这项工作得到了国家卫生研究院的资助, R35 GM118177 PND。

1. 准备解决方案 <ol> <li> 使用以下方法准备 HL-5 介质:10 克/升葡萄糖, 10 克/升 proteose 蛋白胨, 5 克/升酵母提取物, 0.965 克/升 Na 2 HPO 4 和 #183; 7H 2 O, 0.485 克/l. 2 PO 4 , 除非另有说明, 0.03 克/L 链霉素在去离子水中。在室温下蒸压和贮存. 
	注: 由于 proteose 蛋白胨与从不同供应商获得的酵母提取物以及个别批次之间的差异, 介质的 pH 值可能需要调整到6.4 到6.7 的典型范围. </li>
	<li> 使用以下方法准备 SM 板:10 克/升葡萄糖, 10 克/升蛋白胨, 1 克/升酵母提取物, 2.31 克/升, 2 PO 4 , 1 克/升 K 2 HPO 4 , 和在去离子水中20克/l 琼脂。高压釜, 冷却, 并倒入每 10 cm 培养皿30毫升的琼脂溶液。琼脂凝固后, 将盘子储存在4和 #176; C 为1月. </li>
	<li> 使用13.4 克/升 Na 2 HPO 4 和 #183, 7H 2 O 和6.8 克/l. 2 PO 4 在去离子水中准备10x 磷酸盐缓冲 (PB)。将10x 库存存储在4和 #176; c. 用去离子水稀释至 1x. </li>
	<li> 用 2 mm MgSO 4 和 0.2 mm CaCl 2 来补充 1X PB 的数据库缓冲区。
	<li> 在去离子水中准备100毫米咖啡因。存储在 #8722; 20 和 #176; C. </li>
	<li> 在去离子水中溶解一水合物营钠盐, 准备100毫米的营地库存解决方案。在去离子水中准备一个1毫米的工作库存。存储两个股票解决方案在和 #8722; 20 和 #176; c. 准备最后的阵营解决方案在实验当天在 DB 所需的浓度. 
	注: 股票解决方案可以冷冻解冻几次. </li>
	<li> 在 1x PB 中制备25毫米叶酸。加入几滴 1 N 氢氧化钠, 直到粉末完全溶解。存储在 #8722; 20 和 #176; C. </li>
	<li> 使用以下方法准备3x 示例缓冲区: 187.5 毫米三盐酸盐 pH 6.8, 6% (含钒) 十二烷基硫酸钠 (SDS), 30% 甘油, 126 毫米糖, 0.03% (w/v) 溴蓝色。分和 #8722; 20 和 #176; C. 
	<ol> <li> 在使用前, 在37和 #176 中进行解冻; C 水浴, 并通过稀释3x 样品缓冲液至 1x, 通过蛋白酶和磷酸酶抑制剂制备样品缓冲液, 并加入50毫米氟, 2 毫米 Na 3 VO 4 , 25 mm 焦磷酸钠, 和1x在去离子水中完成无 EDTA 蛋白酶抑制剂鸡尾酒。在启动步骤 3.1. 2-3. 1.3 中描述的过程之前, 立即添加抑制剂. </li>
	</ol> </li> <li> 在亚甲基亚砜中准备 5 mM Latrunculin 解决方案。分和 #8722; 20 和 #176; C. </li>
</ol> 2。 D. 柄 单元格的准备 注意: 在组织培养板或悬浮培养中维护 d. 柄 细胞 HL-5 介质, 如前所述 23 。必要时, 根据标准的电穿孔协议 24 转换带有荧光标记的生物传感器的单元格。不同的 柄 菌株, 以及质粒编码生物传感器或其他感兴趣的基因可以从 Dicty 的股票中心 (dictybase.org) 获得. 
<ol> <li> 生长和收集营养物 D. 柄 细胞 <ol> <li> 用于分析营养细胞, 在细菌存在下生长细胞以减少 macropinosomes 的数量, 这通常在axenically 生长的细胞 25 。
		<ol> <li> 通过从甘油储存或从以前的培养基中接种少量的细胞, 并在不使用抗生素的情况下, 将杆菌的培养基培养成所需的 HL-5。在室温 (20-22 和 #176; C) 上, 在 200 rpm (0.42 x g) 的轨道振动筛上孵育细菌培养 16-18 h. 
			注意: K 。此处使用的 杆菌 应变是致病 (请参见 材料表 )。
		</ol> </li> <li> 在指数增长阶段从组织培养板或悬浮培养中收集 D. 柄 , 并根据制造商和 #39 的说明使用例计数单元格。将 1 x 10 5 D. 柄 单元格扩展为260和 #181; L K. 杆菌 悬浮在 SM 板上。第二天把盘子倒过来。在室温下生长细胞直到 柄 细胞开始清除细菌草坪, 但在它们开始聚集之前 (约36-48 小时). </li>
