这项工作是 MaxSynBio 财团的一部分, 由德国联邦教育和研究部和马克斯·普朗克协会联合出资。

作者没有什么可透露的。

对称和非对称盐条件下的相态观测 GUVs 的成功生产
这里提出的协议介绍了一种策略来评估盐缓冲和溶液不对称对膜相状态的荷电 GUVs 在广泛的组合物的影响。实现这一目标的主要挑战之一是在盐缓冲区中生产带电的 GUVs。
我们成功地生产了 GUVs 在蔗糖溶液和 high-saline 缓冲的简单自发肿胀方法, 其中包括 pre-hydration 步骤的沉积脂质膜和隔夜最后水化步骤的囊泡生长。重要的是要注意, 脂质沉积应在粗糙的聚四氟乙烯板, 以确保均匀的脂质传播, 以产生 unilamellar 泡。此外, 它是必不可少的执行每一个步骤, 在囊泡准备在一个温度下, 脂质膜是在一个均匀和液相状态。另外, 囊泡的数量可能是多的, 并对最终的种群阶段状态分析产生偏见。GUVs 自发肿胀的具体协议产生一个囊泡丛, 它一方面提供了 re-suspend 小体积以获得高度浓缩的囊泡分散体的可能性, 另一方面提供了不对称的溶液通过膜的条件, 同时尽量减少泡的稀释8,28。重要的是, 在囊泡稀释或外部溶液交换, 内外 osmolarities 保持匹配的形态学变化引起的渗透不匹配可能诱发或防止 Lo + Ld 相分离41或, 在低渗的情况下条件, 可能导致囊泡爆裂。
在这里, 试图优化 electroformation 协议在盐的解决方案导致生产无 GUVs, 而 PVA 辅助肿胀产生多层泡。尽管它需要更长的准备时间和结果在小泡批次的低质量10,17, 成功生产的带电泡由自发肿胀带来了额外的优势。它要求最小的努力, 而导致足够的收益率统计批次分析, 不像 electroformation, 不需要先进的设备或优化。此外, 没有任何污染物的脂质氧化已观察到42,43。根据文献, 囊泡的脂质组成与它们生长的相应种群之间没有差异7,17。此外, PTFE 衬底上的泡形成不提示包含任何污染物与凝胶辅助肿胀方法, 在这种情况下, 外分子可能由基底23引入。Electroformation 在创造不对称的溶液条件时会伴随着过多的囊泡稀释而产生更多的缺陷。GUVs 产生的 electroformation 通常是作为一个均匀的分散 (与高度集中的囊泡悬浮在自发肿胀形成的形式形成对比)。任何稀释的外部溶液也会极大地稀释囊泡的数量。此外, 在这项工作的过程中观察到, electroformation 在蔗糖中产生的 DOPG/eSM/GUVs 在盐缓冲液中稀释后变得不稳定。显微镜下的脂质片上的荧光表明, 囊泡在视力检查之前会破裂。这种不稳定性可归因于 electroformation 制备的囊泡膜张力升高, 与自发肿胀的10相比。
虽然囊泡稀释是一种简单而快速的方法来创建不对称的老爹解决方案条件, 它只完成了部分外部解决方案交换, 虽然是一个很高的分数 (这里: 95%,图 2), 作为稀释后的痕迹肿胀解决方案将保持。外部溶液交换的程度的选择是在移与肿胀溶液 (2 节) 之间的平衡, 而不是稀释太多。因此, 我们介绍了一种替代的微流控方法, 在其他地方详细讨论了37 , 允许在老大阶段状态观测期间进行快速和完整的外部解决方案交换, 以验证为稀释而进行的相位状态观测泡.在从对称到非对称溶液条件的变化时, 观测到的相位状态变化确实是一致的。此外, 这两种生成非对称解决方案条件的方法都是比较快速的 (对比 Ref.8), 并没有任何已知的本地组合更改风险 (比较引用30、31),增加膜张力 (比较参考32), 或局部加热 (比较参考33), 至于介绍中讨论的替代方法。在微流体捕获过程中, 囊泡内部与外部的渗透平衡不仅是避免上述阶段状态伪影的必要条件, 而且是高渗溶液条件引起的通货紧缩, 可能导致被困 GUVs 滑倒通过外部解决方案交换后的帖子。
