1. 小脂囊泡的制备
<ol><li>用 79/20/1 的质量混合比制备氯仿中脂 SPE/DOPS/ATTO488-DOPE 的溶液. 氯仿中脂类的最终浓度为1毫克/毫升, 最终质量为0.6 毫克 (通常少于1毫克)。使用一次性圆形底部玻璃瓶 (圆底玻璃管, 10 x 75 毫米), 当准备的脂质溶液没有预先清洁步骤。<ol>
<li>使用前至少5次用氯仿填充和冲洗玻璃注射器 (25 µL)。要填充和冲洗玻璃注射器至少5次, 3-4 毫升的氯仿是足够的。 注意: 在处理氯仿时, 使用玻璃注射器, 并在任何时候佩戴适当的手套和防护眼镜, 并在油烟机中执行所有程序。在处理氯仿时不要使用任何塑料烧瓶、注射器或吸管。</li>
	</ol>
</li>
	<li>用金属箔包裹含有脂质混合物的玻璃瓶, 以保护环境光中的内容, 并在旋转蒸发器中蒸发溶剂3小时以除去氯仿, 压力达到7帕真空度 80 rpm。在玻璃瓶的底部蒸发后形成干脂膜 (图 2A)。 注: 增加几次, 使用大量的脂质可能需要更长的旋转蒸发时间, 以确保完全清除氯仿。</li>
	<li>缓慢地添加600µL 磷酸盐缓冲盐水 (PBS) 缓冲液 (7.8 pH 值, 不稀释) 在干脂膜上。在增加了缓冲后, 轻轻地添加6µL 甘油, 以保护样品从完全脱水期间, 在 MLV 的形成步骤 (达到最后浓度甘油 1 wt%)36。用 parafilm 和金属箔盖住玻璃瓶子以保护周围光线。在冰箱里过夜4摄氏度。 注: PBS 缓冲器解决方案包括0.151 克 Trizma 基, 1.592 克 K3po4, 1.021 克的 MgSO 2 po 4, 0.062克4· 7H2O , 和0.0465 克 EDTA 在250毫升纯净水。将溶液贮存在摄氏4摄氏度, 并在使用前温暖室温。10毫米 HEPES 缓冲器的等效量可以代替 PBS 解决方案使用。HEPES 缓冲液是通过稀释1米 HEPES 溶液和纯净水来制备的。</li>
	<li>第二天, 油脂实验在室温下使用超声波浴的玻璃瓶, 其中含有1分钟的溶液。除去 parafilm 和吸管, 直到形成一个视觉上均匀的小脂囊泡的溶液 (图 2B)。整除30µL 的小脂囊泡溶液成单独的0.5 毫升塑料管。将这些股票整除数-18 摄氏度, 最多6月。 注: 如果小脂囊泡溶液没有视觉上的均匀, 用涡流混合器以最大速度涡旋溶液4次。</li>
</ol>2. MLV 配合物的制备
<ol><li>解冻到室温一个塑料管含有整除的冰冻悬浮的小脂泡。涡流管4倍, 1-2 s 使用涡流搅拌机在最大速度。</li>
	<li>将5µL 的小脂囊泡悬浮在玻璃罩滑动 (24 x 60 毫米) 的表面, 形成一个小圆滴。使用玻璃盖子滑动没有任何预清洁步骤。</li>
	<li>将玻璃罩滑入真空干燥 (< 100 帕真空), 20 分钟。</li>
	<li>将干脂膜在室温下贮存4分钟, 然后缓慢地将50µL 的10毫米 HEPES 缓冲器放在干脂膜上进行补液。等待5分钟预先形成的 MLV 复合体 (图 2C)。</li>
	<li>在显微镜台上放置一个玻璃罩滑块, 然后将300µL 的 HEPES 缓冲器放在上面, 并将液滴的中心放在目标上方 (图 3B)。</li>
	<li>将预成型的 MLV 溶液的50µL 转移到 HEPES 溶液中 (300 µL)。等待25分钟, 允许疏生的 MLV 配合物牢固地附着在玻璃盖板的表面上 (图 2D)。 注意: 偶尔, MLV 复合物不形成在玻璃盖子滑动, 而不是油脂补丁或仅 MLVs 被观察。这可以解释为在步骤2.4 或 2.6, 或许多冻融循环的小囊泡悬浮的样品意外晃动。重复步骤 2.1-2.6 或使用刚准备好的脂质库存解决方案 (步骤 1.1) 获得 MLV 配合物。</li>
</ol>3. 微制备和显微注射
<ol><li>在硼硅酸盐玻璃毛细血管的边缘, 轻轻地将毛细管端放到蜡烛的火焰中, 以防止微被折断, 同时将其附着到微持有者身上。</li>
	<li>使用程序集抽取至少3个玻璃毛细血管使用自动激光拉拔: 热 = 400, 滤 = 4, 威 = 50, Del = 225, 普勒 =;热 = 400, 滤 = 4, 威 = 60, Del = 150, 普勒 = 120。必须输入程序集的第一个值为左空。使用内直径的硼硅酸盐玻璃毛细血管 (id) 1.00 毫米和外径 (外径) 0.78 毫米导致一个微, 尖端开口约0.3 µm 直径。</li>
	<li>用 microloader 在 HEPES 缓冲器 (10 毫米) 中, 用8µL CaCl2解来填充每个微。使用 0.2-0.5 µm 注射器过滤器在使用前过滤 CaCl2溶液, 以避免堵塞吸管尖端。 注意: 确保 CaCl2在 HEPES 缓冲解决方案中完全溶解, 它也用于步骤 2.4-2.6。</li>
	<li>将微持有者连接到机器人。将微装入微夹紧。使用供应管连接注射泵和毛细管支架。</li>
	<li>启动注射泵。将注射泵上的设置调整为 20 hpa (注入压力), 并设置一个5的 hpa 补偿压力, 自动模式。暂停注射泵, 直到微准备好进行微注射。</li>
	<li>将显微镜设置为 brightfield 模式。将其配置为差异干扰对比度 (DIC)。采用63X 或100X 高 NA (1.3) 目标实现最佳膜边缘分辨率。</li>
	<li>使用粗机器人将微放在含有 MLV 配合物的液滴上方。在目标上方找到微的尖端。</li>
	<li>把显微镜聚焦在 MLV 的中点, 然后将大约50µm 重新对焦在老大上面。使用机器人的粗模式将微轻轻地带入焦点。</li>
	<li>将机器人切换到精细模式。慢慢地重新专注于老大的表面, 同时翻译微的尖端和保持尖端的焦点。将微笔尖从µm 表面定位大约20个。如果微提示是由于意外接触玻璃盖子滑动 (例如图 5B), 立即更换, 以避免不必要的钙离子释放到样品散装溶液。</li>
</ol>4. 利用钙离子源台胞的形成和翻译
<ol><li>将显微镜设置成荧光模式。设置 dichroics 激发在 488 nm 和检测的排放量在 505-550 nm。</li>
	<li>打开注射泵, 启动钙离子注入。使用机器人的精细模式, 慢慢接近老大与微小费。将微尖端定位在3µm 从膜表面的距离, 同时从微中注入 CaCl2溶液, 以允许台胞形成 (图 4)。</li>
	<li>要将台胞沿老大面平移, 请使用精机器人 (图 6) 在µm 表面周围缓慢移动吸管尖端 (速度为 0.1-0.3)。在继续钙离子注入的同时, 保持µm 表面与微尖端之间的距离 (3)。 注: 在微笔尖 (0.7 µm 以上) 的高平移率下, 台胞不会与微同步迁移。相反, 它们会分散和缩短, 而新的台胞将在微尖端的新位置形成 (图 7)。</li>
	<li>要停止微注射, 请关闭注射泵, 将微从老大表面移开。</li>
</ol>

