这项工作是支持日本的上原纪念基金会（ES）以及白厅基金会，国立神经疾病与中风和斯坦 - 奥本海默基金会奖（BSK）。

实验步骤实验过程由两个关键部件，是明智的方式在一个步骤如下所述。 第1部分：准备海马星形胶质细胞的培养简单地说，混合海马星形胶质细胞神经元的文化准备使用一个完善的协议1,2,3。我们优化的过程，产生健康的培养的星形胶质细胞。下面列出的所有程序应在无菌层流罩，如环境进行。 准备盖玻片 <ul><li> 22毫米的盖玻片（VWR，48380-068） </li><li>聚- D -赖氨酸（PDL，Sigma公司P0899），分装（1毫克/毫升） </li><li>层粘连蛋白等分（20微克/毫升）：1毫克/毫升层粘连蛋白溶液（Sigma公司L2020），添加49毫升无菌水1.2毫升分装和储存在-20 ° C </li></ul><ol><li>在无菌水溶解的PDL（50微克/毫升） </li><li>高压灭菌的盖玻片，用无菌水，并在客运专线（100盖玻片20毫升）冲洗。在室温下离开过夜。 </li><li>用无菌水（3广泛洗）替换的PDL。然后干在4盖玻片和存储° C。 </li><li>一天前在无菌以及电镀的星形胶质细胞，个别地方盖玻片的6孔​​板（多孔平底板有盖无菌从屋宇署生物科学）。每个盖玻片放入400μL层粘连蛋白，在培养箱中孵育过夜，以避免蒸发。 </li></ol> 解剖 夹层媒体： <ul><li> 500毫升厄尔的平衡盐溶液（EBSS）（1X），液体（Invitrogen公司，14155-063） </li><li> 5毫升HEPES溶液（Sigma公司，H0887） </li></ul> 海马媒体： <ul><li> 412.5毫升的最低限度的基本培养基（MEM）（1X），液体中含有Earle的盐，但没有L -谷氨酰胺或酚红（Invitrogen公司，51200-038） </li><li> 10毫升的葡萄糖（Sigma公司，D -（+） - 葡萄糖无水SIGMA超G7528）在MEM 1米</li><li> 5 ml青霉素，链霉素（Invitrogen公司，15140-122） </li><li> 5毫升丙酮酸钠溶液（Sigma公司，S8636） </li><li> 12.5毫升的HEPES溶液1 M（Sigma公司，H0887） </li><li> 5毫升的N - 2补编（100X），液体（Invitrogen公司，17502-048） </li><li> 50毫升马血清，热灭活（Invitrogen公司，26050-088） </li></ul> 木瓜蛋白酶的解决方案： <ul><li>沃辛顿木瓜蛋白酶- 022瓶（LK003178;终浓度为20 U /毫升）中加入5毫升清扫媒体和孵育在37 ° C为60分钟。 </li></ul><ol><li>杀头P1（通常为2幼仔）按照国家法规和本地机构的动物护理和使用委员会的审核和批准的准则岁的幼鼠。 </li><li>取下的皮肤在培养皿和颅骨和地方的大脑充满了冷夹层媒体。 </li><li>删除脑膜，解剖两个半球的解剖海马。 </li><li>斩〜1X1毫米件和消化的木瓜蛋​​白酶溶液在37℃11-13分钟彗星的海马。 </li><li>一旦组织的相继落户，取出木瓜仔细，并添加5毫升海马介质的酶中删除所有痕迹。海马液（2毫升）重复此步骤和悬浮。 </li><li>直到有没有留下的团块逐渐变小的孔（1000微米〜500微米〜300微米）的三个火焰抛光移液器，磨碎的细胞〜5倍。 </li><li>磨碎后，通过一个细胞过滤器（孔径70微米，BD生物科学）的细胞。计数在10μL悬浮细胞。调整海马中等音量，以有40万细胞/ ml。 </li><li>吸过的盖玻片层粘连蛋白，前盖可以干，板每盖玻片的细胞悬液200μL。 </li><li>他们重视孵化器在60分钟，然后加入2毫升每口井的海马中等许可。 </li><li>第二天，吸老海马介质，去除死细胞和碎片，加2毫升预热新鲜海马中等。 </li></ol> 维修 Neurobasal媒体： <ul><li> 500毫升neurobasal（Invitrogen公司，12348-017） </li><li> 10毫升B27的无血清的补充（Invitrogen公司，17504-044） </li><li> 5 ml青霉素，链霉素（Invitrogen公司，15140-122） </li><li> 1.25毫升L -谷氨酰胺（Invitrogen公司，25030-149） </li></ul>每周两次的星形胶质细胞神经​​元文化与neurobasal中型饲料，电镀后开始的4天。 Preincubate通风烧瓶中的孵化器约30分钟的媒体，以平衡的温度和CO 2。 第2部分：钙成像海马记录缓冲液：氯化钠110毫米，5.4毫米氯化钾， 氯化钙 1.8毫米，0.8毫米MgCl 2的 D -葡萄糖，10毫米，10毫米的HEPES（Sigma的所有化学品），pH值7.4（用NaOH调整）。 装入星形胶质细胞钙指示剂 <ol><li>广场上海马2毫升含2.5微米荧光4，AM（Invitrogen公司的F - 14217）和0.05％聚醚®在DMSO溶液中的F - 127 20％的记录缓冲区充满了6孔板（Invitrogen公司在一个良好的文化， P - 3000MP）和孵育室温为10-30分钟。 </li><li>删除FLUO - 4解决方案和洗盖玻片海马记录的缓冲区，并在室温30分钟的孵育3倍。 </li><li>广场上腔盖玻片，用镜头清洁液和清洁盖玻片的另一侧。 </li><li>的目标之一浸油的小水滴（Cargille DF型浸油）和室（华纳仪器25毫米的圆形盖玻片打开商会）放在显微镜（奥林巴斯IX71）的阶段。 </li><li>看看使用透射光的细胞到细胞的外观，并让他们成为关注的焦点。 488纳米，然后照亮细胞从单色（多彩V，从早忙到视觉），并确定是否统一和星形胶质细胞检测荧光信号荧光4。 </li><li>使用EPI和TIRF照明措施星形胶质细胞中的钙。 </li></ol> TIRF显微镜 简而言之，我们使用奥林巴斯IX71显微镜与安道尔IXON DV887DCS EMCCD相机配备。激发和图像采集控制使用TILLVision软件实现。 454/488/515 nm的氩气（100兆瓦）和442 nm的固态激光器（45兆瓦）的梁相结合，并控制与一个至折线激光合成，TIRF双端口冷凝器和acoustoptical可调谐滤波器和控制器（AOTF的;所有从直到光子）和美联储成Kineflex宽带光纤进入TIRF冷凝器。我们使用了奥林巴斯60X 1.45 NA透镜实现TIRF。摄像机的增益调整为每个星形胶质细胞提供了最佳的信号噪声的图像。 TIRF显微镜的背景和原则，最近已经审查4，5 。大部分我们所使用的光学元件均购自至光子，这是安捷伦科技公司（http://www.till-photonics.com/）的一部分。 TIR穿透深度可以从下面的公式计算。 <img style="float:left;" src="/files/ftp_upload/1142/equation.png" alt="方程" /> D =穿透深度 N1 =玻璃的折射率 N2 =折射率细胞 =角的发病率 NAI =数值孔径的发病率 为了确保激光TIRF最佳对齐，我们发现它很有用，观察100 nm的荧光珠（Invitrogen公司，F8803）。我们目前还珠与EPI和TIRF显微镜的框架和录像。在TIRF，观察一个信号噪声的急剧增加和珠显示布朗扩散。我们发现它很有用的100纳米珠定期（每周）〜一次，以确保最佳TIRF发生，而不是损害斜照明即会发生与TIRF显微镜观察的行为，如果不等于临界角α（见图1）。 G蛋白偶联受体激动剂的应用星形胶质细胞表达的Gq蛋白偶联受体6，7，包括多种代谢型谷氨酸受体和P2Y受体（受体激动剂，ATP，ADP） 。这些受体的激活，导致内星形胶质细胞在细胞内钙离子水平的显着增加。例如，人们可以很容易地在ATP（30微米），以星形胶质细胞8-10中的应用观察细胞内钙升高。我们使用一个快速的解决方案的切换华纳仪器称为VC - 77SP快速步灌流系统（http://www.warneronline.com/index.cfm）。用这种方法解决方案可以应用在小于10毫秒。 <img src="/files/ftp_upload/1142/Fig 1a.jpg" alt="图1" /> <img src="/files/ftp_upload/1142/fig1c.jpg" alt="" /> 图1：卡通和成像的照片集。答：显示一个安装在显微镜上airtable的照片，而（B）显示激光器组件，控制器和梁框的照片。 C.显示安装成像腔显微镜阶段的照片。在左侧的快速灌注设备（与步进电机和θ管）可以看出。看到右侧的Axopatch 200A放大器的探头。卡通schematizes设置光通路，以及如何TIRF是实现。激光是集中在1.45 NA的60X物镜，它的立场后焦平面调整偏离中心，并使其出现在临界角α浸油。此时梁患有低折射率的介质，从全内反射和衰减与距离λ（见正文方程）。在这种情况下，这是录音缓冲区周围的星形胶质细胞和星形胶质细胞本身。结果是〜100 nm的质膜内的分子激发光（因而成像）。在动画片的细胞，这是显示为绿色的“兴奋”的膜受体，而在细胞内或细胞的顶面，这些都不是兴奋。已经提供了一个充分考虑TIRF显微镜Steyer和Almers 4。 <img src="/files/ftp_upload/1142/fig2.png" alt="图2" /> 图2。EPI和TIRF显微镜获得的100 nm的荧光珠的图片。 A. EPI图像显示领域的几十个100 nm的荧光珠。红色箭头指向纷纷落户到玻璃盖玻片，而蓝色箭头指向在水中扩散的珠子珠。 B. TIRF图像显示了在相同的视野，在此视图中显示只在A是可见的红色箭头所示的贴壁珠的。这是，因为这些已经解决了的玻璃coverlsip上，并因此在〜100 nm的渐逝场。蓝色箭头所示的珠子是不是这个区域内，因此在TIRF图像看不见的。下图显示的图像的3D渲染。它是明确的大量增加，在信号噪声珠渐逝领域内发生时TIRF显微镜观察。事实上，这些图像的EPI信号与噪声为7.1 ± 0.6，而TIRF为20 ± 0.7。 <img src="/files/ftp_upload/1142/fig3.png" alt="图3" /> 图3。荧光钙指标与EPI和TIRF显微镜收购的染料加载星形胶质细胞的图像。 A.五个星形胶质细胞领域的扩大免疫规划的图像。 B.一个在注意，在图像A和B有明显的不同图中的同一领域的TIRF形象。这是因为只有血浆膜紧密并置到玻璃盖玻片地区TIRF照明成像。较低的面板显示的ATP诱发细胞内钙瞬变与EPI和TIRF显微镜成像。