即使自发肿胀已成功地应用于生长不泡从 DOPC/eSM/系统, 在其他情况下, 没有收费可能会损害老爹肿胀, 由于导致缺乏排斥之间的个人双层44.延长 pre-swelling 期或引入笨重的脂质 headgroups 可能会解决此问题45。此外, 在不同于肿胀的溶液中, 其稀释后的囊泡稳定性可能因不同的脂质成分和稀释介质而不同, 这是我们没有研究过的。我们也没有探索的可能性, 调整的平均老大直径与准备方法在这里提出。但囊泡成分和溶胀液等参数可能影响结果。替代方法的应用46可能会产生更大的脂肪泡和在这里使用的解决方案, 但是, 它们可能会带来与该方法相关的其他缺点。在对称和非对称盐条件下产生 GUVs 的上述方法为进一步研究由不同的脂质组成和分散在不同的培养基中的囊泡提供了一个潜在的工具。由于我们没有探索这些可能性, 未来的试验将显示如何一般的老爹准备和稀释方法可以应用。
在不同温度下观察相分离
不同的实验设置适合研究 GUVs 在不同的温度下存在。虽然这些设置在文献中并不经常被详细描述, 但当前的工作呈现了一个适用于此类研究的基本组件。
_content ">> 控制测量表明, 该 in-house 设计和建造室的温度精确控制的恒温器和温度梯度内的会议厅是在实验温度的决议。确保实验热条件与恒温器读数一致。
在广泛的温度范围内对老大相状态进行评估时, 观察到的泡在温度变化后平衡很重要。一个可能的方法, 以确保这是检查滞后。如果存在滞后, 则应降低温度步骤并/或平衡时间增加。由于温度控制在这工作由一个水基恒温器建立, 工作温度的范围理想地被限制到 0-100 ° c。这个范围可以通过使用其他温度控制液体, 如油或通过其他设置,例如一个珀尔贴设备扩展。在实践中, 工作温度也受到可能的凝结或蒸发的限制。此外, 对于远离室温的温度, 整个观察室的稳态温度梯度的发生变得更有可能。此外, 成像设备可能会在极端温度下受到伤害。为典型的温度范围适当为脂囊研究 (~ 10-50 ° c7,9) 对观察设备的损坏应考虑, 但通常不预期。
囊泡领域观察工件
有许多来源的观察文物使用广域荧光显微镜。首先, 我们应该知道, 应用于囊泡相状态目视检查的对象的最大分辨率r决定了脂域的检测限度, 其依据是: 
<img alt="Equation 2" src="/files/ftp_upload/56034/56034eq2v2.jpg"> 其中, λ是发射波长, NA 是目标的数字光圈。一个典型的目标与40x 放大率和 NA 的 0.6, 检测绿色发射光约 560 nm 将达到0.6 µm 的光学分辨率. 因此, 比较不同的特定脂质混合物的囊泡相状态的研究对于相同的脂质混合物, 条件应使用相同的目标。
另一个工件是脂质领域的发生, 由于脂质光氧化的结果, 由于延长暴露于激发光41。光损伤优先于不饱和烃类脂基。事实上, 对于某些脂类成分, 在经过长时间的激发光照射 (~ 三十年代) 之后, 在这里观察到了最初均匀泡的区域形成。为了解决这个问题, 励磁灯被集中在一个视野, 仅几秒钟的时间状态评估。因此, DiIC18适用于我们的目的。然而, 其他染料, 可能会更敏感, 可能需要在较低的激发强度和更短的励磁光照射时间处理。
机械剪切应力从吸管转移的囊泡可能暂时混合领域, 从而扭曲明显的囊泡相行为。在某些批次中, 不同的囊泡在吸管转移到显微镜片后表现出不同的相行为0分钟和5分钟。在微流控装置中流体流动引起的剪切应力也被证明在域混合47。在观察之前, 囊泡应该被不受干扰地留出足够的时间平衡。在这项研究中, 被困在微流控装置上的囊泡在观察前的囊泡加载和溶液交换后未受干扰1小时。
为了避免一些 above-mentioned 的困难以及光衍射极限施加的限制, 诸如核磁共振光谱学48或超分辨率显微镜技术的替代方法49可以使用。
结论与展望
所介绍的工作演示了一套方法, 允许分析盐对称和非对称溶液条件对膜相分离的影响。所提出的方法都适用于其他应用。以微流控器件为例, 给出了在溶液不对称诱导下的区域形成和消失动力学的研究平台。此外, 领域的外观作为一个盐浓度的功能可以检查的方式。所有的方法也可以用来观察对相位行为的影响使用任何其他的解决方案的利益。

