作者感谢瑞典研究理事会 (349-2007-8680) 资助和 Dalsjöfors Kött AB (瑞典 Dalsjöfors) 捐赠牛肾上腺。

1. 肾上腺酶消化分离牛嗜细胞的细胞培养
<ol><li>为了分离从牛肾上腺收集的嗜细胞, 用70% 乙醇溶液消毒细胞。用手术刀修剪脂肪和结缔组织。除去血液, 用洛克的溶液 (氯化钠154毫米, 氯化钾5.6 毫米, NaHCO3 3.6 毫米, 羟乙基 piperazineethanesulfonic 酸 (HEPES) 5.6 毫米在 pH 7.4) 冲洗肾上腺静脉自动温控到37摄氏度。</li>
	<li>要消化组织, 充气每个腺体通过注射大约2.5 毫升温暖无菌过滤0.2% 胶原酶 P 溶液通过肾上腺静脉使用注射器和孵化组织20分钟在37°c。检查腺体以确保髓质的消化过程完成。组织应感到柔软而不紧张。</li>
	<li>收集经消化的髓质, 通过切断腺体的纵向方向。用手术刀将髓质组织剁碎。过滤组织悬浮在钢筛子和稀释以洛克的解答到大约50毫升容量减少胶原酶 P 的活动。</li>
	<li>在室温下将 300 x g 的细胞颗粒放入离心机中10分钟, 并将其收集成50毫升不育试管。在20毫升的溶液中重新悬浮获得的颗粒, 并将溶液在无菌100µm 尼龙网中过滤成管子。</li>
	<li>混合嗜细胞悬浮与无菌 Percoll (1:1) 和离心机的细胞溶液在 18600 x g 20 分钟室温。收集密度梯度的顶层, 并在100µm 尼龙网格上过滤溶液。</li>
	<li>要排除 Percoll, 请用洛克溶液稀释细胞悬浮液, 在室温下以 300 x g 为10分钟, 然后在洛克溶液中重新悬浮颗粒。分离细胞悬浮液的估计细胞密度约为400万细胞/毫升。</li>
	<li>对于胞吐作用和细胞内细胞仪测量的安培记录, 胶原蛋白 IV 的嗜细胞, 涂覆60毫米塑料碟, 密度约为 17.5 x10 3 细胞/厘米2, 在 37CO 5% 中孵化出2°c 的细胞。环境.在细胞培养1-3 天内进行电化学实验。</li>
	<li>对于 TEM 成像实验, 75 厘米2细胞培养皿中的板嗜细胞在每瓶7-8 元细胞的密度下, 在 5% CO2环境中孵育37°c 1 天。</li>
</ol>2. 单细胞胞吐作用探头实验24
<ol><li>为进行这些实验, 制备碳纤维电极, 取一个外径为电极的玻璃毛细管, 该外直径适用于这些实验所使用的磁头阶段。使用硼硅酸盐玻璃毛细管, 外径为1.2 毫米, 内径为0.69 毫米。<ol>
<li>将毛细管一端连接到吸水管。将5µm 直径的碳纤维放在某物上, 如一张白纸, 以增强可视性。</li>
		<li>确定一根碳纤维, 用手指将纤维放在一端, 保持碳纤维到位, 同时将毛细管的开口端定位到碳纤维的自由端附近。将碳纤维轻轻地吸进玻璃毛细管中, 使碳纤维通过毛细管两端伸出。移开吸入力。</li>
	</ol></li>
	<li>将毛细管与碳纤维放入微拉拔器中, 将玻璃毛细管拉出两个独立的玻璃吸管小贴士。要断开两个玻璃尖之间连接的碳纤维, 请使用剪刀切割碳纤维并获得两个碳纤维电极。</li>
	<li>在显微镜下, 将碳纤维放在较厚的显微镜滑梯上, 允许手工切割碳纤维的边缘, 纤维从玻璃毛细管涂层延伸到使用手术刀。</li>
	<li>切割碳纤维后, 留下一个玻璃涂层碳纤维尖端, 插入几毫米的电极尖端到环氧树脂溶液中10分钟拉起环氧树脂和密封任何可能的开放空间之间的碳纤维和周围的玻璃。将电极缓慢地从环氧树脂溶液中提起, 以防止在电极尖端形成粗大的胶滴。</li>
	<li>将电极放在持有者 (例如, 一个木制扁棍) 上, 用两条边粘着的耐热胶带将电极连接在一起。将环氧树脂处理过的电极在烤箱中隔夜烘烤100摄氏度。电极提示容易打破, 如果他们接触到表面, 所以确保电极始终是安全储存使用的持有人在那里电极固定到位, 并提示不冒险被触摸。</li>
	<li>要获得平面圆盘电极表面, 请将电极放在 microgrinder 的支架上, 并在45°的天使上将每个碳纤维电极斜角。在斜边后, 用一个永久性的标记标记其上表面的毛细管, 后来知道如何在将电极靠近电池胞吐作用测量时, 在45°角定位圆盘电极曲面。 注意: 这一步很重要, 因为在这些实验中, 椭圆圆盘电极需要放置在与培养皿表面平行的细胞顶部。此外, 斜边电极是在同一天进行的实验, 以确保一个新鲜和清洁电极表面。</li>
	<li>使用前, 将每个碳纤维微电极放在测试溶液中 (例如, PBS 缓冲器中的0.1 毫米多巴胺 (pH 7.4)) 以循环伏安法监测稳态电流。对于循环伏安扫描, 将三角形电位波形从-0.2 v 到 +0.8 V 与 Ag/AgCl 参考电极在 100 mV/秒中, 以确保良好的反应动力学数据得到与理论上计算的值为5µm 直径圆盘碳纤维微电极25。</li>
	<li>对于胞吐作用的探头记录, 将培养皿嗜细胞放到倒置显微镜上。重要的是要屏蔽与法拉第笼建立的显微镜, 以消除电子噪音在探头记录, 由于非常小的电流被测量。在细胞实验期间, 使用显微镜加热阶段保持37°c 的温度。</li>
	<li>使用低噪声膜片钳仪器, 在工作电极上应用 +700 mV 的恒定电位, 与在工作电极之外的培养皿中放置的 Ag/AgCl 参考电极。对于记录胞吐作用的嗜细胞, 数字化的信号在10赫和应用内部低通贝塞尔滤波器在2赫, 以过滤记录的信号。</li>
	<li>要在单个单元格上执行安培记录, 请在与恒电位仪一起使用的头部阶段的电极支架上安装新的斜面和经过测试的碳纤盘微电极。<ol>
<li>轻轻地将电极与扁椭圆形圆盘形状电极表面朝下, 朝向细胞的顶端表面, 然后用机器人将电极与细胞膜接触。</li>
		<li>通过监测电极在细胞膜顶部放置时所引起的细胞变形, 将电极与电极的距离进行调整, 然后仔细地将焊条缩回到一个距离, 使细胞恢复接近其原始细胞形状的形状。.理想的动力学和定量记录是创建一个100纳米薄液膜, 以分离电极和细胞表面, 这是类似的条件, 突触后检测的化学释放在突触。</li>
	</ol></li>
	<li>要刺激细胞胞吐作用, 请将玻璃微的顶端大小为2-3 µm 直径, 用5毫米BaCl 2 溶液在离细胞至少20µm 的距离内填充, 以防止从吸管尖端漏出的任何溶液影响实验和应用5秒的注入脉冲5毫米 BaCl2溶液在细胞表面刺激细胞胞吐作用。</li>
	<li>为了研究渗透压对胞吐作用过程的可逆影响, 准备一个等位缓冲液 (150 毫米氯化钠, 5 毫米氯化钾, 1.2 毫米 MgCl2, 5 毫米葡萄糖, 10 毫米 HEPES, pH 7.4) 与 310 mOsm/千克 iso 渗透压力和一个高渗缓冲由调整与 730 mOsm/千克对应的渗压缓冲液的 NaCl 浓度。</li>