		<li> 收集 D. 柄 5 mL DB 缓冲区中的单元格, 用玻璃吊具将其刮掉。将电池转移到50毫升聚丙烯管。用另外5毫升 DB 缓冲液冲洗盘子, 并与原来的悬浮液池。用 DB 缓冲器将管子装入50毫升. </li>
		<li> 离心 D. 柄 悬浮在 360 x g 处, 3-4 分钟, 吸气上清, 重50毫升 DB 缓冲液中的颗粒。重复洗涤步骤, 直到清液清除 (〜3至4洗)。重在 DB 缓冲区中的最后一个单元格, 最终密度为 ~ 5 x 10 6 单元/mL. </li>
	</ol> </li> <li> 准备聚合-称职的 d. 柄 单元格 <ol> <li> 开发 d. 柄 细胞, 由1小时饿死, 然后是4小时的饥饿和脉冲 50 nM 营地每6分钟按标准协议 23 . </li>
		<li> 后续开发, 测量单元悬浮的最终体积. </li>
		<li> 基于用于开发的单元格的初始数目, 计算最终悬浮的单元格密度. 
		注: 如果营地是在100和 #181 交付; L 每6分钟, 细胞体积增加1毫升, 每小时的阵营脉冲。因此, 对于在4毫升 DB 中使用 8 x 10 7 单元格的标准条件, 在 4 h 开发后, 最终的体积为7毫升, 最终的细胞密度是 ~ 1.1 x 10 7 单元/ml. </li>
	</ol> </li> </ol> 3。细胞对机械或化学刺激反应的生化分析 <ol> <li> 急性机械刺激细胞其次是细胞裂解 <ol> <li> 板 2 x 10 6 聚合-主管单元格 (从步骤 2.2) 在 4 h 之后开发在35毫米的菜肴与2毫升 DB。允许细胞在室温下附加10分钟。用1毫升 DB 冲洗两次细胞。在 DB 中孵育2.5 毫米咖啡因的细胞, 30 分钟 #160; 没有任何干扰的板块. 
		注意: 如果细胞需要用一种药理抑制剂来治疗, 在 basalation 中加入适量的抑制剂或相同数量的适当的车. </li>
		<li> 应用机械刺激, 将板 (一次一次) 放在轨道振动筛上, 并立即将其以 150 rpm (0.24 x g) 为5秒. 
		注意: 该板也可以手动刺激的运动以横的方式约 5 s. </li>
		<li> 在刺激开始后的指定时间内抽吸缓冲区。立即溶解细胞, 加入100和 #181; 用蛋白酶和磷酸酶抑制剂进行样品缓冲. </li>
		<li> 将板放在冰上, 然后将裂解液收集到1.5 毫升的管子中。#39; 时间0和 #39, 溶解细胞, 不摇晃。把管子转到95和 #176; C 热阻滞后立即溶解10分钟。在-20 和 #176 进行免疫或存储裂解; C. </li>
	</ol> </li> <li> 具有趋化因子 <ol> <li> Basalate 聚合能力的单元格的激发, 在 4 h 的开发后, 通过在 200 rpm (0.42 x g) 上的 5 mM 咖啡因在轨道振动筛上迅速晃动, 以30分钟。
		<ol> <li> 将细胞悬浮在 360 x g 上, 3-4 分钟吸入上清液, 重10毫升 ice-cold DB 缓冲液中的颗粒。重复洗涤步骤. </li>
		</ol> </li> <li> 根据用于开发单元格的初始单元格编号, 将 ice-cold DB 缓冲区中的最终单元格重为 4 x 10 7 单元格/mL 的最终密度 (请参见步骤 2.2)。在 stimul 前保持细胞悬浮在冰上ation.
		<ol> <li> 吸管50和 #181; 10 和 #181 的 L; 我营或车辆进入一个在轨道振动筛放置的聚苯乙烯杯. </li>
		</ol> </li> <li> 要激发单元格, 请添加450和 #181; L ice-cold 细胞悬浮在同一杯中, 并立即打开在 200 rpm (0.42 x g) 的振动筛。在刺激开始后的不同时间 ( 例如 10, 30、45、60、120s), 简单地停止振动筛, 取出50和 #181; 等分细胞悬浮, 溶解细胞, 加入1.5 毫升管, 含25和 #181; l 3X 样本缓冲器。
		<ol> <li> #39; 时间0和 #39; 使用刺激 ice-cold 单元格中的单元格. 
			注: 在刺激过程中, 细胞悬浮的最终温度是 ~ 9 和 #176; C 26 。在这种情况下, 峰值反应发生的时间稍晚于在室温下进行的刺激所观察到的峰值 (例如, 在显微镜上对附着细胞的趋化刺激, 或对附着细胞的机械刺激). </li>
		</ol> </li> <li> 将管道传输到95和 #176; C 热块在溶解后立即进行10分钟。在-20 和 #176 进行免疫或存储裂解; C. </li>
