUV）的膜重塑。使用冷冻EM对与脂质囊泡结合的隔膜丝的排列和超微结构进行分析。使用SEM对沉积在可变曲率的波浪基板上的固体负载脂质双分子层结合的隔膜蛋白丝的行为，对隔膜曲率敏感性进行分析，从而可以分析正曲率和负曲率的曲率敏感性。与之前的分析20，24相比，本文建议结合使用多种方法，彻底分析隔膜自组装、协同变形膜和曲率敏感。该方案被认为是有用的，并且适用于任何对膜具有亲和力的丝状蛋白质。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine</td>
			<td>Avanti Polar Lipids</td>
			<td>850725</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phospho-L-serine</td>
			<td>Avanti Polar Lipids</td>
			<td>840035</td>
			<td></td>
		</tr>
		<tr>
			<td>Bath sonicator</td>
			<td>Elma</td>
			<td></td>
			<td>Elmasonic S10H</td>
		</tr>
		<tr>
			<td>Bodipy-TR-Ceramide</td>
			<td>invitrogen, Thermo Fischer scientific</td>
			<td>11504726</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals: NaCl, Tris-HCl, sucrose, KCl, MgCl2, B-casein, chloroform, sodium cacodylate, tannic acid, ethanol</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>nikon</td>
			<td></td>
			<td>spinning disk or confocal</td>
		</tr>
		<tr>
			<td>Critical point dryer</td>
			<td>Leica microsystems</td>
			<td></td>
			<td>CPD300</td>
		</tr>
		<tr>
			<td>Deionized water generator</td>
			<td>MilliQ</td>
			<td>F1CA38083B</td>
			<td>MilliQ integral 3</td>
		</tr>
		<tr>
			<td>Egg L-&#945;-phosphatidylcholine</td>
			<td>Avanti Polar Lipids</td>
			<td>840051</td>
			<td></td>
		</tr>
		<tr>
			<td>Field Emission Gun SEM (FESEM)</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Gemini SEM500</td>
		</tr>
		<tr>
			<td>Glutaraldehyde 25 %, aqueous solution</td>
			<td>Thermo Fischer scientific</td>
			<td>50-262-19</td>
			<td></td>
		</tr>
		<tr>
			<td>High vacuum grease, Dow corning</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IMOD software</td>
			<td>https://bio3d.colorado.edu/imod/</td>
			<td></td>
			<td>software suite for tilted series image alignment and 3D reconstruction</td>
		</tr>
		<tr>
			<td>Lacey Formvar/carbon electron microscopy grids</td>
			<td>Eloise</td>
			<td>01883-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipids</td>
			<td>Avanti Polar Lipids</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-&#945;-phosphatidylinositol-4,5-bisphosphate</td>
			<td>Avanti Polar Lipids</td>
			<td>840046</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal evaporator</td>
			<td>Leica microsystems</td>
			<td></td>
			<td>EM ACE600</td>
		</tr>
		<tr>
			<td>NOA (Norland Optical Adhesives), NOA 71 and NOA 81</td>
			<td>Norland Products</td>
			<td>NOA71, NOA81</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetraoxyde 4%</td>
			<td>delta microscopies</td>
			<td>19170</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmometer</td>
			<td>L&#246;ser</td>
			<td></td>
			<td>15 M</td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Alcatel</td>
			<td></td>
			<td>pascal 2005 SD</td>
		</tr>
		<tr>
			<td>Plasma generator</td>
			<td>Electron Microscopy Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plunge freezing equipment</td>
			<td>leica microsystems</td>
			<td></td>
			<td>EMGP</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>Thermofischer</td>
			<td></td>
			<td>Tecnai G2 200 kV, LaB6</td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Electron Microscopy Science</td>
			<td>22451</td>
			<td>this product is not available for purchase any longer</td>
		</tr>
		<tr>
			<td>Wax plates, Vitrex</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

bottom-up in vitro methods to assay the ultrastructural organization, membrane reshaping, and curvature sensitivity behavior of septins