	<li>将细胞放在等位缓冲中, 通过应用钡注入脉冲, 并在初始胞吐作用活动期间记录安培电流瞬变约3分钟, 刺激细胞胞吐作用。<ol>
<li>为了将等渗条件下的胞吐作用反应与高渗条件进行比较, 在高渗缓冲液中孵化出10分钟的细胞, 随后应用钡注入脉冲刺激胞吐作用, 执行3分钟安培记录。</li>
		<li>对于细胞的可逆反应, 在等渗缓冲液中再次孵化细胞10分钟, 通过应用5s 钡注入脉冲来刺激细胞, 并执行3分钟记录细胞胞吐作用反应。</li>
	</ol></li>
	<li>作为控制实验, 以确定对多钡刺激对胞吐作用活动的影响, 执行安培记录胞吐作用释放从三连续 BaCl 2 刺激对同一细胞放置在等渗缓冲和采用同一时间协议对不同渗透的连续细胞孵化实验。</li>
</ol>3. 细胞内电化学细胞术24,26
<ol><li>为了制造囊泡量子尺寸测量用电极, 使用相同的材料, 并开始准备5µm 直径碳纤维电极根据2.1 和2.2 节的说明, 电极制造的安培胞吐作用测量.</li>
	<li>在显微镜下, 使用手术刀切割从玻璃尖端延伸出来的碳纤维, 使从玻璃尖端伸出的碳纤维长度从30到100µm。</li>
	<li>要准备一个碳纤维电极的火焰蚀刻尖端, 使用丁烷火焰。要实现均匀蚀刻的圆柱形电极尖端, 请在旋转时按住圆柱形碳纤维电极, 将碳纤维从玻璃上延伸到丁烷火焰的蓝色边缘, 直到碳尖端形成红色。颜色。这通常需要少于2秒。如果成功, 这会导致蚀刻电极尖端的直径为 50-100 nm (如图 5B中所示的 SEM 图像)。火焰蚀刻后, 将电极置于显微镜下, 以评估电极尖端。</li>
	<li>将圆柱形 nanotip 微电极插入环氧树脂溶液中3分钟, 然后将电极尖端的十五年代浸渍到丙酮溶液中。这允许环氧树脂密封在碳纤维和中空玻璃毛细管壁之间的潜在间隙空间, 而丙酮清除环氧树脂的蚀刻碳纤维电极表面。要治疗环氧树脂, 在烤箱中烘烤电极在100摄氏度过夜。</li>
	<li>使用前, 测试每一个碳纤维微电极的稳态电流, 如2.7 节中使用循环伏安法所解释的。在实验中, 仅使用显示 1.5-2.5 nA27的高原电流的电极。</li>
	<li>对于细胞间探头测量, 将细胞置于显微镜下, 如2.8 所述, 并使用2.9 中所述的恒电位仪相同的实验设置。</li>
	<li>为了进行细胞内安培测量, 并防止对细胞造成重大的物理损伤, 通过添加一个温和的机械力, 将 nanotip 圆柱微电极插入细胞中, 刚好足以推动电极通过细胞等离子体膜和进入细胞胞浆使用机器人。</li>
	<li>插入后, 用细胞膜密封在圆柱电极周围, 开始在活细胞原位进行安培记录。在电极的氧化电位下, 囊泡会吸附到电极表面并随机破裂。 因此, 没有必要采取任何形式的刺激措施来启动这一进程。</li>
	<li>为了确定对囊泡量子大小的渗透效应, 从一组已在等渗和高渗缓冲中孵化出的细胞中收集到的胞内细胞术测量, 使用2.13 节所述的实验条件。</li>
</ol>4. 探头记录的数据分析
<ol><li>为了分析胞吐作用和胞内囊泡量子尺寸分析中记录的探头数据, 使用一个软件程序, 允许在记录的电流与时间轨迹上分析电流瞬态, 使运动峰值参数和可以确定单个电流峰值的综合总电荷。对于数据分析, 我们使用了在苏尔寿实验室中开发的用于数据分析程序28的软件。</li>
	<li>在分析安培峰值时, 选择每个记录的噪声的平均平方 (RMS) 标准偏差的峰值三倍的阈值限制。</li>
	<li>手动收集每个单元记录的安培峰值, 以防止不跟随高斯形状的假峰值, 可以是合并胞吐作用事件的结果的双峰值, 并且只接受具有阈值为三倍的高斯形峰值高于 RMS 噪声。</li>
	<li>收集每单安培峰值总电荷的数据, 并使用 Faradays 法 (n = QnF) 计算每胞吐作用或胞内单囊破裂事件中检测到的儿茶酚胺神经递质的摩尔量, 其中 N 是摩尔神经递质通过电极表面的氧化还原反应, Q = 每穗下检测到的总电荷, n = 在氧化还原反应中转移的电子数 (n=2 为儿茶酚胺) 和 Faraday´s 常数 F=96485 C/摩尔。</li>
	<li>对收集的数据进行统计分析, 首先计算每个单元每个事件检测到的平均电荷。然后, 对于细胞之间的差异, 计算细胞之间的平均电荷, 并使用学生的 t 测试之间的细胞假设不等方差。本研究采用 MATLAB 程序进行统计分析。</li>
</ol>5. TEM 成像对囊泡大小的分析
<ol><li>为了在不同的渗透条件下对细胞进行透射电镜成像, 首先在 5% CO2环境中, 在细胞化学固定前, 先在10分钟内将等位或高渗的细胞孵化为37摄氏度。</li>
	<li>使用 Karnovsky 固定方法29执行单元格定位。使用此方法, 用含有0.01% 叠氮化钠、1% 甲醛和1.25% 戊二醛的溶液孵育细胞, 样品可以储存在4摄氏度。</li>
	<li>固定后, 用0.15 米常使用钠缓冲细胞洗涤。</li>
	<li>为了染色细胞标本后固定, 并在准备 TEM 成像, 孵化细胞与1% 锇毒气溶液为2小时4摄氏度, 其次是 1 h 孵化在0.5% 的醋酸铀溶液在室温下在黑暗中。</li>
	<li>在最后的固定步骤中, 先用100% 乙醇冲洗细胞, 然后用丙酮冲洗细胞, 使细胞脱水。</li>
	<li>在环氧树脂中嵌入细胞样本, 然后以 4000 x g 为30-40 分钟进行离心, 并允许细胞样品在40摄氏度聚合为 15 h, 其次为48摄氏度孵化。</li>
	<li>在制备成像时, 将琼脂100树脂中嵌入的细胞样本分成 60 nm 的切片, 使用 ultramicrotome。</li>
	<li>将细胞以4% 铀 acetate/25%ethanol 溶液为4分钟, 然后用清水冲洗二十年代。用雷诺铅柠檬酸3分钟冲洗细胞, 然后用清水冲洗二十年代。</li>
	<li>对细胞样品进行透射电镜成像。在我们的实验中, 我们使用了一个透射电镜, 已经运行了120伏。</li>
	<li>从每一个细胞切片的记录图像中, 可以显示细胞胞浆中的囊泡的细胞超微结构, 首先要通过将像素与每个 TEM 图像中记录的刻度条相关, 来校准图像像素的大小。使用成像软件来确定每一个细胞在等渗或高渗条件下的图像中的囊泡大小。在实验中, 我们使用软件 ImageJ 进行图像分析。值得注意的是, 对于细胞的透射电镜图像进行细胞超微结构的图像分析, 这些测量的大小调整是基于细胞剖面的厚度需要考虑的, 以前是由 Almers´s 实验室30来描述的。</li>
</ol>