仿生细胞系统允许研究膜的行为后, 暴露在外部刺激, 如离子, 蛋白质, 或纳米粒子。GUVs 是这样一个模型, 可以通过调整形状来响应化学环境的变化, 这通常涉及管状结构的形成和内陷24、41、42、43.
本文提供了一种在非接触方式下产生台胞的方法, 通过在表面注入钙离子的情况下, 通过重塑老大表面。该协议描述了 MLV 复合体的制备, 它模仿细胞等离子膜, 以及如何使用微注射技术产生接近老爹表面的钙离子梯度形成台胞。以往的大多数实验研究, 解决钙离子膜的相互作用暴露脂囊泡到大块钙离子浓度14,17。根据实验条件, 这种大块接触可能导致不同的膜反应, 没有管状突起形成25。
MLV 配合物的形成是相当简单的, 只需要标准的实验室设备, 如旋转蒸发器、超声波浴和真空干燥。不过, 在囊泡准备过程中还有几个关键步骤需要考虑。重要的是要确保囊泡的脱水是完整的 (在协议的步骤 2.3) 和一个只含有少量盐晶体的干循环脂膜在玻璃盖板的表面形成。在实验过程中, 用氯仿中的新鲜脂质溶液和新鲜的 HEPES 缓冲液对囊泡溶液进行细致的处理, 对 MLV 配合物的成功制备至关重要。此外, 安全附着 MLV 复合物的玻璃覆盖滑移面是至关重要的微操作系统和显微注射。为了确认 MLV 复合体的足够附着力, 微 (不带喷射流) 可以用来轻轻地推向老大表面。牢固附着的囊泡不会沿表面滑动直接接触。由于实验是在一个开放的缓冲液滴, 这可能被审问几个小时, 必须考虑蒸发。从缓冲液滴蒸发将改变渗透条件, 这可能会影响和稳定的囊泡。为了恢复渗透条件, 定期向样品中添加纯净水还原原体积, 使系统恢复稳态。
在修改脂质膜成分时, 关键是以 MLV 复合体的形式产生囊泡, 因为 MLV 允许在膜整形过程中将脂质材料转移到老爹身上。以前的研究表明, 用纯脂代替 SPE 混合物的成分, 或者增加5-30% 的胆固醇, 也允许 MLV 复杂的形成28,44。被准备的 GUVs 的多数是 unilamellar45。
此外, 当测试其他价阳离子, 如镁离子, 台胞的形成严重依赖于存在的负电荷扣在脂质混合物。没有扣, 台胞在本议定书所描述的囊泡中不形成。此外, 单价阳离子, 如钾和钠, 不会导致台胞的形成, 即使在含扣的囊泡25。
除了在 GUVs 的准备和操作方面的关键步骤外, 还有几个重要的因素需要考虑在注射过程中。成功地显微注射钙离子严重依赖于正常运行的玻璃 micropipettes, 这是在实验当天准备。有几个因素可能导致微的故障。一个常见的原因是堵塞的提示打开。小的脂质微粒, 是囊泡制剂的副产品, 分散在溶液中, 倾向于坚持微尖端, 从而产生堵塞。清洁吸管尖端最好的方法是把它从囊泡溶液中抬起, 然后放回靠近老大的表面。必须避免使用微注射泵的吹出功能, 因为它会导致大量的钙离子注入到散装溶液中。此外, 微内包裹的小气泡防止了适当的显微注射, 在这种情况下, 微应该换成新的。通过将实验装置放置在减震工作台上, 最大限度地减少尖端的振荡, 可以显著减少尖端断裂。
此外, 在选择观察方案时需要注意, 尽量减少漂白, 同时获得最佳的时间分辨图像。本协议采用了广域激光诱导荧光显微术, 因为它允许在目标有限探针深度上获得相对较高的图像采集速率。此外, 使用倒置显微镜可以同时注射和观察脂囊和台胞。
提出的方法的主要局限性之一是广泛的手工工作和足够的微操作系统技能的要求。由于复合体是通过自发的膨胀过程形成的, GUVs 和 MLVs 的大小是无法控制的。此外, 本议定书不允许控制准备好的 MLV 复合体的膜张力, 这可能是必要的, 以收集有关膜重塑的更多细节。GUVs 与 MLVs 连接, 后者提供脂质材料, 用于台胞的大量生长, 在一定程度上无法完全利用从 GUVs 获得的膜。MLVs 还有助于降低 MLV 复杂44内的任何侧向表面张力变化, 这将使试图通过微吸入控制囊泡的张力复杂化。这种基于 MLV 的模型确实提供了一种更低的张力, 可以更好地模拟与膜油藏相连的细胞膜结构中所发现的张力机制, 如膜褶皱和内陷46。同时, 微抽吸技术可以成功地应用于单 GUVs 膜张力的控制。例如, 格雷勃等人的工作提供了关于单 GUVs 中的膜管状内陷的形成的详细情况, 在不同张力状态下, 在散装条件下将钙离子与膜结合在一起。最后, 在局部和大量接触钙的情况下, 膜的行为的比较需要改进对表面的膜黏附力的控制, 这超出了本协议的范围。
综上所述, 所提出的技术允许非接触膜重塑和形成的台胞在局部刺激钙离子。该方法的未来应用中心的翻译从合成囊泡系统到当地的生物膜, 如细胞泡。所提出的方法可以与其他单细胞审讯方案, 如补丁钳或微电极探头, 或结合局部加热策略31,47,48。测试其他离子或分子的效果是直接的, 只需要用感兴趣的分子来代替钙离子。此外, 复杂的合成脂泡可以通过膜功能化的跨膜蛋白产生, 这可能扩大我们对细胞重塑的生物物理学和细胞膜动力学的认识, 与当地的传感相关化学梯度。最后但并非最不重要的是, 非接触性刺激的脂质膜也可以转化为高分子软物质系统, 为新的非接触操作平台提供了基础。