	</ol> </li> <li> 分析由免疫 <ol> <li> 运行裂解 (从步骤3.1.3 或 3.2.4) 对4-15% 的三盐酸聚丙烯酰胺凝胶, 转移到聚偏氟乙烯膜, 块, 免疫与磷-PKC#950; Thr410 (用于检测 phospho-PKBR1 和磷 PKBA)。用辣根过氧化物酶-共轭兔二次抗体进行孵化, 其次是化学发光法, 利用增强的化学发光基底检测信号. </li>
		<li> 用于检测多种蛋白质, 用剥离缓冲条剥去印迹, 崩与 phospho-p42/44 MAPK Thr302/304 的主要抗体 (检测 phospho-ERK2)。用马斯亮艳蓝对聚偏氟乙烯膜进行染色, 确定等量的蛋白质负荷. </li>
	</ol> </li> </ol> 4。显微镜下单细胞的急性机械刺激和活体成像 <ol> <li> 使用流设备对急性机械刺激反应的评估 <ol> 收集和稀释营养或聚集能力按步骤2.1 和2.2 分别描述的 DB 中的单元格为 1 x 10 6 单元/mL。加载〜600和 #181; L 进入幻灯片与通道 (请参见 材料表 以了解详细信息)。允许单元格附加10分钟, 确保幻灯片中的所有入口都已完全填满。如有必要, 顶部有额外的缓冲器. 
		注: 用于分析的特定幻灯片有三输入, 它们将进窄、1 mm 通道, 然后合并到一个宽、3 mm 通道。海峡的高度是0.4 毫米。虽然单元格在整个通道中都是电镀的, 但只有宽部分是成像的. </li>
		<li> 通过流体单元的前右阀将连接到50毫升油藏的一条线传递给该管道 (请参阅 材料表 了解详细信息)。使用该泵的软件, 确保关闭阀门 ( 即 到左侧)。用 DB 填满水库。
		<ol> <li> 将压力设为50毫巴, 并将其打开。通过单击阀门打开它, 用 DB 填充和冲洗线。将压力恢复到 0, 并在三十年代后关闭阀门. </li>
		</ol> </li> <li> 将该行连接到幻灯片一侧的三个入口之一, 而不会捕获任何气泡。插入其他两个入口。将管线从排水管连接到幻灯片第二面的单个入口. 
		注: 此设置中用于输入和排泄管线的油管内径为 1.6 mm. </li>
		<li> 将幻灯片放在显微镜下20X 空气目标下。要冲洗通道, 请切换阀门以打开线路, 然后单击和 #34;P ressure #34;, 从更高的压力 (〜50毫巴或〜 40 dyn/cm 2 ) 开始, 将液体推入排水管。
		<ol> <li> 一旦液体从排水管流出, 将外部压力降低到零 ( i. e . 重力流只, 〜 15 dyn/cm 2 ), 并继续漂洗为〜三十年代. 将阀门切换到相反的位置以停止流。
		</ol> </li> <li> 以3秒为间隔在40X 油目标下的倒置荧光显微镜下获取带有 RFP 或 GFP 光照的图像。与阀门在闭合的位置, 转动压力在期望压力, 典型地在15和 40 dyn/cm 之间 2 (0-50 毫巴). </li>
		<li> 在获得5帧后, 通过将阀门切换至 #34 来提供刺激; 打开和 #34; 位置。通过将阀门切换到相反的方向, 在2-5 秒后关闭流. 
		注意: 对于某些较弱的生物传感器 ( 例如 PTEN、CynA 和 RalGDS), 可能需要使用装有40X 油目标的共聚焦显微镜来成像细胞. </li>
	</ol> </li> <li> 测试药理抑制剂对急性机械刺激反应的影响使用流设备 <ol> <li> 板在幻灯片中的单元格, 如步骤4.1.1 中所述。设置两条线: 通过一条线通过前右阀为步骤 4.1.2, 第二个通过后左阀。钳住左线, 将阀门放在 #34; 闭合和 #34; (左) 位置。用包含适当的车辆的 DB 填充一条线, 第二个包含有兴趣的药理抑制剂的溶液 ( 例如 5 和 #181; M Latrunculin a). 
		注: 由于 #34; 闭合和 #34; 左位置为该线打开阀门, 因此需要对左侧线进行物理钳. </li>
		<li> 通过在50毫巴上打开压力并将阀门切换至 #34, 来填充和冲洗右线; 打开和 #34; (右) 位置。三十年代后将阀门向左切换. 通过取下钳为〜三十年代填补和冲洗左线. 用通道将两条线连接到幻灯片一侧的两个入口, 插入剩余的入口, 并将排水管连接到第二侧的单个入口。<li> 在正确的行中用缓冲区清洗幻灯片, 并在上面的步骤4.1.3 和4.1.4 中描述的相同缓冲区中执行刺激. 
		注意: 虽然在本设置中选择了右行作为第一个, 但该顺序可以反转. </li>
		<li> 下面的刺激, 开关阀门打开在零压力的右线 ( 即 重力流只, 〜 15 dyn/cm 2 )。从左线卸下夹具, 并将阀门向左切换以打开该线。夹住第一条线. </li>
		<li> 经过3-5 毫升的缓冲液通过抑制剂, 开关阀门的权利, 以停止流动, 并孵育的细胞与抑制剂为所需的时间长度 ( 如 15 分钟)。通过切换阀门每〜10分钟〜十五年代, 以防止氧气匮乏的流量。重复刺激与第二行. </li>
