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, 以趋化梯度。
尽管每种方法都有明显的优缺点, 但生物化学和显微方法却能产生互补的数据。刺激后, 细胞裂解和免疫的 PKBs, KrsB, 和 ERK, 例如, 分析了整个种群的细胞, 从而平均细胞变异, 不同的检测, 检查个别细胞的行为。然而, 基于人群的化验往往掩盖了细胞行为的细微变化, 通过分析单个细胞可以很容易地观察到。此外, 这里所描述的生化检测只对聚集能力的细胞有效, 原因将在稍后讨论。相比之下, 孵育 RBD, RalGDS, PH 裂纹, 石灰Δcoil和 PTEN, 例如, 皮质和胞之间的 microscopy-based 测定对营养和聚集能力的细胞都是有效的, 因此可以观察广泛适用于具有不同偏振度的细胞。使这两种方法相辅相成的是, 他们研究了不同的可利用的前缘/滞后边缘标记, 从而扩大了信号转导和肌动蛋白骨架网络的覆盖范围。
目前还不清楚为什么营养细胞, 它的反应非常强劲的 microscopy-based 化验, 不响应刺激后, 细胞裂解和免疫。一种可能性是, 当板块旋转时对细胞施加的剪切力与流室中的细胞所经历的力不匹配。相反, 发达细胞可能有较低的激活阈值, 因此, 在任何一种情况下都可以进行响应。这是不可能的生物传感器的活动是测量的生化检测没有表达或没有激活的营养细胞, 因为刺激的营养细胞与叶酸导致强健的激活 PKBR1/PKBA 和 ERK2 (数据不显示)。此外, 营养细胞显示明确招聘 PH 裂纹, 这反映了 PIP3 的积累, 在 microscopy-based 测定。由于 PKBA 的招募也依赖于 PIP3, 所以似乎不太可能 PKBA 不会对急性机械刺激作出反应, 除非上述的生物化学检测条件不理想。这仍然是一个积极调查的领域。
对趋化调控的生物化学分析要求在整个实验中存在咖啡因。最初添加咖啡因是为了防止细胞因分泌 cAMP 而导致细胞信号传导。然而, 似乎咖啡因有另一个作用, 除了抑制腺苷 (aca), 因为 aca-空细胞仍然需要咖啡因的磷酸化 PKBR1 反应急性机械刺激。先前已显示咖啡因可阻止 TorC2 活动26。这是可能的抑制 TorC2 阻断了一些基底运动的细胞, 从而降低了基底活化 PKBR1 和其他组成部分的信号转导和肌动蛋白骨架网络。低基底活动是观察剪切流响应的必要条件。事实上, 当细胞在发育的早期时间点被收集时, 它们表现出增加的基底活动和减少的剪切流诱发反应。有趣的是, 与发达细胞31相比, 营养细胞的 PKBA 活性和较少的 PKBR1。它是可能的基本的状态被调控有些不同地在营养和聚集能干细胞, 进一步导致缺乏反应营养细胞在生物化学的试验。奇怪的是, 当细胞被收集在后来的发展点, 他们也有一个减少的反应, 很可能是由于极性的强烈影响的本地化和激活的各种趋化调控剂在本试验中检查。
细胞在显微镜室中的刺激显示, 刺激的强度和持续时间对最大反应的贡献为22。换言之, 即使使用了很长的时间 (十年代与2秒), 也可以实现一个低剪切力的最大响应。这一观察解释了为什么细胞在第一次接触到持续而微弱的刺激, 导致剪切流诱导的迁移时, 有一个初始的全球反应。与目前的设备, 我们无法产生足够低的切变流调查的下限范围。
也许, 使用急性机械刺激进行的研究最重要的含意是, 机械和化学刺激似乎都能进入普通的信号传导和肌动蛋白骨架网络。虽然我们表明, 这两种刺激结合使用散装缓冲添加剂的机械刺激, 未来的研究将致力于开发一个系统的趋化蛋白可以迅速交付在流经渠道, 这将是一个更多功能和强大的方法来评估机械和化学刺激的整合。不幸的是, 使用当前两线设置的趋化因子加法与第二剪切流刺激有关, 因为趋化因子必须在流的存在下传递。一个可行的选择可能是激光刺激释放的趋化因子从笼前体, 成功地显示为营地的圣约翰et al。32. 在未来, 它也将是有趣的研究其他刺激的集成, 例如, 剪切流和可变电场, 或剪切流和可变衬底刚度。后一个例子很有趣, 因为它涉及两种类型的机械刺激, 虽然最初的信号接收可能会有所不同。确认所有诱发定向迁移的刺激物共享相同的信号转导网络, 并且只在刺激被感知的方式上有所不同, 这将解释细胞如何在复杂的环境中导航。最后, 我们已经证明, 急性刺激与剪切流导致的人嗜中性粒细胞的传播22。未来的工作将进一步研究的定位和激活的各种组成部分的信号转导和肌动蛋白骨架网络与机械刺激的哺乳动物细胞。