钙离子在生物过程中的作用, 特别是它们参与信号、细胞分裂和膜融合, 是许多机械学研究的重点1。胞内胞浆钙离子浓度为 100 nM, 而细胞器中的钙, 如内质网、分泌囊泡、线粒体等, 均达到 millimolars 浓度的水平。这将产生陡峭的钙离子浓度梯度级阶横跨胞内膜2,3,4,5,6,7,8 ,9。胞外钙离子水平约为2毫米, 因此, 钙离子浓度的变化发生在细胞外和细胞内的水平。此外, 最近的研究提供证据表明, 细胞内钙离子信号事件和神经元活动可能发生在局部波动的细胞外钙离子浓度, 表明同步的重要性细胞内和胞外钙离子变异10。
为了了解钙离子与生物膜的相互作用, 本文成功地实现了用脂质双层泡代替本机细胞膜的合成方法。将囊泡暴露在钙离子溶液中会导致脂质头组和烃链填料的变化, 膜张力增加, 囊泡聚集, 以及脂质和膜相转变11,12 的分离. ,13,14,15,16。利用 X 射线、 1h-核磁共振、光谱或热力学研究11、16等实验技术, 研究了脂质膜暴露于钙离子时的性质,17,18. 在这些研究中, 膜的组成经常被调整为类似于本机细胞膜, 含有磷脂酰胆碱 (PC)、脑磷脂 (PE) 和磷脂 (PS) 等生理脂质。PS 在人工囊泡制备中特别重要, 因为它是许多细胞过程中必不可少的成分, 包括胞内膜的贩运、胞吐作用和凋亡19、20。
合成脂囊的大小通常从纳米到几微米不等。在不同的囊泡制剂中, 巨大的 unilamellar 囊 (GUVs), 其直径为数微米, 特别重要, 因为它们的体积相对较大, 与单个细胞的尺寸紧密相似21,22,23. GUVs 的可用表面积使局部化学梯度对膜生物物理特性的影响得以研究。通过将部分膜表面暴露给外部刺激, 可以对膜动力学进行更深入的研究。例如, 已经表明, 化学或 pH 梯度在 GUVs 表面的局部应用导致管状突起的形成, 在散装暴露24,25没有观察到。在膜行为上观察到的差异要求进一步的方法发展单囊的审讯计划, 以获得一些对细胞膜重塑机制的深入了解。
建立在从 1900s26,27早期的显微注射和微操作系统的方法, 与最近的进展, 单囊泡操作方案从 2000s23,28, 本文提出了用钙离子的局部应用来生成台胞膜中膜管状突起 () 的膜重构和形成方法。
我们的方法利用一个囊状复合体, 由一个与 multilamellar 囊 (MLV) 连接的老爹作为仿生膜模型系统 (图 1A)。MLV 被要求作为一个脂质的水库为复合体提供脂质材料给老大在接触到钙离子梯度。这种连接使得复合体能够补偿在诱导重塑和人的心房形态转换过程中膜张力的增加, 为中期生长提供脂质。此外, MLV 有助于表面固定化, 因为它的质量比老大的大。MLV 配合物, 当固定在一个坚实的基底上, 以前曾用于生产管状囊泡网络, 研究聚合物膜相互作用, 并模仿胞吐作用29,30后期阶段, 31,32,33。
以前的协议利用大豆极性脂质提取物 (SPE) 制备 MLV 配合物28。SPE 包括了磷脂的混合物, 包括 PC (45.7%), PE (22.1%), 磷酸肌醇 (PI, 18.4%), 磷脂酸 (PA, 6.9%), 和其他脂 (6.9%) 的混合物。在本协议中, SPE 混合物掺入20% 的 12-dioleoyl-磷脂酰-3-磷-l-丝氨酸 (钠盐) (扣), 以模拟细胞膜的内小叶。另外1% 的阿托 488-1, 2-dioleoyl 锡-磷脂酰 3-乙醇胺 (ATTO488-DOPE) 被用来染色脂双层, 使监测膜重塑使用荧光显微镜。GUVs 在双层中有对称的脂质成分, 并且在当地暴露于5毫米的氯化钙浓度 (CaCl2)。这种实验条件下, 钙离子浓度升高, 也模仿凋亡细胞外膜小叶, 其中 PS 分子表达34。MLV 配合物的形成需要使用 Criado 和凯勒35最初开发的改良的补液脱水方法。囊泡制备协议包括形成一个干脂层, 然后用来在溶液中形成小泡。这个解决方案然后脱水和水化形成最终的 MLV 复合体。图 2AD说明了准备典型的 MLV 复合体的主要步骤。
囊泡制剂完成后, 囊泡复合物固定在玻璃基板上, 采用微注射技术, 通过开尖端玻璃微, 将少量钙离子输送到老爹的外小叶中。在头膜表面, 钙溶液的流动产生了局部钙离子梯度, 导致膜的重塑和台胞的产生。台胞的方向远离钙离子源, 并在老大体内生长。这一中期计划的形成可以直接监测使用荧光显微镜和记录使用数码相机。图 3显示了用于产生膜重塑的实验装置。在本协议中, 台胞 (图 2E和图 4) 的形成表明了在大体积条件下进行的钙离子暴露实验的对比结果。在散装条件下, GUVs 破裂和形成膜补丁, 可以观察到坚持玻璃表面25。
本文还详细介绍了 MLV 配合物的形成, 以及对钙离子进行显微注射的程序。这些协议主要集中在钙离子显微注射;然而, 这种方法可以很容易地被修改, 以用于研究膜反应由于局部接触其他离子或蛋白质。此外, 在膜重构过程中, 可以调节囊泡的组成, 以分离脂质成分的作用。所提出的协议不需要任何复杂的设备来生产 MLV 配合物, 具有高度的重现性。