	</ol> </li> <li> 替代方法, 用于评估对急性机械刺激的响应使用微 <ol> <li> 根据标准协议 23 设置微填充数据库。将补偿压力保持在1500磅. </li>
		<li> 收集和稀释2.1 步中描述的营养细胞。放置25和 #181; 7.5 x 10 5 单元/ml 在1井室中, 允许至少坚持10分钟, 并覆盖 3 mL DB. </li>
		<li> 将腔室放在装有40X 油目标的倒置荧光显微镜上。找到单元格。轻轻地把微到视野的中间, 直到它第一次触及到房间的底部. 
		注: 由于补偿压力设置为 1500 Psi, 因此细胞将持续暴露在微的非常缓慢的 DB 流中. </li>
		<li> 以3秒的间隔开始获取带有 RFP 或 GFP 照明的图像。应用 #39; 清洁和 #39; 功能从微中释放出一团液体。继续映像 i由于单独的补偿压力, 流动的存在. </li>
	</ol> </li> <li> 一种用于评估对急性机械刺激的响应的替代方法, 使用批量缓冲区添加 <ol> <li> 收集和稀释营养细胞, 如步骤2.1 所述。放置20和 #181; L 在 1 x 10 6 单元/mL 中的单元格中, 从8井室中的一个井的中间滴下。允许单元格至少保持 10 min. </li>
		<li> 以3秒为间隔, 在倒置荧光显微镜上开始获取带有 RFP 或 GFP 特定光照的图像。专注于靠近水滴边缘的区域. </li>
		<li> 快速添加430和 #181; L DB 到井的一侧。继续成像. </li>
		<li> 为分析机械和化学刺激之间的相互作用, 12 或四十五年代以下机械刺激, 轻轻地添加50和 #181; L 的叶酸 (最终浓度 20 nM) 或车辆 (DB), 而不诱发机械反应。继续成像. </li>
	</ol> </li> <li> 响应的量化 <ol> <li> 在图像分析软件中打开32位 TIFF 图像 (请参阅 材料表 以了解详细信息) 27 。
		<li> 下 #39; 分析和 #39; tab, 转到 #34; 设置测量和 #34;。检查框 #39; 平均灰度值和 #39;。确保未选中其他框。单击并 #34; #34... </li>
		<li> 下 #39;P 工艺和 #39; 选项卡上, 单击和 #34; 减去背景和 #34;。保持滚动球半径为50像素, 并且不要检查菜单中列出的任何选项。使用和 #39 放大一个单元格; 放大玻璃和 #39; 工具. </li>
		<li> 使用 #39; 矩形和 #39; 工具, 在胞中绘制一个框 (〜 2.5 x 2.5 和 #181; m 2 ), 确保不将其绘制在核或等离子膜上。#34; #34; #34; #34; 钥匙或转到 #39; 分析和 #39; 选项卡并单击 #34; 测量和 #34; 确定框的平均灰度值。前进到后续帧和测量再次, 确保该框停留在胞. 
		注意: 由于 D. 柄 单元格移动非常快, 因此该框可以从一个框架移动到下一个帧, 以将其保留在胞中. </li>
		<li> 在分析完所有框架后, 将这些值复制到电子表格中。为了解释各种生物传感器的表达水平的细胞变化, 对0时观测到的平均灰度值进行规范化。计算值的倒数以反映信号在皮层上的积累. </li>
	</ol> </li> </ol> 5。显微镜下单个细胞的连续机械刺激和活成像 <ol> <li> 按照上述方法设置精确的单元格, 以便在步骤 4.1.1-4.1.3 中进行急性机械刺激分析。打开的插头覆盖的水库与缓冲区, 使音量可以突破, 如果响应分析超过几分钟的时间. 
	注: 由于储油层在实验期间保持开放状态, 因此该方法在没有外部压力的情况下进行, 仅依靠重力流。为了增加重力的流量, 排水管可以放在显微镜下的桌子上. </li>
	<li> 在倒置荧光显微镜上使用 RFP 或 GFP 特定的光照, 在3秒的时间间隔内开始成像细胞, 用于分析生物传感器的反应。或者, 在十年代间隔使用20X 目标的图像进行相位对比, 以分析整个细胞迁移. </li>
	<li> 在零压力设置的几个帧之后打开流 ( 即, 通过将阀门切换到打开位置, 可使 流仅由于重力或 15 dyn/cm 2 )。当流体的水平降到5-10 毫升, 在水库, 顶部与更多的缓冲区。一定要小心地添加液体, 这样气泡就不会被困在线路中. 
	注意: 在细胞中添加相同温度的液体是很重要的, 以避免休克反应. </li>
	<li> 根据制造商和 #39 的说明, 使用迁移分析软件对单元格速度和持久性进行量化 (参见 材料表 获取详细信息). </li>
</ol>