膜重塑不断发生在质膜和细胞器内。为了充分剖析环境的作用（离子条件，蛋白质和脂质组成，膜曲率）以及与特定膜重塑过程相关的不同伙伴，我们采用 体外 自下而上的方法。近年来，人们一直对揭示与主要疾病相关的隔膜蛋白的作用有着浓厚的兴趣。隔膜是与质膜相互作用的必需且无处不在的细胞骨架蛋白。它们与细胞分裂，细胞运动，神经形态发生和精子发生以及其他功能有关。因此，重要的是要了解隔膜上如何相互作用和组织以随后诱导膜变形，以及它们如何对特定的膜曲率敏感。本文旨在破译分子水平上隔膜的超结构与微米级膜重塑之间的相互作用。为此，对萌芽酵母和哺乳动物隔膜复合物进行重组表达和纯化。然后使用 体外 测定的组合来分析隔膜处的自组装。使用支撑的脂质双分子层（SLBs），巨型单层囊泡（GUV），大型单层囊泡（LUV）和波浪基质来研究隔膜自组装，膜重塑和膜曲率之间的相互作用。

无人机变形 在隔膜蛋白聚合的条件下， 图3显示了与隔膜蛋白一起孵育后重塑的GUV的典型共聚焦荧光图像。裸露的GUV（图3A）是完全球形的。在与超过50 nM的萌芽酵母隔膜丝孵育时，囊泡出现变形。浓度高达100nM的芽酵母隔膜八聚体，囊泡出现小平面，变形保持静态，因此不波动（图3B）。高于200nM的发芽酵母隔板，?...

Watch this Scientific Journal Video about Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins at JoVE.com

Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

败类是细胞骨架蛋白。它们与脂质膜相互作用，可以感测但也会产生微米级的膜曲率。我们在该协议中描述了用于分析膜变形，曲率敏感的隔膜结合和隔膜长丝超微结构的自下而上的 体外 方法。

自下而上的 体外 方法测定隔膜的超微结构组织、膜重塑和曲率敏感性行为

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic ...