<table><tbody><tr><td>Soy bean polar lipid extract</td><td>Avanti&lt;br/&gt; Polar Lipids, Inc. (Alabaster, USA)</td><td>541602C</td><td>100 mg</td></tr><tr><td>1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) DOPS</td><td>Avanti&lt;br/&gt; Polar Lipids, Inc. (Alabaster, USA)</td><td>840035C</td><td>1x25 mg</td></tr><tr><td>ATTO 488- 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)</td><td>ATTO-TEC (Germany)</td><td>AD 488-31</td><td>1 mg</td></tr><tr><td>Hamilton&amp;nbsp;syringe, 700 series, fixed needle, 702N, volume 25&amp;nbsp;&amp;mu;L, needle size 22s ga (bevel tip), needle L 51&amp;nbsp;mm (2&amp;nbsp;in.)</td><td>Sigma Aldrich (Missouri, USA)</td><td>20735&amp;nbsp;SIGMA-ALDRICH</td><td></td></tr><tr><td>Pyrex Tube, culture, disposable, rimless, 10x75 mm, Borosilicate glass 250/pack</td><td>Corning Incorporated (Corning, NY 14831)</td><td>99445-10</td><td></td></tr><tr><td>Chloroform&amp;nbsp;CHROMASOLV&amp;nbsp;Plus, for HPLC, &amp;ge;99.9%, contains amylenes as stabilizer</td><td>Sigma Aldrich (Missouri, USA)</td><td>650498-1L-D</td><td></td></tr><tr><td>Rotary evaporator</td><td>B&amp;uuml;chi Rotavapor R-144 Switzerland</td><td></td><td></td></tr><tr><td>Kalciumklorid purum torkad minimum 95% medelkornig 5-10 mm</td><td>KEBO lab (Sweden)</td><td>MA00360500</td><td></td></tr><tr><td>Magnesium chloride hexahydrate reagent grade ACS, ISO</td><td>Sharlau Chemie S.A. (Spain)</td><td>P9333-500G</td><td></td></tr><tr><td>Potassium chloride, SigmaUltra, minimum 99.0%</td><td>Sigma Aldrich (Missouri, USA)</td><td>S7653-1KG</td><td></td></tr><tr><td>Sodium chloride, SigmaUltra, minimum 99.5%</td><td>Sigma Aldrich (Missouri, USA)</td><td>G5516-1L</td><td></td></tr><tr><td>Glycerol, for molecular biology, minimum 99%</td><td>Sigma Aldrich (Missouri, USA)</td><td>T-1503 2050 g</td><td></td></tr><tr><td>Trizma base</td><td>Sigma Aldrich (Missouri, USA)</td><td>P5629-500G</td><td></td></tr><tr><td>Potassium phosphate tribasic (K3PO4)</td><td>Sigma Aldrich (Missouri, USA)</td><td>P5655</td><td></td></tr><tr><td>Potassium phosphate monobasic (KH2PO4)</td><td>Sigma Aldrich (Missouri, USA)</td><td>5886</td><td></td></tr><tr><td>MgSO4</td><td>Merck (USA)</td><td>34549-100 g</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich (Missouri, USA)</td><td>H0887 Sigma&amp;nbsp;</td><td></td></tr><tr><td>HEPES solution 1&amp;nbsp;M, pH 7.0-7.6, sterile-filtered, BioReagent, suitable for cell culture</td><td>Sigma Aldrich (Missouri, USA)</td><td>Z260282-1PAK</td><td>24x60 mm</td></tr><tr><td>Acrodisc&amp;nbsp;syringe filters,&amp;nbsp;PVDF membrane, diam. 13&amp;nbsp;mm, pore size 0.2&amp;nbsp;&amp;mu;m</td><td>Sigma Aldrich (Missouri, USA)</td><td>631-1339&amp;nbsp;</td><td></td></tr><tr><td>Menzel Gl&amp;auml;zer #1, glass cover slip</td><td>VWR (USA)</td><td></td><td></td></tr><tr><td>Diaphragm vacuum pump for the desiccator</td><td>Vacuubrand (Germany)</td><td></td><td></td></tr><tr><td>Ultrasonicate bath</td><td>Bandelin Sonolex (Germany)</td><td></td><td></td></tr><tr><td>VX-100 Lab vortexer vortex mixer</td><td>Labnet International (USA)</td><td></td><td></td></tr><tr><td>488 nm laser line&amp;nbsp;</td><td>Cobolt MLD-488 nm (Solna, Sweden)</td><td></td><td></td></tr><tr><td>Leica Microsystems&amp;nbsp;immersion oil for microscopes</td><td>Leica (Germany)</td><td>12847995&amp;nbsp;</td><td></td></tr><tr><td>Inverted fluorescence microscopy system&amp;nbsp;</td><td>Leica DM IRB (Wetzlar, Germany)</td><td></td><td></td></tr><tr><td>Camera (Prosilica Ex 1920, Allied Vision)</td><td>Technologies GmbH (Thuringia, Germany)</td><td>300038</td><td></td></tr><tr><td>PatchStar Micromanipulator</td><td>Scientifica (Uckfield, UK)</td><td>612-7933</td><td></td></tr><tr><td>Borosilicate glass capillaries, GC100TF-10,&amp;nbsp; 1.00mm O.D. X 0.78mm I.D.&amp;nbsp;</td><td>Harvard Apparatus U.K</td><td></td><td></td></tr><tr><td>Eppendorf microloader (pipette tips)</td><td>VWR (USA)</td><td></td><td></td></tr><tr><td>P-2000 CO2 laser-puller&amp;nbsp;</td><td>Sutter Instruments (Novato, USA)</td><td></td><td></td></tr><tr><td>Femtoliter automatic injection pump, Eppendorf Femtojet</td><td>Eppendorf (Germany)</td><td></td><td></td></tr></tbody></table>