<ol>
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作者没有什么可透露的。

这里描述的方法提供了一个方便的方法来评估人口和个别细胞的行为, 以响应剪切流。重要的是, 虽然以前的研究分析了在迁移过程中领先和滞后边缘标记的定位, 但目前的方法可以通过应用切变流来研究直接的影响。使用这种方法我们证明, 事实上, 细胞对切变流的初始响应不需要细胞迁移。相反, 对初始切变流刺激的快速和瞬态响应之前, 长期暴露于切变流的方向迁移, 类似于初始的全球响应, 以?...

真核细胞的迁移在环境中有不同的化学和物理暗示, 包括可溶解的或基质结合的化的梯度, 基板的变刚度, 电场, 或剪切流。虽然我们对推动趋化的分子机制的理解有许多进展, 但对于其他类型的定向迁移以及这些不同的信号是如何整合在细胞层面以产生统一的迁徙响应.
定向迁移向日益集中的趋化因子涉及三行为成分: 运动, 定向传感, 和极性1。运动是指由伪足突起所实现的细胞的随机运动。定向传感是细胞检测趋化因子的来源的能力, 即使在固定细胞中也能发生。极性是指在细胞的前缘和后缘之间更稳定的胞内成分的不对称分布, 从而导致运动中的持续性增加。
细胞趋化反应取决于四概念上定义的调控网络的活动: 受体/G 蛋白, 信号转导, 肌动蛋白骨架, 和极性1。趋化与 g 蛋白偶联受体结合, 通过聚 g 蛋白α和βγ向下游信号转导网络传递信号, 从而放大方向性信号。信号传导网络中的多个通路在平行的作用下, 进入肌动蛋白骨架网络, 以偏压肌动蛋白聚合, 并随之伪足突起, 在梯度方向。在趋化的重要调控者中有 Ras 酶、TorC2、肌3激酶 (PI3K)、磷酸酶和张力同源 (PTEN) 和 guanylyl 苷。信号传导网络中的反馈机制以及信号转导与肌动蛋白细胞骨架网络之间的联系进一步放大了反应。最后, 定义差的极性网络接收来自肌动蛋白骨架的输入, 并进一步偏向信号转导网络, 以促进梯度方向的持续迁移。
我们对趋化性的许多机械理解之所以成为可能, 是因为开发了荧光标签的生物传感器, 用于监管网络的各个组成部分。许多趋化调控分子本身或其活动的不对称分布。例如, 能够识别小酶 ras 和 Rap1 的激活版本的生物传感器 (此处称为 RBD) 和 RalGDS 的 ras 绑定域, 分别定位到 chemotaxing 单元的前导边缘2,3。同样, PI3K 及其产品醇 (34、5)-trisphosphate (PIP3), 由 pleckstrin 同源 (PH) 域识别, 也显示在单元格45前面的本地化。相比之下, 3-磷酸酶 PTEN, 它将 PIP3 回醇 (45)--1,6-, 本地化的滞后边缘的细胞6。重要的是, 这些生物传感器改变他们的本地化响应全球刺激与趋化。领先的边缘标记, 这是胞浆或在休息细胞突起的提示, relocalize 到皮层, 而滞后的边缘标记, 其中有皮质定位, 并没有从突起的提示在休息细胞, 成为胞浆后刺激.生物传感器在全球刺激作用下的分布分析最小化运动和极性的贡献, 这往往混淆的意见。全球或统一的刺激细胞悬浮与趋化因子也被用来作为一个工具, 以评估整个种群的蛋白质活化变化, 通常检测蛋白磷酸化7,8,9.这种生化检测主要用于获取时间信息, 而显微镜则用于收集有关监管网络各组成部分行为的时间和空间信息。
信号转导网络包含易激动的系统的特征10,11。对超阈值趋刺激的反应是 "全有-无" 和显示耐火期。响应也会随机触发, 并且可以显示振荡行为。信号转导事件是本地化的皮层的区域, 传播为波12,13,14,15。前面, 或后面, 标记被招募, 或脱离, 活动区域的传播波。由于在活动区尾部的耐火区域, 相反的定向波湮灭, 因为他们满足。传播信号传导波的基础上的细胞突起, 调解细胞迁移10。
上述关于趋化性的许多信息来自于对社会阿米巴的研究网柄, 虽然类似的调节机制也适用于中性粒细胞和其他哺乳动物细胞类型16 .网是一个成熟的模型生物体, 在饥饿时有一个强健的趋反应, 当数以千计的单细胞向聚集中心迁移时, 最终形成一个含有孢子的多细胞子实体。在这个有机体的单细胞生长阶段, 趋化性也是至关重要的, 用于定位细菌的食物来源。重要的是, 单个网细胞的迁移非常类似于哺乳动物中性粒细胞或转移性癌细胞的迁移, 所有这些都经历了非常迅速的变形型迁移。事实上, 在网和哺乳动物白细胞17之间, 调控网络的整体拓扑以及参与趋化的各个信号转导通路都是守恒的。此外, 其他细胞, 如成纤维蛋白, 使用受体酪氨酸激酶 (RTK) 而不是受体;然而, 受体可能会进入类似的网络。
与趋化性相反, 对驱动各种其他定向迁移模式的信号机制的透彻理解是缺乏的。类似于在趋化梯度渐变中迁移的细胞, 一些研究报告了典型的前缘标记的活化和/或定位, 包括肌动蛋白聚合、PIP3 和/或胞外信号调节激酶 (ERK) 1/2, 在在响应剪切流或电场变化的情况下接受定向偏移的单元格前面18,19,20,21。然而, 在这些研究中, 持续接触刺激物也会导致细胞迁移, 这就导致了一个问题, 例如, 领先的边缘标记是否专门针对刺激的反应而定位, 或者它们是否只是在前沿因为迁移单元前面的 pseudopods 数量增加。
我们开发的化验, 让我们观察细胞对急性机械摄动的反应, 作为剪切流在人口水平和作为单个细胞22。类似于全球刺激与趋化, 急性 stimula剪切流允许研究的细胞反应的机械刺激, 而不是混杂的贡献, 从运动或极性。结合这些生物化学和微观的检测与遗传或药理的扰动, 让我们了解如何机械刺激的感知和传播。此外, 这种方法还提供了一种新的方法, 以攻入系统下游的趋化因子受体在没有一个趋化因子, 从而隔离信号转导和肌动蛋白骨架网络从受体/G蛋白质网络。
使用下面描述的技术, 我们最近证明, 急性剪应力导致趋信号转导和肌动蛋白骨架网络的多个组成部分的激活22。通过在不同的时间间隔应用急性机械刺激, 我们证明, 类似于化, 对机械刺激的反应也表现出兴奋系统的特征, 包括在饱和条件和不耐期的存在。最后, 通过结合机械和化学刺激, 我们发现, 这两个刺激共享信号转导和肌动蛋白细胞骨架网络, 这可能允许集成的多重刺激的偏见细胞迁移。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dextrose</td><td>Fisher</td><td>D16-1</td><td>Use to prepare HL-5 media and SM plates (steps 1.1, 1.2)</td></tr><tr><td>Proteose peptone</td><td>Fisher</td><td>DF0120-17-6</td><td>Use to prepare HL-5 media (step 1.1)</td></tr><tr><td>Yeast extract</td><td>Fisher</td><td>50-550-445</td><td>Use to prepare HL-5 media and SM plates (steps 1.1, 1.2)</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher</td><td>S373-500</td><td>Use to prepare HL-5 media, and 10X Phosphate Buffer (steps 1.1, 1.3)</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>P386-500</td><td>Use to prepare HL-5 media, SM plates, and 10X Phosphate Buffer (steps 1.1-1.3)</td></tr><tr><td>Streptomycin (dihydrostreptomycin sulfate)</td><td>Fisher</td><td>ICN10040525</td><td>Use to prepare HL-5 media (step 1.1)</td></tr><tr><td>Peptone (Bacto)</td><td>Fisher</td><td>DF0118-17-0</td><td>Use to prepare SM plates (step 1.2)</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>P288-100</td><td>Use to prepare SM plates (step 1.2)</td></tr><tr><td>Agar</td><td>Fisher</td><td>BP1423-500</td><td>Use to prepare SM plates (step 1.2)</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>M65-500</td><td>Use to prepare DB Buffer (step 1.4)</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>C79-500</td><td>Use to prepare DB Buffer (step 1.4)</td></tr><tr><td>Caffeine</td><td>Fisher</td><td>O1728-500</td><td>Use to prepare caffeine (step 1.5)</td></tr><tr><td>cAMP (EMD Millipore Calbiochem Adenosine 3 ft.,5 ft.