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作者没有什么可透露的。

我们这里提出了一个协议和三互补分析方法结合分析分泌性囊泡和胞吐作用过程的优点, 以更好地了解渗透压等物理力如何影响分泌性囊泡分泌细胞中的胞吐作用过程。这些方法包括碳纤维微电极探头, 它是一种记录胞吐作用活性的建立方法, 细胞内电化学细胞术, 用于确定量子在其当地环境中的囊泡大小, 并透射电镜图像分析监测分泌囊泡体积。在这项工作中, 通过收集探头记录的定量数据, 在暴露于等渗和高渗细胞外环境的嗜细胞中的单泡胞吐作用事件, 我们证实渗透休克显著降低在胞吐作用期间释放的量神经递质, 这些细胞可以可逆地恢复和释放原始数量, 如果放回一个等渗环境(图 3)。
安培记录提供了胞吐作用事件频率与时间的信息, 因此也可以用来监测不同渗透环境中细胞胞吐作用活动的变化。这些数据可以解释和用来辨别活动的变化是立即发生的, 还是作为时间的函数。正如我们在以前的工作中所显示的, 虽然渗透应力阻碍了 exocytotic 活动, 但它似乎没有阻碍容易发布池的融合24。胞吐作用活动数据还可用于分析记录时间内的总 exocytotic 活动。在这里, 我们提出了累积数的胞吐作用事件从3分钟的记录胞吐作用在嗜细胞暴露在两个不同的渗透条件。这些数据显示有渗透应力的细胞的胞吐作用活性的抑制, 并在细胞返回到细胞外的等渗环境后, 可以实现部分恢复。然而, 非常重要的是, 控制实验表明, 在这些实验所用的时间范围内, 多巴2 +刺激导致了细胞每次被激发到分泌物时胞吐作用活性的显著降低。由第三个连续的 Ba2 +刺激, 只保持了胞吐作用活动的第三个, 并且显然钡, 并且刺激的时间在这些实验中, 影响了囊泡周期。这可能与解释为什么在渗透性休克后放置在等渗溶液中的细胞中 exocytotic 活性的原因有关, 为什么这些细胞在三后与等渗条件下的细胞表现出类似的相对减少。连续 Ba2 +刺激。因此, 探头记录胞吐作用提供有关分泌性囊泡的信息, 从囊泡被触发与等离子膜融合, 通过融合毛孔释放神经递质。从而可以收集单个囊泡神经递质释放的定量数据和胞吐作用活动的信息。
释放的神经递质的数量可以由触发的胞吐作用模式来调节, 其中要么是完全的囊泡含量, 要么是部分内容被释放。通过仅研究胞吐作用释放, 使用碳纤维探头, 只有检测出什么是从囊中排出, 很难区分是否检测到释放的神经递质量的变化与触发模式的改变有关胞吐作用, 影响囊泡含量释放的细胞生物物理特性的变化, 或囊泡量子大小的变化。因此, 通过使用细胞内电化学细胞仪对胞吐作用的安培记录进行补充,原位测量活细胞中的囊泡量子大小可以被描绘出来, 从而用来比较胞吐作用26 期间囊泡含量释放的分数。
在这项研究中, 为了探讨囊泡量子大小是否受渗透胁迫的影响, 我们应用该技术对暴露在等渗和高渗条件下的细胞进行囊泡量子大小的定量和评价。当插入 nanotip 电极入活细胞的细胞质被认为相当侵入性, 这些实验只执行一次每细胞并且没有被重复在同一个细胞。因此, 这些实验优先作为个体测量, 从随机选择的细胞组暴露在等渗和高渗细胞外环境。为了比较细胞内电化学细胞术对胞吐作用释放的量神经递质的改变, 量子大小的变化, 应考虑匹配细胞内记录的实验条件和同时执行探头记录胞吐作用在单独的随机细胞。如果在同一单细胞上进行研究, 在这些实验协议中也必须考虑连续 BaCl2刺激的影响, 并确保实验条件与细胞内细胞术所使用的情况相匹配。测量.同样值得注意的是, 在插入细胞时很难控制电极的精确位置和深度。因此, 每个细胞提供一个随机样本的囊泡量子大小分析。此外, 使用这种方法探测囊泡量子大小并不能区分, 例如, 囊泡成熟度的差异, 因此也可能增加测量的变化。细胞内实验表明, 在细胞外渗透压下, 囊泡量子大小降低。将该方法检测到的量子尺寸相对减少, 与胞吐作用过程中神经递质释放量相对变化的观察进行比较。本研究发现, 量子大小的相对下降与神经递质释放在细胞传感渗透胁迫下的下降顺序相同。
为了验证渗透应力是否改变了囊泡神经递质的浓度, 用透射电镜对已暴露于等渗和高渗条件下的化学固定嗜细胞进行 TEM 成像分析, 评价囊泡体积。TEM 成像分析表明, 当细胞暴露于渗透休克时, 囊泡会收缩, 相对减小的大小与囊泡量子大小一起调整, 以维持恒定的神经递质浓度24。TEM 图像分析, 提供了纳米图像分辨率, 可以区分两个阶段, 致密的核心蛋白基质和周围的光环溶液 LDCVs 内, 从而使其能够确定致密的核心蛋白基质体积和计算晕解的体积。从这一分析中, 囊泡体积的下降, 主要是与围绕致密核心蛋白基质24的光晕溶液体积递减有关。
总之, 这项研究提出了一个协议, 演示如何组合三分析方法, 并允许在神经递质释放之前的分泌性囊泡的特征, 以及当细胞被触发时从这些囊泡释放什么胞吐作用, 以更好地了解分泌性囊泡和细胞功能, 如胞吐作用是如何影响细胞外应力。该协议还可用于帮助回答关于胞吐作用和囊泡量子大小的神经递质释放如何受物理环境中其他改变或可能影响分泌细胞的潜在药物的影响的问题。