membrane remodeling of giant vesicles in response to localized calcium ion gradients

在多种基本细胞过程中, 如膜的贩运和细胞凋亡, 细胞膜形态的转变同时发生于钙离子浓度的局部变化。确定了这些过程中所涉及的主要分子成分;然而, 钙离子梯度与细胞膜内脂质之间的具体相互作用远不为人所知, 主要是由于生物细胞的复杂性质和观察方案的困难。为了弥合这一缺口, 成功地实施了一种合成方法, 以揭示钙离子对细胞膜模拟剂的局部作用。建立一个类似于细胞内条件的模拟是一个几倍的问题。首先, 需要一个适当的维度和膜组成的适当的仿生模型来捕获细胞的物理特性。其次, 需要一个微操作系统设置, 以提供少量的钙离子到一个特定的膜位置。最后, 需要一个观察方案来检测和记录脂质膜对外界刺激的反应。本文提供了一个详细的仿生方法来研究钙离子膜相互作用, 其中一个脂囊系统, 包括一个巨大的 unilamellar 囊 (老爹) 连接到一个 multilamellar 囊 (MLV), 暴露在局部钙使用显微注射系统形成的梯度。用荧光显微镜观察了离子对膜的影响动力学, 并记录了视频帧率。由于膜刺激, 高弯曲膜管状突起 (台胞) 形成内部的老大, 面向远离膜。所述方法以完全非接触性和控制性的方式诱导脂质膜和中期生产的重构。该方法介绍了一种处理钙离子膜相互作用细节的方法, 为研究细胞膜整形机制提供了新的途径。