-cyclic Monophosphate, Sodium Salt)</td><td>Fisher</td><td>11-680-1100MG</td><td>Use to prepare cAMP (step 1.6)</td></tr><tr><td>Folic acid</td><td>Sigma</td><td>F7876</td><td>Use to prepare folic acid (step 1.7)</td></tr><tr><td>Tris base</td><td>Fisher</td><td>BP152-500</td><td>Use to prepare 3X sample buffer (step 1.8)</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Fisher</td><td>BP166-500</td><td>Use to prepare 3X sample buffer (step 1.8)</td></tr><tr><td>Glycerol</td><td>Fisher</td><td>G33-500</td><td>Use to prepare 3X sample buffer (step 1.8)</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Bio-Rad</td><td>1610610</td><td>Use to prepare 3X sample buffer (step 1.8)</td></tr><tr><td>Bromophenol blue</td><td>Bio-Rad</td><td>1610404</td><td>Use to prepare 3X sample buffer (step 1.8)</td></tr><tr><td>NaF</td><td>Fisher</td><td>S299-100</td><td>Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8)</td></tr><tr><td>Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>50-994-911</td><td>Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8)</td></tr><tr><td>Sodium pyrophosphate decahydrate</td><td>Fisher</td><td>S390-500</td><td>Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8)</td></tr><tr><td>Complete EDTA-free protease inhibitor cocktail (Roche)</td><td>Sigma</td><td>11873580001</td><td>Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8)</td></tr><tr><td>Latrunculin A</td><td>Enzo Life Sciences</td><td>BML-T119-0100</td><td>Use to prepare Latrunculin A (step 1.9)</td></tr><tr><td>4-15% Tris-HCl polyacrylamide gel</td><td>Bio-Rad</td><td>3450029</td><td>Use for immunoblotting (step 3.3)</td></tr><tr><td>Polyvinylidene fluoride membrane</td><td>Bio-Rad</td><td>1620177</td><td>Use for immunoblotting (step 3.3)</td></tr><tr><td>Phospho-PKC (pan) (zeta Thr410) (190D10) Rabbit antibody</td><td>Cell Signaling</td><td>2060</td><td>Primary antibody for use in immunoblotting (step 3.3)</td></tr><tr><td>Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP antibody</td><td>Cell Signaling</td><td>4370</td><td>Primary antibody for use in immunoblotting (step 3.3)</td></tr><tr><td>Rabbit IgG HRP Linked Whole Ab (from Donkey)</td><td>GE Healthcare</td><td>NA934-100UL</td><td>Secondary antibody for use in immunoblotting (step 3.3)</td></tr><tr><td>Enhanced chemiluminescence substrate (Clarity Western ECL Substrate)</td><td>Bio-Rad</td><td>1705060</td><td>Use for immunoblotting (step 3.3)</td></tr><tr><td>Stripping buffer (Restore Plus Western blot stripping buffer)</td><td>Pierce</td><td>PI46430</td><td>Use for immunoblotting (step 3.3)</td></tr><tr><td>Coomassie Brilliant Blue</td><td>Fisher</td><td>PI20278</td><td>Use for immunoblotting (step 3.3)</td></tr><tr><td>K. aerogenes strain (non-pathogenic)</td><td>Dicty Stock Center (dictybase.org)</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials and Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Orbital shaker (model G-33, or a comparable alternative)</td><td>New Brunswick Scientific</td><td></td><td>Use to grow &lt;em&gt;K. aerogenes&lt;/em&gt; (step 2.1.1), and to basalate and stimulate &lt;em&gt;D. discoideum&lt;/em&gt; cells (step 3). The radius of gyration for this shaker is 9.5 mm.