自从用荧光显微镜观察液液相分离巨 unilamellar 泡 (GUVs) 中的微米畴后, GUVs 已被用作研究脂筏假说的模型系统1,2,3. 由于它们的独立双层的区域位于从生物细胞的范围内, 它们是适合假设的筏的等离子体膜的模拟。大量的研究对这种 GUVs 已进行了泡在纯净水, 蔗糖或盐分解决方案4,5,6,7,8。然而, 这些条件并不反映生物膜对盐环境和反式膜溶液不对称性的生理相关暴露, 这是细胞的条件。
在这项工作和在我们的小组的早先出版物9, 被审查的被充电的多组分 GUVs 的阶段状态作为盐和解答不对称的存在的作用在细胞膜。GUVs 是从不同比例的 dioleoylphosphatidylglycerol (DOPG), 鸡蛋磷脂 (eSM), 和胆固醇 (在蔗糖溶液 (与渗透 210 mOsm/千克) 或盐缓冲 (100 毫米氯化钠, 10 毫米三, pH 7.5, 210 的混合物制备。mOsm/千克)。脂质选择由在这个混合物的相图已经获得的数据辩解了6,8。
在文献10,11,12 (注意, 在这里, 我们将不考虑那些涉及脂质从油基转移到水相的方法)。13,14因为膜中剩余的油残留物可能会影响相行为的固有危险。盐缓冲区中 GUVs 的制备与特定的挑战有关。对于低离子强度的解决方案, electroformation 方法15,16提供了一种快速的方法来准备 GUVs 的高收益率和小缺陷10,17。该方法的基础是在导电表面 (电极) 上沉积一层脂质, 将其烘干, 并在应用交流电场时用水溶液对其进行保湿。但是, 如果盐水存在于水溶液18,19中, 则此方法需要进行调整。假定 electroformation 的泡生长驱动力为电16 , 在高电导率20中受阻。因此, GUVs 在盐溶液中的 electroswelling 不是一个简单的方法, 因为它需要对溶胀溶液中存在的不同盐浓度进行优化。凝胶辅助囊泡肿胀21,22是一个潜在的替代 electroformation 与更快速的形成时间。这种方法建立在增强的脂质膜水合时, 凝胶 (琼脂糖或聚乙烯醇 (PVA)) 作为基板使用。然而, 这些方法, 在 agarose-based 肿胀23和/或温度限制的情况下, 出现膜污染的风险, 如 PVA-based 肿胀。同样, 在纤维素纸基材上生长 GUVs 的协议最近已经建立了24。该方法的一般问题是缺乏对基质纯度的控制以及大量的脂质的使用。在这项工作中, 我们将介绍和提出的优势, 最传统的方法为老爹准备, 即自发肿胀方法25,26。它包括在拒水基板上干燥一个脂质层, 在水汽的大气中使其水化, 并在所需的溶胀溶液中随后膨胀 (请参见图 1和协议部分中的详细信息)。这种方法不提供控制的囊泡大小分布和结果的整体较小的小泡相比, 方法的生产是辅助的电场, 聚合物基板或微流控手段。然而, 囊泡的质量和大小是适当的检查膜相状态的探讨这里。
在囊泡膜的溶液之间产生不对称也与某些挑战有关。一个常用的方法是直接稀释泡悬浮在所需的外部解决方案27,28。然而, 这也减少了囊泡分布密度。另一种策略是在流单元的底部缓慢地交换外部解决方案, GUVs 可以解决 in-and outflux。为了避免干扰或甚至失去流动的小泡, 低流速应用了8, 这使得这种方法 time-inefficient。此外, 这两种方法都不能保证完全的外部解决方案交换。一个明显的解决办法是固定囊泡以避免在外部溶液交换过程中失去它们。例如, 化 GUVs 可以被拴在一个亲和涂层的表面29上。然而, 这种方法可能导致在粘附的和因此 non-adhered 膜段30,31的成分变化。磁场或电场在陷阱泡中的应用导致膜张力32。使用光镊诱捕囊泡需要有一个手柄附加 (即珠), 而光学担架的用途可能涉及局部加热33。GUVs 的捕获也可以通过在没有最终剥离34的情况下在铂线上生长来实现。然而这导致囊泡没有被隔绝, 并且通常连接到导线或其他小泡由稀薄的油脂管 (系)。
所介绍的工作突出了克服上述限制的战略。我们首先介绍了自适应和优化的自发肿胀法的 GUVs 在盐缓冲区生产的详细描述。然后, 我们引入了两种方法, 通过简单的稀释或利用微流体设备来有效地创建非对称的溶液条件。由于我们的目标是分析 GUVs 在不同的溶液条件下的膜相状态, 随后的章节描述了成功的统计分析的标准, 并提出了避免错误分类的提示。
在等温条件下以及在不同温度下进行了分析。温度 c控制是普遍使用的, 关于实验温度控制室的细节很少被描述。在这里, 一个 in-house 建立在不同温度条件下观察 GUVs 的设置。