在这项工作中, 我们展示了 MLV 复合体的创建, 以及它们如何可以用来阐明钙离子梯度对细胞膜动力学的影响。表面附着的 MLV 配合物是需要这些实验, 以允许一个固定的细胞大小的模拟, 而建立一个离子梯度通过显微注射。老大膜通过台胞从钙离子源的形成来响应局部的钙浓度。此外, 台胞可以通过移动微尖端在膜表面周围, 以非接触方式在老大周围进行翻译。
图 2显示了 MLV 准备过程的示意图。虽然在提出的协议中, MLV 复合体已经在步骤 2.4 (图 2C) 中预先形成, 但将囊泡溶液转移到另一个300µL 的缓冲液滴 (协议的步骤 2.6) 允许稀疏形成的 MLV 复合物来充分坚持玻璃基板。这样, 就建立了适合微操作系统和显微注射的复合体 (图 2D)。偶尔, GUVs 包含一个或几个被包裹的脂囊, 不影响中期计划的形成, 如图 4A所示。这些 GUVs 产生台胞;然而, 如果包裹的囊泡很大, 成像可能会受阻。大多数准备好的 GUVs (最多 75%) 出现半球状, 并连接到 MLVs, 如图 1A所示。这些囊泡是本协议的主要重点, 适用于微注射程序。其余 GUVs 的约25% 出现起伏 (图 1B), 这表明膜张力较低。这些起伏的水泡也允许形成管状突起;然而, 它们表现出不同的动力学和形成的台胞的不同形态, 这超出了本议定书25的范围。所制备的囊泡的典型直径在 MLVs 的2到15µm 之间变化, GUVs 2 到40µm。钙离子暴露的最佳老爹尺寸为5µm 或更大。
Micropipettes 已经被用于单细胞审讯计划以及需要操纵合成 GUVs 几十年37,38的程序。这些应用大多涉及微与细胞表面或囊泡之间的直接物理接触。例子包括补丁钳技术或拉膜束缚从老大膜, 除了建立纳米管-囊泡网络23,28,39。最近, micropipettes 也被用来产生局部梯度的离子和分子围绕 GUVs 重建异构细胞微环境24,25。在所提出的协议中, 一个正常运行的微 (图 5A) 是在老大表面建立钙离子梯度的关键因素, 方法是释放内包含的溶液。正确定位微在老大表面和避免接触脂质膜是成功的显微注射的关键。微尖端是保持在距离约3µm 从老大表面, 这是一个最佳的距离, 以产生台胞, 同时模仿钙在细胞膜附近的变化。如果微提示意外损坏 (图 5B), 则需要更换。先前测试的浓度范围内的 CaCl2内的微, 这允许的中期计划的形成, 是介于2和5毫米25之间, 相当于胞外钙浓度。在较低的 CaCl2浓度,即1 毫米, 没有台胞的观察。
台胞在钙微注射后的形成始于小膜内陷的形成, 在暴露部位生长成台胞 (图 4B)。只要台胞膜仍然暴露在钙离子中, 就会继续生长。他们波动和指向远离钙离子源 (图 4C,补充电影 1)。当钙离子供应被终止时, 台胞分散在老爹表面上, 使得很难断定台胞是保持还是最终消失。
台胞的形成可以用局部钙离子结合的形式来解释, 并将其与暴露在外小叶的老大膜和触发自发曲率 (m), 直接相关的形成自发张力σ = 2κm2 (κ是弯曲刚度), 诱导膜弯曲, 形成内台胞。以前的报告已经表明, 膜的变形可以解释为在钙离子与膜的结合上的负电荷脂质的凝结和/或聚类, 导致负自发曲率40。另一种情况是, Ca2 +到负电荷双层表面的强约束中和暴露的脂质双层小叶的表面电荷密度。这导致电荷密度的差异横跨双层, 导致非零自发曲率, 足以弯曲膜25。
观察到的台胞的形成表明了脂质膜对钙离子的敏感性, 为膜管结构的制备提供了一种新的非接触方法。阐明这一膜 tubulation 的起源, 对于了解不同细胞功能和细胞重塑过程中膜形态转换的动力学是很重要的, 并且有助于在细胞内获得脂质组织的洞察力。膜。
观察到的台胞总是在膜上发生钙离子暴露的部位产生。因此, 通过选择微尖端的位置, 可以定义凸度曲面的区域, 以实现凸起的生长。接下来, 如果微是缓慢 (0.1-0.3 µm/秒) 移动的老大表面, 同时保持分离距离从膜, 台胞是翻译与微串联。图 6和相应的辅助影片 2演示了台胞在老大表面附近的这种迁移。这一过程很可能伴随着新的台胞在连续钙离子注入25的形成。在高翻译率 (超过0.7 µm/秒), 台胞无法遵循微提示。相反, 新的突起是在微尖端的新位置形成的 (图 7)。
观察到台胞表面的非接触钙离子引导平移, 可以进一步了解细胞内膜管结构动力学背后的驱动力。此外, 该方法还提供了一种新的非接触模式, 用于控制材料在软物质系统中的传输, 采用化学梯度。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57789/57789fig1.jpg"> 图 1: 有代表性的荧光显微图像, MLV 配合物固定在玻璃罩的表面上.(a). 球面老大的例子。老大以 MLV 的半球的形式出现。(B). 一个起伏的老大的例子。黑色箭头表示膜的变形区域。图像的增强和倒置, 以改善荧光标记的老爹膜的可视化。刻度条代表5µm.<a href="https://www.jove.com/files/ftp_upload/57789/57789fig1large.jpg" target="_blank">请点击这里查看这个数字的大版本</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57789/57789fig2.jpg"> 图 2: MLV 准备和微注射方案的示意图.