肾上腺中的嗜细胞是神经内分泌细胞, 释放神经递质分子进入血流。这是通过一个细胞过程, 其中包括对接和融合的神经递质囊泡, 导致内容释放从囊泡到细胞外空间的过程称为胞吐作用。嗜细胞中的神经递质 (肾上腺素和去甲肾上腺素) 被膜蛋白主动输送到大致密核囊泡 (LDCVs) 中, 并存储在高浓度 (~ 0.5-1 米)1,2。LDCVs 内神经递质的积累是由儿茶酚胺分子与由 chromogranin 蛋白 (169 毫克/毫升)3,4,5 组成的 intravesicular 致密核心蛋白基质的亲和性完成的. 、6和一个 intravesicular 鸡尾酒解决方案, 其中包含用于儿茶酚胺加载和存储的关键组件, 如 ATP (125-300 mM)7、Ca2 + (解决方案中的50-100 µM 和 ~ 40 毫米绑定到蛋白质矩阵)8, Mg2 + (5 毫米)9, 抗坏血酸 (10-30 毫米)10, pH 值为 ~ 5.511,12。LDCVs 保持一个 iso 渗透条件与细胞细胞质 (310 mOsm/千克)13, 即使理论溶质浓度在囊泡总和高达750毫米。intravesicular 组分的组成不仅对儿茶酚胺的加载和贮存至关重要, 而且对溶质聚集到致密的核心蛋白基质中也是必不可少的。这大大减少了囊泡的渗透, 并可能影响在胞吐作用5, 6 中释放的儿茶酚胺数量.
通过安培记录对细胞外渗透压对胞吐作用过程的影响的研究报告说, 高胞外渗透压抑制胞吐作用活性, 并减少从单一的神经递质分泌的数量囊泡间隔4,14,15,16,17,18,19。对这些观察的解释推测了在细胞胞浆抑制囊泡融合事件中大分子拥挤的可能增强, 以及膜生物物理特性的改变影响了神经递质在胞吐作用期间释放。这些想法假设高胞外渗透应力不影响囊泡量子大小, 这定义了在触发胞吐作用的前一阶段囊泡室中储存的神经递质分子的数量14,15,17,19,20,21. 在单细胞胞吐作用释放量的安培测量中, 碳纤盘微电极与细胞表面紧密接触, 形成模拟突触构型的实验设置, 其中安培电极作为突触后检测器 (图 1)22,23。通过刺激细胞胞吐作用, 可以诱导神经递质囊泡与细胞膜融合, 释放部分或全部囊泡含量进入细胞外空间。在电极表面释放的这些神经递质分子可以电化学检测, 如果神经递质是活性 (例如, 儿茶酚胺) 通过应用氧化还原电位 +700 mV vs 银/AgCl 参考电极.因此, 一系列的电流峰值标志着对个别胞吐作用事件的检测。从安培记录中的电流与时间轨迹, 每一个安培峰值下的区域表示每胞吐作用事件检测到的电荷, 并且可以转换为释放的神经递质的摩尔, 使用法拉第方程。因此, 安培记录提供定量信息的数量的神经递质从单一的胞吐作用事件和报告的频率胞吐作用事件, 但不提供定量信息的分泌性囊泡在囊泡融合和神经递质释放之前的内容已经开始。
因此, 为了更好地理解细胞细胞质分泌囊在细胞被触发接受胞吐作用前对细胞外渗透应力的反应, 其他互补分析方法可以用来丰富这些信息。例如, 研究渗透应力是否改变囊泡体积, 透射电镜 (TEM) 成像分析可用于测定化学固定后细胞的囊泡大小。为探讨渗透胁迫对囊泡量子大小的影响, 一种新近开发的称为胞内电化学细胞术的安培技术, 可用于在分泌性囊泡中定量测定囊泡神经递质含量。在活细胞的细胞质中仍然居住的本机状态26。在细胞内电化学细胞技术中, nanotip 柱状碳纤维电极轻轻地插入活细胞的细胞质中, 并将 +700 mV 电位应用于该电极 (vs a Ag/AgCl 参考电极),在囊泡中儿茶酚胺含量可以通过检测单个囊泡的氧化还原电流峰值来进行量化, 然后在电极表面进行随机破裂, 从而释放出它们的内容。电极表面26。因此, 在电流与时间的跟踪中, 每一个囊泡破裂事件都可能导致电流瞬变, 通过整合每个电流穗的面积, 可以用 Faraday´s 法计算囊泡量子大小。
因此, 通过将囊泡尺寸测量所得的定量信息与囊泡量子大小分析相结合, 经胞内电化学细胞仪记录, 囊泡神经递质浓度也可被确定。这允许囊泡的特征, 当细胞暴露于不同的渗透条件, 并提供了一个更好的看法, 如何水泡可能对细胞外渗透应力反应在胞吐作用之前的阶段。结合这些方法的结果表明, 在胞外高渗透压的存在下, 囊泡收缩和调整其量子大小, 并比较这些测量的相对变化的定量信息表明,收缩时, 囊泡调整其含量和大小以维持恒定的神经递质浓度24。因此, 这种理解对于连接到渗透胁迫下细胞中神经递质释放的观察是有价值的。在这些协议中, 我们描述了使用这三互补方法, 允许描述在他们的家乡环境中分泌的囊泡对细胞外渗透的反应以及这种反应对胞吐作用的影响。过程.除了我们以前关于高渗透压对胞吐作用24 影响的观察外, 我们还提出了进一步的实验, 描述渗透休克后细胞的恢复和多钡刺激在嗜中的作用。细胞.