<table><tbody><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phospho-(1&#039;-rac-glycerol), sodium salt</td><td>Avanti Polar Lipids</td><td>840475C</td><td>abbreviated as DOPG in the text</td></tr><tr><td>chicken egg sphingomyelin</td><td>Avanti Polar Lipids</td><td>860061</td><td>abbreviated as eSM</td></tr><tr><td>cholesterol (ovine wool, &gt; 98 %)</td><td>Avanti Polar Lipids</td><td>700000</td><td>abbreviated as Chol</td></tr><tr><td>1,1&#039;-dioctadecyl-3,3,3&#039;,3&#039;-tetramethylindocarbocyanine perchlorate</td><td>Molecular Probes</td><td>D-282</td><td>abbreviated as DiIC&lt;sub&gt;18&lt;/sub&gt;</td></tr><tr><td>Chloroform, HPLC grade (&amp;ge; 99.8 %)</td><td>Merck</td><td></td><td></td></tr><tr><td>NaCl (&gt; 99.8 %)</td><td>Roth</td><td></td><td></td></tr><tr><td>HCl (37 %)</td><td>Roth</td><td></td><td></td></tr><tr><td>Tris (&amp;ge; 99.9 %)</td><td>Roth</td><td></td><td></td></tr><tr><td>Sucrose (&amp;ge; 99.5 %)</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Parafilm</td><td></td><td></td><td></td></tr><tr><td>Threaded vial 45x27 mm, 15 mL</td><td>Kimble</td><td></td><td>Soda flat bottom, white screw cap</td></tr><tr><td>pH meter</td><td>Mettler Toledo</td><td>MP220</td><td></td></tr><tr><td>Osmometer</td><td>Gonotec</td><td>Osmomat030</td><td></td></tr><tr><td>Epi-fluorescence microscope</td><td>Zeiss</td><td>Axio Observer D1</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Leica</td><td>TCS SP5</td><td></td></tr><tr><td>Objective 40x, 0.6 NA</td><td>Zeiss</td><td>LD Achroplan</td><td></td></tr><tr><td>Objective 40x, 0.75 NA</td><td>Leica</td><td>506174</td><td></td></tr><tr><td>Objective 63x, 0.9 NA</td><td>Leica</td><td>506148</td><td></td></tr><tr><td>Microscope slide, 56x26 mm, 0.17 &amp;plusmn; 0.01 mm</td><td>Menzel-Gl&amp;auml;ser</td><td></td><td></td></tr><tr><td>Cover slip, 22x22 mm, 0.17 &amp;plusmn; 0.01 mm</td><td>Menzel-Gl&amp;auml;aser</td><td></td><td></td></tr><tr><td>Parafilm &quot;M&quot;</td><td>Bremix Flexible Packaging</td><td></td><td></td></tr><tr><td>Syringes, 5 mL, 10 mL</td><td>Braun</td><td></td><td></td></tr><tr><td>0.45 &amp;micro;m syringe filter</td><td>GVS North America</td><td>Cameo 25AS, 1213723</td><td>Acetate, sterile</td></tr></tbody></table>