(a) 在玻璃瓶底部形成干脂层, 其结果是从脂质溶液中氯仿的旋转蒸发。(B) 水化脂层为微气泡, 形成小的脂囊。(C). 囊泡溶液的小水滴 (5 µL) 放置在玻璃盖板的表面上, 并转移到干燥中, 以脱水脂质溶液, 形成干脂膜 (不显示此步骤)。再水化50µL 缓冲液的干脂膜产生预成型的 MLV 配合物。(D). 将预形成的复合物转移到较大的缓冲容积后, 就形成了附着在玻璃盖板表面上的疏生 MLVs。(E) 将微靠近老大的表面, 释放钙离子, 触发台胞的形成。插图不绘制为缩放。<a href="https://www.jove.com/files/ftp_upload/57789/57789fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57789/57789fig3.jpg"> 图 3: 钙离子显微注射实验装置.(A) 在 GUVs 中生成台胞所需的实验装置的组成部分。(B). 微注射设置的细节。玻璃盖玻片与 MLV 配合物的溶液放置在显微镜阶段。微与玻璃盖板表面之间的夹角为 30° (用白色标记)。<a href="https://www.jove.com/files/ftp_upload/57789/57789fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57789/57789fig4.jpg"> 图 4: 台胞在老爹表面注射钙离子时形成的.(A) 在接触钙离子梯度之前, MLV 复合物。箭指的是在老大体内包裹的脂囊, 不妨碍观察台胞。小台胞是形成后, 钙离子的显微注射 (5 毫米浓度 CaCl2在微)。(C) 台胞在连续接触钙离子时的生长。荧光图像的增强和倒置, 为可视化目的。刻度条代表5µm.<a href="https://www.jove.com/files/ftp_upload/57789/57789fig4large.jpg" target="_blank">请点击这里查看这个数字的大版本</a>。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57789/57789fig5.jpg"> 图 5: 用于钙离子显微注射的玻璃 micropipettes.(a) 一个正常运作的微的例子。(B). 微的例子 (损坏的区域用黑色箭头表示)。两个图像都增强和反转, 以更好的可视化。刻度条代表5µm.<a href="https://www.jove.com/files/ftp_upload/57789/57789fig5large.jpg" target="_blank">请点击这里查看这个数字的大版本</a>。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57789/57789fig6.jpg"> 图 6: 钙离子引导翻译台胞的表面周围的老大.(A). 台胞是在5毫米 CaCl2溶液的显微注射后在老大表面形成的。(B)。在膜表面周围的微尖端 (0.2-0.3 微米/秒) 的平移触发了台胞在钙离子源方向上的运动。黑色箭头突出了微运动的方向。图像的增强和倒置, 改善了膜的可视化。刻度条代表5µm.<a href="https://www.jove.com/files/ftp_upload/57789/57789fig6large.jpg" target="_blank">请点击这里查看这个数字的大版本</a>。
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57789/57789fig7.jpg"> 图 7: 台胞在微尖的高翻译率.(A). 台胞是在 5 mM CaCl2溶液 (白线) 显微注射后在老大表面形成的。(B)。微的高翻译率在老爹表面 (1 微米/秒) 导致新的台胞的形成在微尖 (洋红色线) 的新的位置, 并且以前被形成的台胞 (白色线) 的散射。<a href="https://www.jove.com/files/ftp_upload/57789/57789fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Movie 1" src="/files/ftp_upload/57789/57789mov1.jpg"> 辅助电影 1:台胞在对 Ca 的局部接触时的形成2 +渐变.<a href="https://www.jove.com/files/ftp_upload/57789/Lobovkina_Movie_1.avi" target="_blank">请点击这里下载这部电影.</a> 
<img alt="Movie 2" src="/files/ftp_upload/57789/57789mov2.jpg"> 辅助电影 2:钙离子引导的中期计划在老大表面附近迁移.  <a href="https://www.jove.com/files/ftp_upload/57789/Lobovkina_Movie_2.avi" target="_blank">请点击这里下载这部电影.</a>