<ol>
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它是行之有效的，星形胶质细胞显示细胞内钙升高。这些自发地发生，可引发神经元的活动，或通过受体激动剂的应用程序激活星形胶质细胞表面的受体11。其中一个重要的和有争议的问题是是否星形胶质细胞内钙升高可触发释放信号分子，激活受体对神经元的11，12。因为有支持和反对这种观点的证据， 海顿7，第13和麦卡锡 11实验室在评论强调，这是有争议的。根据我们最近的?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>VWR&#174; Micro Cover Slips, Round, No. 1</td>
<td>Tool</td>
<td>VWR</td>
<td>48380-068</td>
<td>&nbsp;</td>
<tr>
<td>Poly-D-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P0899</td>
<td>&nbsp;</td>
<tr>
<td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L2020</td>
<td>&nbsp;</td>
<tr>
<td>Earle&#x2019;s Balanced Salt Solution (EBSS) (1X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>14155-063</td>
<td>&nbsp;</td>
<tr>
<td>Minimum Essential Medium (MEM) (1X), liquid Contains Earle's salts, but no L-glutamine or phenol red</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>51200-038</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate solution</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>HEPES solution 1 M</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H0887</td>
<td>&nbsp;</td>
<tr>
<td>N-2 Supplement (100X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17502-048</td>
<td>&nbsp;</td>
<tr>
<td>Horse Serum, Heat-Inactivated</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>26050-088</td>
<td>&nbsp;</td>
<tr>
<td>PAPAIN-022</td>
<td>Reagent</td>
<td>Worthington</td>
<td>LK003178</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal&#x2122; Medium (1X) Liquid without Phenol Red</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>12348-017</td>
<td>&nbsp;</td>
<tr>
<td>B-27 Serum-Free Supplement (50X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine-200 mM (100X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>25030-149</td>
<td>&nbsp;</td>
<tr>
<td>Cell Strainers</td>
<td>Tool</td>
<td>BD Biosciences</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon Multiwell Flat-Bottom Plates with Lids, Sterile</td>
<td>Tool</td>
<td>BD Biosciences</td>
<td>353046</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P3911</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2 hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>21108</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2 hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M2670</td>
<td>&nbsp;</td>
<tr>
<td>HEPES free acid</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>D-(+)-glucose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G7528</td>
<td>&nbsp;</td>
<tr>
<td>Fluo-4, AM 1 mM solution in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F-14217</td>
<td>&nbsp;</td>
<tr>
<td>Pluronic&#174; F-127 20% solution in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>P-3000MP</td>
<td>&nbsp;</td>
<tr>
<td>Immersion Oil TYPE DF</td>
<td>Microscope</td>
<td>Cargille</td>
<td>16242</td>
<td>&nbsp;</td>
<tr>
<td>Open chamber for 25 mm round coverslips, 100 &mu;l volume</td>
<td>Tool</td>
<td>WARNER Instruments</td>
<td>64-0362 (RC-21BDW)</td>
<td>&nbsp;</td>
<tr>
<td>P-2 platform for Series 20 chambers, non-heater</td>
<td>Tool</td>
<td>WARNER Instruments</td>
<td>64-0278 (P-2)</td>
<td>&nbsp;</td>
<tr>
<td>FluoSpheres carboxylate-modified microspheres, 0.1 &mu;m, yellow-green fluorescent (505/515) 2% solids</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F8803</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