</td></tr><tr><td>10 cm Petri dish</td><td>Fisher</td><td>FB0875712</td><td>Use to prepare SM plates (step 1.2)</td></tr><tr><td>Hemocytometer</td><td>Fisher</td><td>02-671-51B</td><td>Use to count &lt;em&gt;D. discoideum&lt;/em&gt; cells (step 2.1.2)</td></tr><tr><td>35 mm dish (Corning Falcon Easy-Grip Tissue Culture Dish)</td><td>Fisher</td><td>08-772A</td><td>Use to plate &lt;em&gt;D. discoideum&lt;/em&gt;cells for mechanical stimulation followed by cell lysis (step 3.1.1)</td></tr><tr><td>Polystyrene cup (5 mL)</td><td>VWR</td><td>13915-985</td><td>Use to stimulate &lt;em&gt;D. discoideum&lt;/em&gt; cell suspension with a chemoattractant (step 3.2.2)</td></tr><tr><td>Fluidic unit with pump (ibidi Pump System)</td><td>Ibidi</td><td>10902</td><td>Use to deliver mechanical stimulation to &lt;em&gt;D. discoideum&lt;/em&gt; cells in a channel (steps 4.1, 4.2, and 5)</td></tr><tr><td>Slide with a channel (&amp;mu;-Slide III 3in1 ibiTreat: #1.5 polymer coverslip, tissue culture treated, sterilized)</td><td>Ibidi</td><td>80316</td><td>Use to plate &lt;em&gt;D. discoideum&lt;/em&gt; cells for mechanical stimulation delivered by a fluidic device (steps 4.1, 4.2, and 5)</td></tr><tr><td>50 mL reservoirs (Ibidi Reservoir Holder for Fluidic Unit)</td><td>Ibidi</td><td>10978</td><td>Use to setup the fluidic device for delivering mechanical stimulation to &lt;em&gt;D. discoideum&lt;/em&gt; cells in a channel (steps 4.1, 4.2, and 5)</td></tr><tr><td>Lines for the fluidic unit (Perfusion Set YELLOW-and-GREEN)</td><td>Ibidi</td><td>10964</td><td>Use to setup the fluidic device for delivering mechanical stimulation to &lt;em&gt;D. discoideum&lt;/em&gt; cells in a channel (steps 4.1, 4.2, and 5). Note: the two lines can also be cut from the 1.6 mm tubing (catalog number 10842).</td></tr><tr><td>Line for the drain (1.6 mm ID tubing)</td><td>Ibidi</td><td>10842</td><td>Use to setup the fluidic device for delivering mechanical stimulation to &lt;em&gt;D. discoideum&lt;/em&gt; cells in a channel (steps 4.1, 4.2, and 5).</td></tr><tr><td>Zeiss Observer.Z1 inverted microscope equipped with a 40X/1.3 oil objective and a 20X/0.3 air objective (or a comparable alternative)</td><td>Zeiss</td><td></td><td>Used to image &lt;em&gt;D. discoideum&lt;/em&gt; cells with phase-contrast or fluorescence illumination (steps 4 and 5)</td></tr><tr><td>UltraView spinning disk confocal microscope equipped with a 40&amp;times;/1.25&amp;ndash;0.75 oil objective (or a comparable alternative)</td><td>Perkin-Elmer</td><td>DM 16000</td><td>An alternative used to image &lt;em&gt;D. discoideum&lt;/em&gt; cells (step 4.1.4)</td></tr><tr><td>FemtoJet Microinjector (Eppendorf; model 5247, or a comparable alternative) with Eppendorf InjectMan</td><td>Eppendorf</td><td>5252000021D and 5192000027</td><td>Use to deliver mechanical stimulation to &lt;em&gt;D. discoideum&lt;/em&gt; cells by a micropipette (step 4.3). Note: catalog number are for the newest version of the equipment (4i).</td></tr><tr><td>Micropipette (Femtotip; 1 &amp;mu;m outside diameter, 0.5 &amp;mu;m inside diameter )</td><td>Eppendorf</td><td>930000035</td><td>Use to deliver mechanical stimulation to &lt;em&gt;D. discoideum&lt;/em&gt; cells by a micropipette (step 4.3)</td></tr><tr><td>Microloader tips (Eppendorf Femtotips Microloader Tips for Femtojet Microinjector)</td><td>Eppendorf</td><td>5242956.003</td><td>Use to fill the micropipette (femtotip) with buffer (step 4.3.1)</td></tr><tr><td>1-well chamber (Thermo Scientific Nunc Lab-Tek Chambered Coverglass)</td><td>Fisher</td><td>12-565-472</td><td>Use to plate &lt;em&gt;D. discoideum&lt;/em&gt; cells for mechanical stimulation delivered by a micropipette (step 4.3.2)</td></tr><tr><td>8-well chamber (Thermo Scientific Nunc Lab-Tek II Chambered Coverglass)</td><td>Fisher</td><td>12-565-338</td><td>Use to plate &lt;em&gt;D. discoideum&lt;/em&gt; cells for mechanical stimulation delivered by bulk buffer addition (step 4.4.1)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Image Analysis Software (Fiji)</td><td>NIH</td><td>https://fiji.sc/</td><td>Use to quantify response to mechanical stimulation (step 4.5)</td></tr><tr><td>Migration analysis software (Tracking Tool PRO)</td><td>Gradientech</td><td>http://gradientech.se/tracking-tool-pro/</td><td>Use to quantify cell speed and persistence (step 5.4)</td></tr></tbody></table>