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>Reagents</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>Na2HPO4&#183;7H2O</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>KH2PO4</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>K2HPO4</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>MgSO4</td>
			<td>Fisher</td>
			<td>M65-500</td>
			<td>Use to prepare DB Buffer (step 1.4)</td>
		</tr>
		<tr>
			<td>CaCl2</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>Na3VO4</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials and Equipment</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 K. aerogenes (step 2.1.1), and to basalate and stimulate D. discoideum 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 D. discoideum 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 D. discoideumcells 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 D. discoideum 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 D. discoideum cells in a channel (steps 4.1, 4.2, and 5)</td>
		</tr>
		<tr>
			<td>Slide with a channel (&#956;-Slide III 3in1 ibiTreat: #1.5 polymer coverslip, tissue culture treated, sterilized)</td>
			<td>Ibidi</td>
			<td>80316</td>
			<td>Use to plate D. discoideum 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 D. discoideum 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 D. discoideum 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 D. discoideum 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 D. discoideum cells with phase-contrast or fluorescence illumination (steps 4 and 5)</td>
		</tr>
		<tr>
			<td>UltraView spinning disk confocal microscope equipped with a 40&#215;/1.25&#8211;0.75 oil objective (or a comparable alternative)</td>
			<td>Perkin-Elmer</td>
			<td>DM 16000</td>
			<td>An alternative used to image D. discoideum 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 D. discoideum 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 &#956;m outside diameter, 0.5 &#956;m inside diameter )</td>
			<td>Eppendorf</td>
			<td>930000035</td>
			<td>Use to deliver mechanical stimulation to D. discoideum 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 D. discoideum 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 D. discoideum cells for mechanical stimulation delivered by bulk buffer addition (step 4.4.1)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</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, 细胞预处理与咖啡因有非常低的基础活动的 PKBR1 和 ERK2, 并显示了强大的增长, 这些激酶的关键残留的磷酸化后, 5 s 刺激与剪切流 (图 1A)。反应是瞬态的, 并达到峰值约 10-15 s PKBR1, 并在三十年代左右, ERK2, 类似于先前公布的结果激活这些蛋白与趋化因子7。
虽然很难评估的效率, 考虑到低基础水平, 初步研究, 导致了这一分析的发展, 比较刺激与剪切流单独 (或与车辆) 剪切流的存在趋化阵营 (图 1B)。这一分析没有显示增加的反应与阵营, 表明信号转导网络对切变流的反应是饱和的。虽然在这个化验营没有增加磷酸化的 PKBR1, 细胞的反应趋化酶可以观察使用标准的刺激协议, 聚集能力的细胞保持在悬浮, 刺激与阵营, 并裂解在各种时间点跟随刺激。如图 1C所示, 在这些条件下, PKBR1 磷酸化的瞬时增加是由于趋化因子, 而不是车辆。在三十年代的峰值反应是观察到的, 因为在该试验中的细胞悬浮温度是〜9° c, 相比, 22 ° c 的机械刺激试验描述以上的26。
超前和滞后边缘标记对急性机械刺激的时空响应
由于上面的人口分析只提供了关于趋化调控者行为的时间信息, 所以细胞也被暴露在短暂的机械刺激中, 同时用显微镜观察。这种测定方法可以是使用聚合能力的或营养的柄细胞, 表达各种荧光标记的生物传感器, 其定位是在以趋化因子刺激的细胞中定义的。两个领先的边缘标记, PH 域的裂纹和石灰Δcoil, 这是由 PIP3 或新的聚合肌动蛋白招募, 都显示了大多数在休息细胞的胞浆本地化, 如先前所报告的 (图 2A,补充电影1)4,29。在基部活动的细胞中, 这些生物传感器也可以在 pseudopods 的提示上看到。在2秒的剪切流刺激下, 生物传感器迅速 relocalized 到6至十年代的峰值, 并由 ~ 三十年代返回到胞。相反, 一个滞后边缘标记 PTEN 在刺激之前定位在皮层 (图 2A)。经过短暂的切变流脉冲, 胞浆 PTEN 强度增加, 虽然比领先的边缘生物传感器的动力学速度慢。生物传感器定位从胞到皮质的变化, 反之亦然, 显然被视为在胞浆强度的变化, 允许简单的定量的反应。