measuring near plasma membrane and global intracellular calcium dynamics in astrocytes

大脑含有神经胶质细胞。星形胶质细胞，神经胶质细胞类型，长期被称为神经元提供了一个被动的支撑作用。然而，越来越多的证据表明，星形胶质细胞可能也积极参与脑功能，通过与神经元的功能的相互作用。然而，星形胶质细胞生物学的许多基本方面仍然争议，职责不清和/或实验未开发。其中一个重要的问题是星形胶质细胞内钙瞬变的动态。这是有关的，因为钙是完善作为一项重要的第二信使，因为它已被提出，星形胶质细胞钙升高可以触发从星形胶质细胞释放发射机。然而，还没有任何近质膜钙信号在星形胶质细胞的详细或令人满意的描述。全内反射荧光显微镜（TIRF）是一个强大的工具来分析有关生理信号事件，约100纳米的活细胞质膜内。在这里，我们使用TIRF显微镜和描述如何几乎同时监测到附近的细胞膜和全球细胞内钙的动态。这种方法的进一步完善和系统应用的潜力，以了解星形胶质细胞钙信号的精确细节。一个星形胶质细胞钙动力学的详细了解可能会提供一个基础，了解是否，如何，何时和为什么星形胶质细胞和神经元接受钙依赖性的功能上的相互作用。

Watch this Scientific Journal Video about Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes at JoVE.com

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

我们描述了如何来衡量膜和全球培养的星形胶质细胞，利用全内反射和epifluorescence显微镜细胞内钙离子动力学附近。

近质膜和全球星形胶质细胞内钙动力学测量

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

measuring-near-plasma-membrane-global-intracellular-calcium-dynamics

Research

JoVE Journal

Biology

13.0K 次观看.  David Geffen School of Medicine, University of California, Los Angeles. 我们描述了如何来衡量膜和全球培养的星形胶质细胞，利用全内反射和epifluorescence显微镜细胞内钙离子动力学附近。

视频: Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

科研

生物学

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

测量心肌细胞的快速钙助焊剂

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

测绘在细胞膜分子扩散，多目标跟踪（MTT）

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

成像质膜变形随着pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

娜功能分析<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt;细胞内的隔间使用质子打死选择器来表达它们的质膜

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

外周相关蛋白血浆膜分型的测定

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.

Necroptosis MLKL 介导的血浆膜破裂特征

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

小鼠胰腺胰岛细胞亚群中的钙动力学成像

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...