phase behavior of charged vesicles under symmetric and asymmetric solution conditions monitored with fluorescence microscopy

相分离的巨型 unilamellar 囊泡 (GUVs) 表现为共存的液序和液体无序域, 是研究脂筏假说的常用生物物理工具。然而, 许多研究忽视了生理解决条件的影响。在该帐户上, 目前的工作提出了盐缓冲和反式膜溶液不对称的影响, 液液相分离的 GUVs 生长从 dioleylphosphatidylglycerol, 鸡蛋磷脂, 胆固醇。研究了等温和不同温度条件下的影响。
本文介绍了在对称和非对称盐溶液条件下, 适用于监测带电泡中共存液域稳定性的设备和实验策略。这包括在高温盐缓冲液中制备带电的多组分 GUVs 的方法。该协议要求在最小化囊泡稀释的同时, 通过一个简单的稀释步骤来进行局部交换的外部溶液的选择。另一种方法是利用微流体设备, 允许一个完整的外部解决方案交换。在不同温度下, 对相分离的溶液效应也进行了研究。为此, 我们提出了一个 in-house 内置温度控制室的基本设计和实用。此外, 我们还考虑了对老爹阶段状态的评估、与之相关的陷阱以及如何绕过它们。

老爹肿胀
在这里描述的自发肿胀的方法, GUVs 组成的 DOPG, eSM, 并在一夜之间生长210毫米蔗糖或100毫米氯化钠, 10 毫米三, pH 7.5 形成一个可见的总和。收获的总和确保高泡的产量。悬浮在溶胀溶液中产生了对称的反式膜溶液条件。为了创建非对称条件, 聚合体分别在 iso-渗蔗糖或盐溶液中悬浮 (图 2)。由此产生的稀释对应于一个 quasi-external 的溶液交换, 同时尽量减少 GUVs 的稀释次数。
用荧光显微镜研究 GUVs 的相图映射
在 GUVs 中存在0.1 摩尔的相位特异性 DiIC18 , 可以通过广域荧光显微镜观察其相状态。通过 63 x/1.2NA 观察到的囊泡表现为 S + L 相分离, 能够解决结构精细的手指状区域。对于所有剩余的病例, 使用了 40 x/0.6 NA 目标。为了避免工件在 GUVs 的视觉检查和最大限度的再现性, 确定了一些标准, 决定了哪些囊泡要考虑的阶段状态分析 (协议步骤 3.6)。
GUVs 制备的三元 DOPG/eSM/与广泛的比例在蔗糖或盐溶液中肿胀的混合物, 在室温下观察到均匀的 lo 或 ld 阶段, lo + ld, 和 S + L 相分离, 请参见图 3。 图 9显示 exemplarily 缺陷的囊泡, 不应包括在数据分析中, 也显示了如何识别多层囊泡。
由于其未知的历史, GUVs 很可能会出现在批次成分变化35。因此, 在统计方法中确定了某一特定成分的整体阶段状态。某种脂质组成的老大群被归因于在一个随机抽样中被观察到占主导地位的相位状态 (协议步骤 3.6)。然而, 靠近 Lo + Ld 共存区的组合物通常会产生批次, 而占主导地位的相状态占少数。对于这种模糊的情况, 目视检查重复了至少三独立样本。在随机样本中, 具有相同相位状态 (如显示 Lo + Ld 相分离) 的小泡的分数平均超过了作为最终结果 (图 10) 的试验次数。
所描述的协议导致在 DOPG、eSM 和盐和蔗糖溶液中的不同比例的大范围内有足够的老大生长。三元相图中的广泛区域可以在对称和非对称盐解条件 (图 11) 下映射。在不同的溶液条件下观察到的囊泡相行为的差异在其他地方详细讨论了9。
用微流控法进行完全缓冲交换后的相行为观察
通过稀释产生不对称溶液的条件会导致外溶胀液的残留。我们的微流控方法允许一个完整的外部解决方案交换。图 5A显示了在共聚焦显微镜舞台上与流体出口和压力控制入口完全组装在一起的微流控设备。管子通过90°金属管子连接, 允许空间从上面传送光成像。
在微流控装置内进行观察, 采用 63 x/1.2NA 水浸物镜的共聚焦显微镜。与以前的观察散装, DiIC18被用来染色的膜。在对被困在装置中的 GUVs 的相状态进行观察时, 必须注意不要曲解数据。由于 GUVs 与柱子的接近, 励磁和发射光路可能被部分阻碍由由的被导致错误出现的领域 (图 12)。在这里, 透射光检测是有用的检查的位置, GUVs 在员额。这种不必要的影响是特别突出的小 GUVs 小于10µm 直径。在这些情况下, 数据被拒绝。在图 13A中的共焦图像显示了一个平面的泡状部分, 它横跨在老爹泡上的 Lo 域上。在这种情况下, 平面剖面扫描进一步高于或低于该节将没有显示的领域, 因为它的小规模相比, 老大, 这就是为什么它会被忽略和囊泡被认为是在同一阶段的状态。因此, 应该使用一个共焦 z 栈来检查整个老爹的表面。对于广域显微镜, 这可能不是一个问题, 因为整个囊泡可以立即成像。
最后, 给出了 GUVs 在外部解的交换之前和之后的例子, 其中清楚地说明了膜的相状态 (图 14)。每台设备有60个腔室 (每台都有一对柱子来诱捕单个的老大), 每台设备允许进行数十次实验 (参见图 4)。但是, 一些 GUVs 可能会在外部解决方案交换过程中丢失。这可以通过以下步骤最小化: 1) 使用牛血清白蛋白 (BSA) 涂层, 以防止在柱子上的老爹粘连/破裂;涂层是通过将腔壁暴露给20毫克/毫升 BSA 进行60分钟的, 随后用工作缓冲器冲洗。另一个可以使用的粘合剂分子是聚 (l-赖氨酸)-接枝聚 (乙二醇)39。2) 仔细的渗透匹配的内外解决方案, 以避免弛缓性 GUVs, 这可能会通过中心的职位或爆裂的 GUVs。3) 优化老爹的准备程序, 以获得直径大于 ~ 8 µm 的囊泡, 以防止通过柱子中心的通道。
温度控制室的设计与表征
我们设计了一个简单的流室, 它具有与外部水温调节器的连接 (图 6)。我们获得了由铝块铣削的室。一般而言, 室内材料不需要热传导, 因为样品是耦合到水浴由较低的盖玻璃, 如图所示, 6B和6C。