<table><tbody><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S7653</td><td></td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P9333</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>S5761</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3375</td><td></td></tr><tr><td>MgCl2</td><td>Sigma Aldrich</td><td>M-2670</td><td></td></tr><tr><td>Glucose</td><td>Sigma Aldrich</td><td>G8270</td><td></td></tr><tr><td>Collagenase P</td><td>Roche, Sweden</td><td>11 213 857 001</td><td></td></tr><tr><td>100-&amp;micro;M Nylon mesh</td><td>Fisher Sientific</td><td>08-771-19</td><td></td></tr><tr><td>Percoll</td><td>Sigma Aldrich</td><td>P1677</td><td></td></tr><tr><td>Collagen IV coated 60 mm plastic dish</td><td>VWR</td><td>354416</td><td></td></tr><tr><td>Centrifuge</td><td>Avanti J-20XP</td><td></td><td></td></tr><tr><td>Borosilicate glass capillary</td><td>Sutter instrument Co., Novato, CA</td><td></td><td></td></tr><tr><td>Micropipette puller</td><td>Narishing Inc., Japan</td><td>PE-21</td><td></td></tr><tr><td>Epoxy solutions (A and B)</td><td>Epoxy technology, Billerica, MA</td><td></td><td></td></tr><tr><td>Beveller</td><td>Narishing Inc.</td><td>EG-400</td><td></td></tr><tr><td>Inverted Microscope</td><td>Olympus</td><td>IX81</td><td></td></tr><tr><td>Patch clamp Instrument</td><td>Molecular Devices, Sunnyvale, CA</td><td>Axopatch 200B</td><td></td></tr><tr><td>Micromanipulator</td><td>Burleigh Instrument Inc., USA</td><td>PCS-5000</td><td></td></tr><tr><td>Butane Flame</td><td>Multiflame AB, H&amp;auml;sselholm, Sweden</td><td></td><td></td></tr><tr><td>Transmission electron microscopy</td><td>Omega</td><td>Leo 912 AB</td><td></td></tr></tbody></table>

monitoring the effect of osmotic stress on secretory vesicles and exocytosis

探头对渗透休克细胞的记录表明, 分泌细胞通过减少单个胞吐作用事件中的胞吐作用活性和囊泡释放的神经递质的数量来应对这一生理压力。有人建议, 减少神经递质的降低是由于细胞膜生物物理性质的变化, 当细胞收缩反应渗透胁迫, 并假设, 分泌泡在细胞胞浆不受影响细胞外渗透应力。探头记录胞吐作用监视器当囊泡与等离子膜融合时, 从细胞中释放出的是什么, 但在触发囊泡聚变之前没有提供有关囊泡含量的信息。因此, 通过结合探头记录与其他补充分析方法, 能够表征分泌囊泡在胞吐作用细胞被触发提供了一个更广泛的概述, 以检查如何分泌囊泡和胞吐作用过程受渗透冲击的影响。我们在这里描述如何补充探头记录与细胞内电化学细胞术和透射电镜 (TEM) 成像可以用来表征变化的分泌囊泡大小和神经递质的内容在嗜细胞与胞吐作用活性有关, 暴露于渗透胁迫前后。通过将从实验中获得的定量信息与所有三种分析方法联系起来, 得出结论: 分泌性囊泡通过缩小大小和减少囊泡量子大小来响应胞外渗透应力, 以保持一个恒定的囊泡神经递质浓度。因此, 这给出了一些解释, 为什么囊泡, 响应渗透应激, 减少释放的神经递质胞吐作用释放。这里的安培记录表明这是一个可逆的过程, 当被放置的细胞恢复到一个等质环境中时, 渗透休克后的囊泡会重新注入神经传递素。