使用该协议，我们可视化了显示各种曲率的图案底物上的细胞骨架丝状蛋白的组织。我们可以测试曲率灵敏度。扫描电子显微镜图像的分辨率足够高，因此纳米级组织是可视化的。我们用于固定的方法足够温和，可以保留蛋白质的结构内组织。这仍然是了解细胞骨架细丝行为的基础研究范围。首先在洁净室环境中设计一种波浪形的诺兰德光学粘合剂或NOA复制品，由波浪形聚二甲基硅氧烷波浪图案制成，波浪形的波浪形聚二甲基硅氧烷波浪图案具有250纳米的振幅和2微米的横向周期性。为此，将五微升液体NOA沉积在直径为一厘米的圆形玻璃盖玻片上，然后将PDMS模板放在液滴上。用320纳米的紫外光处理组件五分钟，将液体NOA光聚合成薄聚合物膜。完成后，用新鲜聚合的NOA从盖玻片上轻轻剥离PDMS模板。使用空气等离子清洁剂处理NOA膜五分钟，使表面亲水。如手稿中所述，制备小单层囊泡或SUV的溶液，并通过在浴超声仪中超声处理下将干燥的脂质膜重悬于观察缓冲液中5至10分钟，直到溶液透明。之后，将支持NOA模式的盖玻片插入细胞培养盒的孔中，并将新鲜发光的NOA模式与100微升每毫升1毫克SUV溶液在室温下孵育30分钟以产生支持的脂质双层。要取出未熔断的SUV，请使用隔膜低盐缓冲液彻底冲洗侧面六次，不要让样品在两次洗涤之间完全干燥。将八聚体隔肽储备溶液在隔膜低盐缓冲液中稀释至终浓度，范围为 10 至 100 纳摩尔，体积为 1 毫升。然后，将蛋白质溶液在载玻片上在室温下孵育一小时。清洗含有融合支持的脂质双层的NOA载玻片，并用隔膜低盐缓冲液孵育蛋白质。在0.1摩尔二甲胂酸钠缓冲液中用2%戊二醛固定液替换隔肽低盐缓冲液，在37摄氏度下预热，让反应进行15分钟，将固定样品洗涤三次，每次用0.1摩尔二甲胂酸钠洗涤5分钟。洗涤后，将样品在四氧化锇固定溶液中孵育，然后是过滤的单宁酸，最后是过滤的乙酸铀酰，每次10分钟。每次溶液后用蒸馏水洗涤样品三次。将样品在从50%到100%的乙醇溶液中连续孵育，每次孵育两到三分钟。将载玻片转移到预装乙醇的临界点干燥器内，并按照制造商的说明进行干燥。由于干燥的样品具有很高的吸水性，因此通过将盖玻片连接到存根上来立即涂覆它们。然后在盖玻片的上表面添加一条银色油漆，而不会产生油漆结块。确保与存根的连接令人满意。当样品完全蒸发时，使用配备等离子磁控溅射头和旋转行星台的设备，并遵循制造商提供的标准协议。在 120 毫安下进行 60 秒的预溅射以去除表面的氧化层。然后用90毫安的薄膜厚度监视器沉积1.5纳米的钨，工作距离为50毫升。之后，将样品储存在真空下，以保护它们免受环境空气的影响，直到并在整个SEM分析过程中。为了通过使用内陆探测器检测二次电子来实现高溶液成像，请将加速电压设置为3kV，将光束电流设置为20微米孔径。对于观察，请使用从每像素 21.25 纳米到每像素 1.224 纳米的分辨率。使用每像素 5.58 纳米的分辨率进行数据分析。对于高分辨率观察，将工作距离设置为1到2毫米之间，如果需要增加景深，则设置在3毫米左右。连续调整扫描速度和线积分，以确保恒定的信噪比，每张图像的采集时间约为 30 至 45 秒。代表性分析说明了沉积在隔膜细丝上的材料对1.5纳米铂和1.5纳米钨的波浪状PDMS图案的影响。据观察，裸露的巨型单层囊泡或GUV是完美的球形。在隔膜诱导的变形后，囊泡出现刻面，变形保持静止，因此没有波动。在较高浓度的隔膜下，观察到囊泡中的周期性变形。用冷冻电子显微镜图像检查与大单层囊泡（LUV）结合的隔膜细丝的自组装，并从倾斜的系列中获得3D重建。隔膜细丝的曲率依赖性排列通过扫描电子显微镜可视化。在低放大倍率下，可以看到具有负曲率和正曲率周期性的波浪图案。隔膜细丝的超微结构组织在更高的放大倍率下观察。NOA必须轻轻剥离以防止降解。成像后，可以进行一些图像分析，以量化SCM可视化的超微结构特征，例如灯丝间距和灯丝的方向。本方法已应用于隔膜素，但它也可用于其他细胞骨架蛋白或组织成长丝的蛋白质，因此对曲率敏感。

Research

JoVE Journal

Biology

Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

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

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

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

蛋白膜覆盖含量：一个协议测试可溶性和不溶性蛋白质之间的相互作用在体外</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

自下而上和猎枪蛋白质组学鉴定一个全面的蛋白质组耳蜗

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks

谁是谁？非侵入性的方法，以个别性和马克晚成小鸡

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal ...

In vitro Methylation Assay to Study Protein Arginine Methylation

In vitro Methylation Assay to Study Protein Arginine Methylation

在体外甲基化分析研究蛋白质精氨酸甲基化

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

微灌技术在调查微血管渗透性的大鼠肠系膜条例

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

体外试验来测量磷脂酰乙醇胺甲基转移酶活

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

方法发现小鼠BCRP1的另类用法子和转录调控

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

完整的工作流程为组蛋白翻译后修饰的分析采用自下而上质谱法：从组蛋白提取数据分析

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

体外sumo 法研究相扑 E3 连接活动

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...

自下而上的 体外 方法测定隔膜的超微结构组织、膜重塑和曲率敏感性行为

自下而上的体外方法测定隔膜的超微结构组织、膜重塑和曲率敏感性行为