独立于精确的设计, 应评估分庭的性能。具体地说, 我们检查了样品温度的线性度与 externally-set 温度, 任何系统的温度偏移量, 并在会议厅内的温度梯度。前两点通过光纤温度探头 (例如FISO FTI-10) 在燃烧室内进行直接温度测量来解决。我们还检查了一个温度梯度在老大悬挂。这种梯度可以从热量流向腔室的外部。空间分辨温度测量可以通过温度敏感荧光40获得, 请参阅图 7。
用于获取相分离的 GUVs 的T混合的数据绘制和拟合
在观测到的温度范围内, 绘制均匀 GUVs 的分数, 形成 sigmoidally 形状的数据点轨迹。我们将数据与玻尔兹曼模型 (协议节 6.8) 相匹配, 从中可以推断出T混合的(图 8)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56034/56034fig1.jpg"> 
图 1: 自发肿胀协议 (顶部面板) 中的实验步骤 (下面板) 与相应的囊泡生长阶段.(a) 均匀的脂质膜分布在粗糙的聚四氟乙烯板上, 并从任何溶剂中干燥。(B) 干燥的脂质膜在封闭容器内的水气氛中 pre-swollen, 以促进双层水化。空玻璃瓶包含 PTFE 板, 并保持开放在这个水化步骤。(C) pre-swollen 脂膜最终通过将所需的溶胀液添加到玻璃小瓶内的脂涂层 PTFE 板上而完全水合。为了避免蒸发, 玻璃瓶在夜间孵化过程中被正确密封。为了使批次中的成分变化最小化, 所有的步骤都需要在脂质混合物完全混溶的温度下进行。<a href="//ecsource.jove.com/files/ftp_upload/56034/56034fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56034/56034fig2v2.jpg"> 
图 2: 老大收割.(a) 一个沉积的脂质膜, 由 DOPG/eSM/在摩尔比率为 20/60/20 与0.1 摩尔 DiIC18是肿胀的盐缓冲组成的100毫米氯化钠, 10 毫米三, pH 7.5。玻璃容器的盖子另外被密封了与石蜡影片。放大的感兴趣区域包含所产生的老爹集合。它的红色外观是 DiIC18存在的结果。(B) GUVs 是用截断的吸管尖端收割的。在这张图中, 聚合体与50µL 溶胀液一起 pipetted, 并被转移到一个新鲜的瓶子里。(C) 创建不对称的反式膜溶液条件时, 聚合体被稀释在另一个等渗外部溶液中。在这里, 聚合连同50µL 溶胀溶液是悬浮在950µL 蔗糖溶液, 导致20x 稀释溶胀溶液。<a href="//ecsource.jove.com/files/ftp_upload/56034/56034fig2large.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/56034/56034fig3v2.jpg"> 
图 3: 老爹影像.GUVs 掺杂0.1 摩尔 DiIC18的广域荧光图像在对称盐或蔗糖溶液中制备和观察, 由 DOPG、eSM 和正离子的不同比例 (在盐缓冲中, 从左到右:40/20/40; 50/20/30; 30/60/10;蔗糖, 从左到右:30/30/40;20/60/20;40/50/10)。图像描述 GUVs 在不同的 (共存) 阶段状态根据标准在协议步骤3.6 评估。在这里, 囊泡通过 40 x/0.6 NA 目标成像。缩放栏 = 5 µm.<a href="//ecsource.jove.com/files/ftp_upload/56034/56034fig3large.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/56034/56034fig4.jpg"> 
图 4: 微流控通道设计的布局.GUVs 通过储层下方的入口进入较低的射流层 (轮廓通道)。过滤器阻止设备中不需要的碎片。然后, 他们输入一个60室 (8 行和15列) 的数组, 每个包含一个单老大捕获的帖子 (参见 insert)。每个腔室都可以被隔离在一个控制层 (填充黑色通道) 驱动的环形阀内的流体层之上。<a href="//ecsource.jove.com/files/ftp_upload/56034/56034fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56034/56034fig5.jpg"> 
图 5: 微流体设备(a) 用于捕获单个 GUVs 的微流体设备的照片, 并完全交换外部解决方案。标签指示水库添加解决方案 (1), 8 x 压力入口连接到压力控制单元 (2), 以及与注射器和泵 (3) 连接的流体出口。面板 (B) 显示的压力控制单元, 每个调节1管连接到微流控装置的8阀。在这里阀门 #1 和 #2 是开放的, 因此相应的环形阀门是闭合的。<a href="//ecsource.jove.com/files/ftp_upload/56034/56034fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56034/56034fig6v2.jpg"> 
图 6: 温度控制室.(A) 分庭在程序集之前。(B) 组装室 (正面朝上), 2 毫米的盖子玻璃粘在顶部和底部。橙色橡胶间隔测量0.5 毫米的高度和密封的0.17 毫米的盖子滑动, 以观察封闭的老大悬挂。在这个图象温度探针入为定标目的 ( 褐色纤维退出房间在右边 ) 。(C) 在无温度探头的倒置显微镜的舞台上进行最终组装。橙色橡胶垫片现在正向下。橡胶是足够的粘合剂来保持样品到位。请注意, 光可以通过样品进行传输, 同时能进行 epi 荧光和明亮的野外观察。<a href="//ecsource.jove.com/files/ftp_upload/56034/56034fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56034/56034fig7v2.jpg"> 