我们在这里描述了如何将 TEM 成像与两种电化学方法、碳纤维探头和细胞内电化学细胞术相结合的协议, 可以提供信息, 得到更广泛的观点, 暗指的效果分泌囊泡的胞外渗透压和胞吐作用过程。通过在单个嗜单元格中使用实验设置 (如图 1所示) 比较胞吐作用释放的代表安培记录, 当细胞接触渗透时, 显示 exocytotic 活动显著减少。与等渗条件下的单元格相比的应力 (图 2A)24。从这些录音和使用 Faraday´s 定律, 每一个安培电流峰值检测到的总电荷被用来计算在暴露于不同渗透的细胞中从单个囊泡胞吐作用事件中排出的分子数量。条件.相比之下, 如图 2B所示, 在高渗溶液中由细胞检测到的平均安培电流峰值的较小面积, 通过细胞传感渗透应力24释放出的神经递质分子较少..
为了确定释放的神经递质分子数量下降的可逆性, 我们在暴露在高渗环境下的细胞中进行了胞吐作用的安培记录, 随后在细胞被放回一个等渗后环境.在这些实验中, 嗜细胞连续三次受到 BaCl2解决方案的刺激: 首先是在一个等位缓冲中, 其次是在高渗溶液中孵化10分钟后的第二次刺激, 最后是第三个在等渗条件下10分钟细胞孵化后的刺激。在图 3A中显示的结果表明, 当细胞暴露于高渗条件下时, 所释放的神经递质的数量减少了 50%, 而与在等渗条件下的第一个 Ba2 +刺激相比。随后, 当细胞被带回一个等渗环境并受到第三 Ba2 +的刺激时, 每胞吐作用事件释放的神经递质的数量逆转回最初的刺激记录的原始量, 这已确认以前的观察14。控制实验与三连续 ba2 +刺激细胞在等渗条件 (见图 3B) 表明, 多序列 ba2 +刺激在等渗条件下没有改变数量神经递质在胞吐作用期间获释。这表明, 囊泡量子大小是快速和可逆调整与细胞外渗透。
然而, 当分析这些实验中的胞吐作用活动的安培迹时,正如图4A 所示, 它变得很明显, 当细胞暴露于高渗应力时, 胞吐作用活性明显受到阻碍。在渗透胁迫期间, 胞吐作用事件减少到12% 的活动在细胞在等渗条件。随后, 渗透性休克和细胞被放回 isosmotic 环境后, 细胞恢复了原来的胞吐作用活动的41%。有趣的是, 在等渗条件下进行的控制实验显示, 如图 4B所示, 在连续三次 BaCl2 刺激后, 胞吐作用事件的频率减少到53% 秒并进一步下降到26% 的第三刺激相比, 第一次刺激。因此, 很明显, 连续 Ba2 +刺激似乎并不影响胞吐作用释放出的神经递质的数量, 但对囊泡释放过程的效率有显著影响。
目的探讨细胞外渗透应激对其原生环境的分泌性囊泡体积或量子大小的影响, 采用 TEM 成像的囊泡大小与胞内电化学细胞术相结合。暴露于等渗和高渗条件下。在细胞内电化学细胞实验中, 当放置在等渗和高渗溶液中时, 将碳纤维 nanotip 电极插入活嗜细胞的细胞质中 (如图 5所示)。所产生的安培电流峰值监测细胞胞浆中的每一个囊泡碰撞, 吸附, 并随机破裂, 并释放在电极表面的囊泡含量在爆裂的26。用 Faraday´s 法计算每个细胞记录的平均囊泡量子大小, 检测了电流与时间痕中每个峰值的综合总电荷。这些细胞内电化学细胞测量 (如图 6和图 7B所示) 表明, 与等渗条件下的细胞相比, 暴露于渗透应力的细胞中的囊泡量子大小明显降低。用胞内电化学细胞仪测定囊泡量子尺寸的变化幅度, 对胞吐作用细胞外渗透胁迫时释放的神经递质的分数下降的比较, 在量子大小和与等渗条件下的细胞相比, 所释放的神经递质的数量相对减少了 60% (图 7)24。为了将囊泡量子大小的调整与渗透压细胞中囊泡神经递质浓度的电位变化联系起来, 本文进行 TEM 图像分析, 以确定暴露于等渗和高渗的细胞的囊泡大小。条件.此外, 在透射电镜图像中, LDCVs 中的致密核心蛋白基质在膜界囊泡中被可视化为暗球形的暗染色, 用于测量这些囊泡中致密核基质的体积。如图 8所示, 在暴露于胞外渗透应力的细胞与等渗条件下的细胞相比, 囊泡大小降低到60%。从 LDCV 和致密核的实测直径计算量计算出了透射电镜图像中 LDCVs 内的清晰解的周围晕解的体积。透射电镜图像分析的总结结果显示, 如图 8所示, 在胞外渗透休克期间, 它主要是 LDCVs 中晕解的体积, 减少了24。