Watch this Scientific Journal Video about Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients at JoVE.com

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

局部钙离子梯度对大泡囊膜重塑的响应

我们提出了一种非接触微操作系统泡的技术, 利用局部钙离子梯度。在巨大的脂质囊的附近注射钙离子溶液, 用于重塑脂质膜, 导致膜管状突起的产生。

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...

membrane-remodeling-giant-vesicles-response-to-localized-calcium-ion

Research

JoVE Journal

Chemistry

7.9K 次观看.  Chalmers University of Technology. 我们提出了一种非接触微操作系统泡的技术, 利用局部钙离子梯度。在巨大的脂质囊的附近注射钙离子溶液, 用于重塑脂质膜, 导致膜管状突起的产生。

视频: Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

在膜上重构隔膜组件以研究生物物理性质和功能

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

科研

生物化学

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

活细胞钙成像相结合的siRNA介导的基因沉默标识CA 2 +泄漏通道内质网膜和监管机制

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

生物学

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

人工脂质纳米管网作为内质神经的底部模型的自发形成与重排

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

生物工程

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

膜靶向 ca 2 + 指标对培养细胞中局部 Ca2 +信号的解剖

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

神经科学

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

直接成像诱导ER钙有针对性的酯酶染料装载（TED）

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

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 ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

跨膜蛋白的重组，电压门控离子通道，KvAP，进入巨人单层囊的显微镜和膜片钳研究

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

从巨 Unilamellar 泡中提取膜纳米管

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Pinching-off of Coated Vesicles

Vesicle budding is orchestrated by distinct cytosolic proteins such as adaptor proteins, coat proteins, and GTPases. To initiate vesicle budding, membrane-bending proteins containing crescent-shaped BAR domains bind to the lipid heads in the bilayer and distort the membrane to form a protein-coated vesicle bud. Adaptors proteins such as AP2 for clathrin-coated vesicles can nucleate on the deformed membrane. Finally, coat proteins such as clathrin or COPI and COPII assemble into a coat forming the coated vesicle.
COP-coated vesicles bud off their organelle membrane with the help of several small GTPases such as SEC, SAR, and ARF proteins. On the other hand, Clathrin-coated vesicles require additional proteins, the most important of which is Dynamin. Dynamin is a GTPase of about 100-kDa that assembles as helical spirals at the necks of vesicle buds. It undergoes a GTP hydrolysis-driven conformational change that results in a twisting motion, which detaches the clathrin-coated vesicle from the membrane.
Once a vesicle buds, it immediately sheds its coat. The energy required for uncoating the clathrin coat is derived through ATP hydrolysis by Hsp70 chaperone protein, an ATPase activated by a co-chaperone named auxin. Auxilin binds Hsp70 and guides it to appropriate locations on the clathrin lattice to stimulate ATP hydrolysis. Auxilin binding creates a detectable distortion in the clathrin coat. It simultaneously contacts another clathrin at its terminal domain, recruiting more Hsc70 to the neighborhood, thus uncoating the entire vesicle. Tight control of vesicular coat formation and shedding facilitates the timing of vesicle budding and fusion with the target membrane.

包被囊泡的夹断

Vesicle budding is orchestrated by distinct cytosolic proteins such as adaptor proteins, coat proteins, and GTPases. To initiate vesicle budding, ...

教育

JoVE Core

细胞生物学

Unit 1

Intracellular Membrane Traffic

关键术语与概念

Fusion of Secretory Vesicles with the Plasma Membrane

Proteins and neurotransmitters in secretory vesicles can be released from a cell upon vesicle docking, priming, and fusion with the plasma membrane. Vesicles are docked and primed in preparation for the quick exocytosis of their contents in response to a stimulus. The fusion process is mainly carried out by a SNAP Receptor or SNARE complex, consisting of synaptobrevin, syntaxin-1, and SNAP-25.
In 1993, Jim Rothman proposed that the antiparallel pairing of vesicular and transmembrane SNAREs, or v- and t-SNAREs, was essential for vesicle docking. However, more recently, it has been shown that vesicle docking can also occur without SNAREs. Additionally, two soluble proteins,&#160; N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAP), bind to the SNARE complex to facilitate fusion.
The v-and t-SNAREs partially fuse in the priming process, forming a fusion-ready state. The protein complexin (Cpx) clamps the SNAREs and holds the vesicles in this partially fused state to prevent premature exocytosis. Vesicular membrane fusion begins when a stimulus, such as an action potential at the axonal terminal of a neuron, opens up a calcium channel, and calcium enters the cell. A total of five calcium ions bind to each synaptotagmin (Syt) &#8211; a vesicular membrane protein on either side of the vesicle. Calcium-bound Syt releases the Cpx clamp from the SNARE complexes and opens up the fusion pore to release the neurotransmitter. After fusion, NSF and SNAP proteins disassemble SNARE complexes for recycling.
Bacterial neurotoxins, such as botulinum from Clostridium botulinum or tetanus from Clostridium tetani, can inhibit secretory vesicle fusion by damaging the SNARE proteins, which prevents the fusion of secretory vesicles with the neuronal plasma membrane. As neurotransmitters are not released, action potentials are not generated, causing paralysis of muscles, and in some cases, death.

分泌囊泡与质膜的融合

Proteins and neurotransmitters in secretory vesicles can be released from a cell upon vesicle docking, priming, and fusion with the plasma membrane. ...

局部钙离子梯度对大泡囊膜重塑的响应

摘要

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