assessment of dictyostelium discoideum response to acute mechanical stimulation

趋化性, 或迁移上升的梯度, 是最了解的定向迁移模式。研究使用社会阿米巴虫网柄显示, 一个复杂的信号转导网络的平行路径放大的反应, 化, 并导致有偏见的肌动蛋白聚合和突起的伪足在渐变的方向。相比之下, 其他类型的定向迁移的分子机制, 例如, 由于接触剪切流或电场, 是未知的。许多趋化性的调控者在迁移细胞的前缘或滞后边缘表现出定位, 以及在全球刺激与趋化因子的作用下, 显示局部或活化的瞬态变化。为了了解其他类型的定向迁移的分子机制, 我们开发了一种方法, 允许在短 (2-5 秒) 接触剪切流的基础上检测细胞对急性机械刺激的反应。这种刺激可以提供一个通道, 而成像细胞表达荧光标记的生物传感器, 以检查个别细胞的行为。此外, 细胞的数量可以刺激在一个板块, 裂解, 和 immunoblotted 使用抗体, 识别活性蛋白的兴趣。通过结合这两种化验, 你可以检查一个广泛的分子组的变化激活的亚细胞定位和/或磷酸化。使用这种方法, 我们确定急性机械刺激触发激活的趋信号转导和肌动蛋白细胞骨架网络。检测细胞对急性机械刺激的反应的能力对于理解剪切流诱导运动所需的启动事件是非常重要的。该方法还为研究趋信号转导网络提供了工具, 而不影响趋化因子受体的混杂作用。

趋化调节剂对急性机械刺激的时间响应
为了评估柄细胞对机械刺激的反应, 粘附聚集能力的细胞被暴露在剪切流的短暂脉冲中。聚合-称职的柄细胞分泌营地, 它可以被相邻的细胞感知。为了克服细胞信号的贡献, 细胞治疗咖啡因, 抑制腺苷在D. 柄, 从而防止难民营分泌的28。事实上, 细胞预处?...

Watch this Scientific Journal Video about Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation at JoVE.com

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

对急性机械刺激的网柄反应的评估

在这里, 我们描述了评估急性机械刺激的细胞反应的方法。在 microscopy-based 试验中, 我们研究了荧光标记的生物传感器的局限性, 并对剪切流进行了简单的刺激。我们还测试了各种感兴趣的蛋白质的活化反应的急性机械刺激生化。

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

assessment-dictyostelium-discoideum-response-to-acute-mechanical

Research

JoVE Journal

Biology

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

6.9K 次观看.  SUNY Oswego. 在这里, 我们描述了评估急性机械刺激的细胞反应的方法。在 microscopy-based 试验中, 我们研究了荧光标记的生物传感器的局限性, 并对剪切流进行了简单的刺激。我们还测试了各种感兴趣的蛋白质的活化反应的急性机械刺激生化。

视频: Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

基于细菌选择和生长的盘状细胞基因工程

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

科研

遗传学

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain2, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS)3,4. Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity5.
A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method6 to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum7 as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria.
CP/CPPS is primarily characterized by the presence of tactile allodynia4. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain8-10. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.

细菌性前列腺炎小鼠模型的测量触觉异常

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

免疫与感染

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

神经科学

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

测量 果蝇 拉瓦的机械结节的改进的检测和工具

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

行为学

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

在小鼠大肠传入结尾的体外功能研究

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

医学

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

生物学

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

体外大鼠结肠传入纤维的电生理特性

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

利用微流体装置进行机械刺激和高分辨率成像的研究C.线虫

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular development program, during which single cells chemotax towards aggregation centers1-4. This process is dependent on the release of cyclic adenosine monophosphate (cAMP)5. cAMP is produced in waves through the concerted action of adenylate cyclase and phosphodiesterases, and binds to G protein-coupled cAMP receptors6,7. A widely used assay to analyze the mechanisms involved in the developmental cycle of the lower eukaryote Dictyostelium discoideum is based on the observation of cell aggregation in submerged conditions8,9. This protocol describes the analysis of the role of coronin A in the developmental cycle by starvation in tissue-culture plates submerged in balanced salt solution (BSS)10. Coronin A is a member of the widely conserved protein family of coronins that have been implicated in a wide variety of activities11,12. Dictyostelium cells lacking coronin A are unable to form multicellular aggregates, and this defect can be rescued by supplying pulses of cAMP, suggesting that coronin A acts upstream of the cAMP cascade10. The techniques described in these studies provide robust tools to investigate functions of proteins during the initial stages of the developmental cycle of Dictyostelium discoideum upstream of the cAMP cascade. Therefore, utilizing this aggregation assay may allow the further study of coronin A function and advance our understanding of coronin biology.

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

调查Coronin A在早期饥饿反应中的作用<em&gt;粘菌</em&gt;通过聚集测定

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

研究蛋白质的质量控制体系<em&gt;Ð。菌</em&gt;使用温控活细胞成像

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

对急性机械刺激的网柄反应的评估

摘要

探索更多视频

此视频中的章节

基于细菌选择和生长的盘状细胞基因工程

细菌性前列腺炎小鼠模型的测量触觉异常

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

测量果蝇拉瓦的机械结节的改进的检测和工具

在小鼠大肠传入结尾的体外功能研究

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌

体外大鼠结肠传入纤维的电生理特性

利用微流体装置进行机械刺激和高分辨率成像的研究C.线虫

调查Coronin A在早期饥饿反应中的作用

研究蛋白质的质量控制体系

对急性机械刺激的网柄反应的评估

摘要

探索更多视频

此视频中的章节

基于细菌选择和生长的盘状细胞基因工程

细菌性前列腺炎小鼠模型的测量触觉异常

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

测量 果蝇 拉瓦的机械结节的改进的检测和工具

在小鼠大肠传入结尾的体外功能研究

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌

体外大鼠结肠传入纤维的电生理特性

利用微流体装置进行机械刺激和高分辨率成像的研究C.线虫

调查Coronin A在早期饥饿反应中的作用

研究蛋白质的质量控制体系

测量果蝇拉瓦的机械结节的改进的检测和工具