生物传感器在响应急性机械刺激时所观察到的行为与在均匀趋化因子作用下的超前和滞后边缘定位动力学是一致的。这种行为不是唯一的三生物传感器显示。如前所述, 还观察到 RBD 和 RalGDS 的前导边缘标记 (分别识别激活的 Ras 和 Rap1) 以及另一个滞后边缘标记 CynA2230中的本地化的类似更改。
一个类似的分析可以用来评估各种抑制剂的影响, 通过简单的修改实验设置从单一的输入线到双。使用此方法, 单元格可以首先公开到控制条件, 然后切换到包含所需测试代理的缓冲区。例如, 肌动蛋白-聚药物的增加阻断了 RBD 的反应, 以及石灰Δcoil, 以急性机械刺激 (图 2B)。
在长时间刺激和剪切流作用下的细胞迁移之前的全球响应
这里描述的方法也可以用来研究剪切流对柄迁移的影响。事实上, 如果流程在2秒后没有关闭, 但在实验期间仍保持不变, 营养细胞就会显示定向迁移 (图 3, 补充电影 2)。应当指出, 引起移徙反应所需的切变流低于通常用于在营养细胞中进行急性刺激的全球反应的压力 (例如, 如图 2B所示)。但是, 即使在较低的压力下, 单元格也会显示一个可靠的一致响应, 如在单元格本身的位置发生变化之前的石灰Δcoil本地化的变化所测量的那样。因此, 这些实验清楚地表明, 1) 全球反应不依赖于细胞的移动, 2) 全球响应在定向迁移之前。
急性机械刺激试验反应的替代方法
虽然使用流动室有明显的优势, 研究的反应, 以急性机械刺激, 我们还验证了两个额外的化验测试个别细胞对急性机械刺激的反应。如图 4所示, 当单元格由微 (图 4A) 或手动使用批量提供的缓冲添加时, 石灰的Δcoil迅速和瞬时 re-localized 从胞到皮层。带吸管的缓冲器 (图 4B)。应该指出的是, 这两种化验的明显缺点是, 传递到细胞的剪切力的确切数量是未知的, 不能轻易改变。然而, 对于需要在机械和化学刺激之间切换的情况, 大容量缓冲器的加入为流动装置的刺激提供了一个坚实的替代品。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56411/56411fig1.jpg"> 
图 1: 对柄细胞对急性机械或化学刺激的反应进行生化评估的代表性结果.聚集能力的细胞暴露在机械或化学刺激, 裂解在指定的时间点, immunoblotted 使用识别磷酸化 PKBR1 或 ERK2 的抗体。(A) 在5秒的轨道振动筛上刺激附着细胞。该膜染色与马斯亮灿烂蓝 (CBB), 以显示相同的蛋白质负荷。(B) 贴壁细胞以横的方式通过手动运动刺激, 约5秒的化学刺激, 由于增加了1µM 营或缓冲 (车辆), 或没有任何化学刺激 (-)。两个 immunoblots 显示了 PKBR1 在0时的基底活化的变化程度。有时, 此技术也可以检测到 PKBA 的激活, 如下面板所示。(C) 悬浮细胞被刺激了与1µM 阵营或缓冲 (车)。该图的 (A) 部分已从 Artemenko et al中修改。(2016)22.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56411/56411fig1large.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/56411/56411fig2.jpg"> 
图 2: 在显微镜下对单个细胞进行急性机械刺激和活体成像的典型结果.(A) 聚合-有能力的D. 柄单元格, 表示 RFP 标记的石灰Δcoil, 或 GFP 标记的 PH 裂纹或 PTEN, 以 15 dyn/cm2的单向层流激发, 2 到5秒. 每3秒收集图像.显示了生物传感器在响应机械刺激时移位的代表性细胞。每个生物传感器在皮层的积累被量化为时间0的平均细胞质强度的逆。平均反应 18 (石灰Δcoil), 20 (PH 裂纹), 或 6 (PTEN) 细胞显示。值是指± SE. (B) 表达 rfp 标记的石灰Δcoil--rfp 和 GFP 标记 RBD 的营养细胞, 用车辆或5µM 至少15分钟处理, 然后用单向层流在 41 dyn/cm2上刺激2美国的图像每3秒收集一次. RBD-GFP 和石灰Δcoil-在皮质上的 RFP 积累被量化为 (A)。价值观是卑鄙的。被分析的细胞的数量在每个情况旁边被表明。缩放条 = 10 µm。此图已从 Artemenko et al.中修改(2016)22.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56411/56411fig2large.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/56411/56411fig3.jpg"> 
图 3: D. 柄细胞响应的代表性结果, 以延长对流的刺激.(A) 表达 RFP 标记的石灰Δcoil的营养细胞暴露在 15 dyn/cm2的连续单向层流流中, 每3秒都能成像. 11 单元的磁道显示在 (B) 中。缩放条 = 10 µm。此图已从 Artemenko et al.中修改(2016)22.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56411/56411fig3large.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/56411/56411fig4.jpg"> 
图 4: 对急性机械刺激的反应进行测试的替代方法的代表性结果.(a) 营养的D. 柄单元格, 表示 RFP 标记的石灰Δcoil被暴露在一个微 (*) 基部释放化验缓冲的速度很慢。在0的时候, 快速地将一团试验缓冲液输送到流速, 从而实现了流量的短暂增加。图像被收集每 3 s. (B) 营养的D. 柄细胞表达 RFP 标记的石灰Δcoil镀作为一个小下落在显微镜室被机械地刺激由大量缓冲的加法到庭.图像收集每 3 s. 缩放栏 = 10 µm. 此图的部件 (A) 已从 Artemenko et al.中修改(2016)22.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56411/56411fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充电影 1: 急性机械刺激触发了石灰Δcoil的快速和瞬态易位--RFP 或 PH-裂纹-GFP 从胞到聚集的皮层-主管D. 柄细胞表达这些生物传感器.单向层流是打开5秒的时间0和细胞成像 3 s 间隔。回放速度为5帧/秒。显示了每个单元格线的两个示例。缩放条 = 10 µm。此影片对应于图 2A , 并已从 Artemenko et al.中进行了修改(2016)22.<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56411/Suppl_Mov_1_060417.avi">请单击此处下载此文件.</a>
补充电影 2: 连续机械刺激触发石灰Δcoil的快速和瞬态易位-从胞到皮层的 RFP, 然后再向流的方向迁移细胞.表达石灰Δcoil--RFP 的营养细胞在 15 dyn/cm2的连续剪切应力下, 从时间0开始, 以3秒的间隔进行成像。播放速度为10帧/秒. 缩放栏 = 10 µm。此影片对应于图 3 , 并且以前已在 Artemenko et al.中发布(2016)22.<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56411/Suppl_Mov_2_060417.avi">请单击此处下载此文件.</a>