图 7: 温度敏感染料的电影测量所获得的观察室内的空间解析温度数据(这里: 500 µM 罗丹明 B)40. 在底部盖板上方的0、10、30、300和400µm 获得的数据点。分别对30° c 和12° c 设置的水浴温度测量了红色和蓝色数据点。黑色数据点表明, 水浴左到平衡到室温。整个会议厅的温度变化较小, 但保持在0.5 ° c 以下 (灰色条)。总房间高度是大约500µm 根据间隔厚度 (参见上述)。误差线表示标准偏差。<a href="//ecsource.jove.com/files/ftp_upload/56034/56034fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56034/56034fig8v2.jpg"> 
图 8: 查找混溶温度.该图显示了同质 (single-liquid 状态) 的单个数据点, 从 DOPG/eSM/GUVs 的比例 30/40/30 掺杂与0.1 摩尔 DiIC18从三独立随机抽样 (N = 20-40) 的分数。误差线表示标准的方法误差。数据点安装在步骤 6.8 (连续黑线) 中描述的玻尔兹曼模型上, 根据 sigmoidal 的半最大值推导出域混合温度T混合(按照红色连续线横坐标)。曲线在纵坐标上 (虚线黑线)。初始和最终值 (1和2) 分别固定为0和1。<a href="//ecsource.jove.com/files/ftp_upload/56034/56034fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56034/56034fig9v2.jpg"> 
图 9: 有缺陷的囊泡.例如, 从 20/60/20 DOPG/eSM/摩尔, 如果 DiIC18不符合在宽场荧光显微镜成像的步骤4.2 中设置的标准, 则为其准备的巨泡的例子。每一次图像的强度显示范围都是针对所描述囊泡的荧光强度而优化的。由于存在额外的膜材料和封装小泡, 在 (A) 中的囊泡的相状态不能被清楚地定义。这种巨型泡的外观不允许对 lamellarity 进行任何可靠的目视检查。面板 (B) 描述了一个泡, 其中的内部挤满了膜材料。因此, 外囊泡膜的荧光信号与其内部信号叠加, 使 lamellarity 和潜在域的可视化成为不可能。(C) 三不同巨泡的荧光强度在同一显示强度范围内直接比较显示。这个图像显示, 巨大的多层泡 (1, 2) 可以识别其增加的荧光信号比老大 (3)。在这里, 多层以及 unilamellar 巨泡展示了 Lo + Ld 相分离。所有图像中的囊泡都是通过 40 x/0.6 NA 目标成像的。缩放栏 = 5 µm.<a href="//ecsource.jove.com/files/ftp_upload/56034/56034fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56034/56034fig10v2.jpg"> 
图 10: Lo + Ld 相分离的 GUVs 在至少三独立样本上的平均分数.囊泡由 30/40/30 DOPG/eSM/近距离边界的 Lo + Ld 共存区组成。对称蔗糖条件的误差条说明了批次散射。纵坐标的标签描述囊泡内/外的溶液;蔗糖: 210 mM 蔗糖;盐: 100 毫米氯化钠, 10 毫米三, pH 7.5;误差线表示标准偏差。<a href="//ecsource.jove.com/files/ftp_upload/56034/56034fig10large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/56034/56034fig11.jpg"> 
图 11: 使用 210 mm 蔗糖 (蔗糖) 和 100 mm 氯化钠、10 mm 三、pH 7.5 (盐) 在不同溶液条件下绘制的 DOPG、eSM 和 GUVs 制备的相图.在蔗糖/蔗糖 (A中探讨了老大相状态; 上部部分), 蔗糖/盐 (入/出) (A; 下部多边形剖面), 盐/盐 (B; 上部), 和盐/蔗糖 (b; 下多边形部分)。这些卡通在突出显示的部分和相应的解决方案条件中说明了主导域模式。在不对称的溶液条件下, 在20x 的老爹稀释下, 观察到低多边形剖面中的囊泡相状态。改编自引用9。<a href="//ecsource.jove.com/files/ftp_upload/56034/56034fig11large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/56034/56034fig12.jpg"> 
图 12: 被困 GUVs 中的工件.一个小老爹的例子, 一个虚假的领域, 可以看到由于接近与职位 (DOPG/eSM/60/20/20)。显示柱子的亮场透射光图像 (gr) 被覆盖的老爹 (橙色) 共焦荧光图像。这些柱子被视为在明亮的图像中产生干涉图案。荧光信号关闭的职位 (右) 减少给予相分离的外观。缩放条 = 5 µm。
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/56034/56034fig13.jpg"> 
图 13: 两个不同的 GUVs 的例子, 它们捕获的是在相位状态清晰可见的情况下.(A) DOPG/eSM/60/20/20 的 Lo-Ld 相分离泡(B) 由 40/30/30 的囊泡制成, 显示 Ld 或 Lo 单相。对整个囊泡的共焦 z 栈进行了检查, 以确认没有 (焦) 域存在。柱子的边缘在图片的右手边被看见 (灰色)。缩放条 = 5 µm。
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/56034/56034fig14v2.jpg"> 
图 14: 使用微流体设备进行完整的射流交换后产生的相位行为.相同的老大是在解决方案交换前和之后显示 (A) 对称盐/盐 (入/出) 到盐/蔗糖 (入/出) (DOPG/eSM/60/20/20, 比例条是2µm) 和 (B) 对称蔗糖/蔗糖 (入/出) 蔗糖/盐 (入/出) (DOPG/eSM/30/40/30, 刻度栏 = 3 µm)。改编自引用9。