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56537/56537fig1.jpg"> 图 1: 单个单元格胞吐作用探头。胞吐作用在单嗜单元格中安培测量的实验设置示意图24。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56537/56537fig1large.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/56537/56537fig2.jpg"> 图 2: 从胞吐作用测量中的安培跟踪。 (a)一个代表安培记录胞吐作用在嗜细胞中的等渗 (黑颜色) 和高渗 (红色) 胞外环境。(B)从胞吐作用测量嗜细胞在等渗 (黑) 和高渗 (红) 胞外环境中的平均安培峰值增大24。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56537/56537fig2large.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/56537/56537fig3.jpg"> 图 3: 渗透休克后胞吐作用释放的儿茶酚胺数量的可逆性。(A)在胞吐作用期间从嗜细胞 (n=4) 中释放的分子数量由三个连续Ba 2 + 刺激, 第一次在等渗作用, 第二个在高渗, 和第三个在等位缓冲。给出了非配对 t 检验的统计结果。p值, 用于比较在与第二 ba 的等渗缓冲中的第一个 ba2 +刺激 (ba2 +传感 1)2 +刺激 (Ba2 +传感 2) 在高渗缓冲中是p= 0.1088 和p值第二个 ba2 +刺激高渗溶液与第三 ba2 +刺激 (Ba2 +传感 3) 在等位缓冲中的比较是 p = 0.059。(B)控制实验, 证明了在等渗缓冲中嗜细胞 (n = 4) 在三连续 Ba2 +刺激下释放的神经递质分子量。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56537/56537fig3large.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/56537/56537fig4.jpg"> 图 4: 渗透压对胞吐作用活性的影响。(A)细胞外渗透对胞吐作用活动的影响表现为胞吐作用事件的频率, 当嗜细胞 (n = 4) 以钡溶液在等渗、高渗、最后在等渗条件下刺激时。这些值被显示为每个单元的平均峰值数和取样的所有单元的平均值 (平均标准误差 (SEM))。通过对未配对数据的 t 检验 (2 +刺激1和高渗 ba 2 +刺激2为p= 0.0126 和p值), 给出了变化的统计意义, 用于比较高渗钡2 +刺激2和等渗 ba2 +刺激3是p= 0.037)。(B)控制实验, 介绍在嗜细胞 (n=4) 在等渗条件下连续三次钡刺激后胞吐作用事件的频率。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56537/56537fig4large.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/56537/56537fig5.jpg"> 图 5: 细胞内囊泡电化学细胞仪监测囊泡量子大小的变化。(a)用于细胞内电化学细胞术的实验设置示意图。(B)一种典型的 nanotip 锥形碳纤维电极24的扫描电子显微图像。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56537/56537fig5large.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/56537/56537fig6.jpg"> 图 6:细胞内电化学细胞仪测量显示(A)代表在嗜细胞内等渗 (黑色) 和高渗 (红) 胞外环境中的细胞内安培细胞内记录的痕迹。(B)在中度 (黑色) 和高渗 (红) 胞外环境中的嗜细胞内, 从细胞内细胞测量中的平均安培峰值增大24。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56537/56537fig6large.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/56537/56537fig7.jpg"> 图 7: 量化胞吐作用释放的儿茶酚胺数量和囊泡量子大小的测定。(A)在等渗 (n=22) 和高渗 (n = 20) 环境中嗜细胞胞吐作用记录期间释放的分子的平均数量。这些值显示为每胞吐作用事件从每个单元格中释放的分子的平均数量, 并从取样的单元格 (如SEM) 中得到平均值。对未配对数据的 t 检验 (p-值 = 0.0003) (B)在嗜细胞内的平均每囊的分子数, 如在等渗 (n= 19) 和高渗 (n=16) 条件。这些值显示为每个单元检测到的每穗分子的平均数量, 并从取样的所有单元格中得到平均值 (如SEM)。对未配对数据 (p-值 = 0.0108)24的 t 检验给出了变化的统计意义。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56537/56537fig7large.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/56537/56537fig8.jpg"> 图 8: 渗透压力对大致密芯囊泡大小的影响.从 attolitres (n=12) 和高渗 (n=9) 缓冲嗜细胞 TEM 图像的图像分析中, 计算了致密核蛋白基质中 LDCVs、致密核心蛋白和光环溶液的计算量。结果是从平均每细胞311囊和平均单细胞 (@ SEM) 收集。p值是从对同渗和高渗缓冲比较的未配对 t 检验中报告的。P值为囊泡体积的 0.0385 (*), 0.3967 用于密芯卷, 0.0047 (**) 用于光晕卷24。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56537/56537fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis at JoVE.com

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

渗透胁迫影响胞吐作用和在这个过程中释放的神经递质的数量。我们演示了如何结合电化学方法和透射电子显微术来研究细胞外渗透压对胞吐作用活性、囊泡量子大小和释放神经递质的影响。在胞吐作用期间。

渗透胁迫对分泌性囊泡和胞吐作用的监测作用

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity ...

monitoring-effect-osmotic-stress-on-secretory-vesicles

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.5K 次观看.  Chalmers University of Technology. 渗透胁迫影响胞吐作用和在这个过程中释放的神经递质的数量。我们演示了如何结合电化学方法和透射电子显微术来研究细胞外渗透压对胞吐作用活性、囊泡量子大小和释放神经递质的影响。在胞吐作用期间。

视频: Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

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 ...

科研

化学

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

调查肥大细胞分泌颗粒;从合成到胞吐作用

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

免疫与感染

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

在肽膜前体的维西图洛合成中,用于自主囊泡生长

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

生物工程

Modulating Shape of Polyester Based Polymersomes using Osmotic Pressure

Polymersomes are membrane-bound, bilayer vesicles created from amphiphilic block copolymers that can encapsulate both hydrophobic and hydrophilic payloads for drug delivery applications. Despite their promise, polymersomes are limited in application due to their spherical shape, which is not readily taken up by cells, as demonstrated by solid nanoparticle scientists. This article describes a salt-based method for increasing the aspect ratios of spherical poly(ethylene glycol) (PEG)- based polymersomes. This method can elongate polymersomes and ultimately control their final shape by adding sodium chloride in post-formation dialysis. Salt concentration can be varied, as described in this method, based on the hydrophobicity of the block copolymer being used as the base for the polymersome and the target shape. Elongated nanoparticles have the potential to better target the endothelium in larger diameter blood vessels, like veins, where margination is observed. This protocol can expand therapeutic nanoparticle applications by utilizing elongation techniques in tandem with the dual-loading, long-circulating benefits of polymersomes.

Polymersomes are self-assembled polymeric vesicles that are formed in spherical shapes to minimize Gibb's Free Energy. In the case of drug delivery, more elongated structures are beneficial. This protocol establishes methods to create more rod-like polymersomes, with elongated aspect ratios, using salt to induce osmotic pressure and reduce internal vesicle volumes.

使用渗透压力调节聚酯聚合物的形状

Polymersomes are membrane-bound, bilayer vesicles created from amphiphilic block copolymers that can encapsulate both hydrophobic and hydrophilic payloads ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

在单酵母细胞MAPK诱导表达的时空定量

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

生物学

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

成像 FITC-葡聚糖作为胞吐作用调节的记者

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 (VAMP7) by total internal reflection fluorescence microscopy (TIRFM) is a straightforward way to explore secretion from the lysosomal compartment. Taking advantage of cell culture on micropatterned surfaces to normalize cell shape, a variety of statistical tools were employed to perform a spatial analysis of secretory patterns. Using Ripley&#8217;s K function and a statistical test based on the nearest neighbor distance (NND), we confirmed that secretion from lysosomes is not a random process but shows significant clustering. Of note, our analysis revealed that exocytosis events are also clustered in nonadhesion areas, indicating that adhesion molecules are not the only structures that can induce secretory hot spots at the plasma membrane. Still, we found that cell adhesion enhances clustering. In addition to precisely defined adhesive and nonadhesive areas, the circular geometry of these micropatterns allows the use of polar coordinates, simplifying analyses. We used Kernel Density Estimation (KDE) and the cumulative distribution function on polar coordinates of exocytosis events to identify enriched areas of exocytosis. In ring-shaped micropattern cells, clustering occurred at the border between the adhesive and nonadhesive areas. Our analysis illustrates how statistical tools can be employed to investigate spatial distributions of diverse biological processes.