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

Nanyang Technological University

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

Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated phenomena such as bone remodeling, muscular hypertrophy, and smooth muscle cell plasticity. However, the forces involved are often extremely complex and difficult to monitor and control in vivo. To better investigate the effects of mechanical forces on cells, we have developed an in vitro method for applying uniaxial cyclic tensile strain to adherent cells cultured on elastic membranes. This method utilizes a custom-designed bioreactor with a motorized cam-rotor system to apply the desired force. Here we present a step-by-step video protocol demonstrating how to assemble the various components of each "stretch chamber", including, in this case, a silicone membrane with micropatterned topography to orient the cells with the direction of the strain. We also describe procedures for sterilizing the chambers, seeding cells onto the membrane, latching the chamber into the bioreactor, and adjusting the mechanical parameters (i.e. magnitude and rate of strain). The procedures outlined in this particular protocol are specific for seeding human mesenchymal stem cells onto silicone membranes with 10 &micro;m wide channels oriented parallel to the direction of strain. However, the methods and materials presented in this system are flexible enough to accommodate a number of variations on this theme: strain rate, magnitude, duration, cell type, membrane topography, membrane coating, etc. can all be tailored to the desired application or outcome. This is a robust method for investigating the effects of uniaxial tensile strain applied to cells in vitro.

It is widely understood that mechanical forces in the body can influence cell differentiation and proliferation. Here we present a video protocol demonstrating the use of a custom-built bioreactor for delivering uniaxial cyclic tensile strain to stem cells cultured on flexible micropatterned substrates.

采用循环单轴应变的干细胞的机械性刺激

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated ...

科研

生物学

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

使用的视动反应研究斑马鱼的视觉功能

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

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 ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

机械性刺激软骨细胞琼脂糖凝胶

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

机械刺激引起的钙波传播的细胞单层:牛角膜内皮细胞的例子

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including 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 ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

分析细胞表面粘附重塑回应机械张力使用磁珠

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...