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Phase Behavior of Charged Vesicles Under Symmetric and Asymmetric Solution Conditions Monitored with Fluorescence Microscopy

相分离巨 unilamellar 囊泡 (GUVs) 的实验常忽略生理溶液的条件。本文研究了盐缓冲液在荷电多组分 GUVs 中液体液相分离中的作用, 作为反式膜溶液不对称和温度的函数。

用荧光显微镜监测对称和非对称溶液条件下带电泡的相行为

Phase-separated giant unilamellar vesicles (GUVs) exhibiting coexisting liquid-ordered and liquid-disordered domains are a common biophysical tool to ...

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9.1K 次观看.  Max Planck Institute of Colloids and Interfaces. 相分离巨 unilamellar 囊泡 (GUVs) 的实验常忽略生理溶液的条件。本文研究了盐缓冲液在荷电多组分 GUVs 中液体液相分离中的作用, 作为反式膜溶液不对称和温度的函数。

视频: Phase Behavior of Charged Vesicles Under Symmetric and Asymmetric Solution Conditions Monitored with Fluorescence Microscopy

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

突触小泡池，利用苯乙烯染料的光转化的研究

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

科研

神经科学

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

电化学和荧光显微镜在质子泵膜酶研究中的单脂质体测定

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

生物化学

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

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

We present a technique for contactless micromanipulation of vesicles, using localized calcium ion gradients. Microinjection of a calcium ion solution, in the vicinity of a giant lipid vesicle, is utilized to remodel the lipid membrane, resulting in the production of membrane tubular protrusions.

局部钙离子梯度对大泡囊膜重塑的响应

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

化学

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

生物工程

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main common features required for this activity resulted from the interactions of CPPs with the plasma membrane (lipids) and more particularly with components of the extracellular matrix of the membrane itself (heparan sulphate). Indeed, independent of the direct translocation or the endocytosis-dependent internalization, lipid bilayers are involved in the internalization process both at the level of the plasma membrane and at the level of intracellular traffic (endosomal vesicles). In this article, we present a detailed protocol describing the different steps of a large unilamellar vesicles (LUVs) formulation, purification, characterization, and application in fluorescence leakage assay in order to detect possible CPP-membrane destabilization/interaction and to address their role in the internalization mechanism. LUVs with a lipid composition reflecting the plasma membrane content are generated in order to encapsulate both a fluorescent dye and a quencher. The addition of peptides in the extravesicular medium and the induction of peptide-membrane interactions on the LUVs might thus induce in a dose-dependent manner a significant increase in fluorescence revealing a leakage. Examples are provided here with the recently developed tryptophan (W)- and arginine (R)-rich Amphipathic Peptides (WRAPs), which showed a rapid and efficient siRNA delivery in various cell lines. Finally, the nature of these interactions and the affinity for lipids are discussed to understand and to improve the membrane translocation and/or the endosomal escape.

The fluorescence leakage assay is a simple method that enables the investigation of peptide/membrane interactions in order to understand their involvement in several biological processes and especially the ability of cell-penetrating peptides to disturb phospholipids bilayers during a direct cellular translocation process.

荧光泄漏测定研究细胞穿透肽对膜的破坏稳定性

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main ...

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An important first step in any membrane-dependent reaction is binding of protein on the phospholipid membrane. An approach to studying protein interaction with lipid membranes has been developed using fluorescently labeled proteins and flow cytometry. This method allows the study of protein-membrane interactions using live cells and natural or artificial phospholipid vesicles. The advantage of this method is the simplicity and availability of reagents and equipment. In this method, proteins are labeled using fluorescent dyes. However, both self-made and commercially available, fluorescently labeled proteins can be used. After conjugation with a fluorescent dye, the proteins are incubated with a source of the phospholipid membrane (microvesicles or cells), and the samples are analyzed by flow cytometry. The obtained data can be used to calculate the kinetic constants and equilibrium Kd. In addition, it is possible to estimate the approximate number of protein binding sites on the phospholipid membrane using special calibration beads.

Here, we describe a set of methods for characterizing the interaction of proteins with membranes of cells or microvesicles.

使用定量流式细胞术分析荧光标记蛋白与人工磷脂微泡的相互作用

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An ...

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤:细胞成像和数据分析

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

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 ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

巨脂质体制剂成像和膜片钳电

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...