Live imaging of lysosomal exocytosis on micropatterned cells allows a spatial quantification of this process. Morphology normalization using micropatterns is an outstanding tool to uncover general rules about the spatial distribution of cellular processes.

定量微模式细胞细胞外细胞异细胞的时态参数

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 ...

Role of ER in the Secretory Pathway

Eukaryotic cells have a special pathway that enables communication between various intracellular membrane-bound compartments and also with the extracellular environment. This pathway is termed as the secretory pathway.
Components of the secretory pathway
About a third of proteins synthesized in the cell are sorted via the secretory route. They shuffle between different compartments in membrane-bound vesicles until they reach their final destination. The main intracellular compartments involved in the secretory pathway are the endoplasmic reticulum, the ER-Golgi intermediate compartment, and the Golgi apparatus. Eventually, the functional protein cargo is delivered within the endomembrane system, exported out of the cell, or inserted into the plasma membrane.
Endoplasmic reticulum: the gateway to the secretory pathway
The endoplasmic reticulum is the primary gateway to the secretory pathway. It is the site where the proteins are folded, modified, and checked for quality before entering the secretory pathway. Various physiological stresses can increase the demand for secretory proteins or cause the accumulation of misfolded proteins in the ER. The disruption in the balance between protein demand and capacity to deliver correctly folded proteins in the ER causes stress.
Unchecked ER stress disturbs ER homeostasis, resulting in cellular dysfunction and disease. The primary characteristics of ER-related diseases are decreased protein folding capacity, failure to recognize and respond to misfolded proteins, and improper activation of responses to misfolded proteins. ER stress is implicated in the pathogenesis of diseases like diabetes mellitus, viral infections, and cancer.&#160; For example, in type II diabetes mellitus, insulin resistance in the peripheral tissues of the liver results in hypersecretion of insulin by the &#946; cells. The secretory overload experienced by the &#946; cells skews the ER balance. It can also result in cell death in later stages of type II diabetes mellitus.

ER 在分泌途径中的作用

Eukaryotic cells have a special pathway that enables communication between various intracellular membrane-bound compartments and also with the ...

教育

JoVE Core

细胞生物学

Unit 1

Transmembrane Transport in Endoplasmic Reticulum and Peroxisomes

关键术语与概念

Exocytosis

Exocytosis is a process that releases molecules outside the cell. Like other bulk transport mechanisms, exocytosis requires energy.
Exocytosis is the opposite of endocytosis, which brings molecules inside the cell. Sometimes, the released materials are signaling molecules. For example, neurons typically use exocytosis to release neurotransmitters. Cells also use exocytosis to insert proteins such as ion channels into their cell membranes, secrete proteins for use in the extracellular matrix, or remove waste.
There are two main types of exocytosis in eukaryotes&#8212;regulated and non-regulated (or constitutive). Regulated exocytosis requires an external signal and is used to release neurotransmitters and secrete hormones. Unlike regulated exocytosis, constitutive exocytosis is carried out by all cells. Cells use constitutive exocytosis to release components of the extracellular matrix or incorporate proteins into the plasma membrane.
Both regulated and constitutive exocytosis occur in a stepwise manner.
The first step is vesicle trafficking, in which vesicles transport material to the plasma membrane. Motor proteins actively move vesicles along the cytoskeletal tracks of microtubules and filaments. The second step is vesicle tethering, in which vesicles are partially linked to the plasma membrane. In the third step, vesicle docking, the vesicle membrane attaches to the plasma membrane, and the two membranes begin to pair with each other.
The fourth step, vesicle priming, occurs only in regulated exocytosis. Vesicle priming includes modifications occurring after the vesicle docks but before releasing its contents. Priming prepares vesicles for fusion with the plasma membrane.
The fifth step is vesicle fusion. Vesicle fusion can be complete or kiss-and-run. In complete fusion, vesicles entirely collapse and become part of the plasma membrane, expelling the contents from the cell in the process. In kiss-and-run fusion, the vesicle is recycled: It only temporarily fuses with the plasma membrane, releases its contents, and returns to the cell&#39;s interior.

胞吐作用

Exocytosis is a process that releases molecules outside the cell. Like other bulk transport mechanisms, exocytosis requires energy.
Exocytosis is the ...

Endocytosis and Exocytosis

Overview of Secretory Vesicles

Secretory vesicles, also known as dense core vesicles (DCVs), are membrane-bound vesicles that transport secretory proteins, such as hormones or neurotransmitters. Regulated secretory vesicles transport proteins from the trans-Golgi network to the exterior of the cell. Proteins present in regulated secretory vesicles are required to be rapidly exocytosed in large amounts upon a specific stimulus.
Various proteins regulate the aggregation of molecules inside the secretory vesicles. Chromogranins (Cgs) A and B are proteins that bind to soluble molecules present in secretory vesicles. This binding aggregates the soluble molecules, forming DCVs. Cgs function optimally at low pH and high intracellular calcium concentration. The low pH is maintained by the continuous pumping of protons by the V-type ATPases. A high concentration of calcium is present in the endoplasmic reticulum and Golgi complex of most mammalian cells. Hence, the secretory vesicles that bud off from the trans-Golgi network are rich in calcium. Calcium induces pH-dependent conformational changes in Cgs A and B. These conformational changes lead to the binding and aggregation of CgsA and B among other vesicular matrix proteins with the vesicular membrane. Aggregation is important for specific proteins to be sorted into regulated secretory vesicles.
The yeast Saccharomyces cerevisiae is a model system for studying the regulation of secretory vesicle-mediated transport. In 1979, Randy Schekman and Peter Novik generated temperature-sensitive mutants of S. cerevisiae. These mutants expressed proteins that were functional only at low temperatures but not at 37&#176;C. Schekman and Novik observed that these mutants accumulated vesicles and internal membranes. These mutants were also defective in different stages of protein secretion and therefore called them sec mutants. Some sec mutants contained vesicles that were unable to fuse with the plasma membrane, while other mutants could not transport proteins to other organelles. Further, molecular analysis of these mutants revealed 23 sec genes that produced proteins with regulatory roles in different transport stages of secretory vesicles.