作者感谢Ann Lavanway的成像支持。作者感谢Wieschaus实验室和De Renzis实验室共享试剂，并感谢Bloomington果蝇种群中心共享苍蝇种群。这项研究得到了NIGMS ESI-MIRA R35GM128745和美国癌症协会机构研究资助 #IRG-82-003-33的支持。

1.设置遗传杂交并准备卵子收集杯
<ol><li>从光遗传系UASp-CIBNpm（I）中选择雌性苍蝇（处女）;UASp-CRY2-Rho1DN-mCherry（III）在体视显微镜下的CO2 垫上，并与来自母体GAL4驱动线67 Sqh-mCherry的雄性苍蝇建立杂交;15 E-钙粘蛋白-GFP。 注意：67和15代表插入第二（II）和第三（III）染色体的母体-微管蛋白-GAL4，分别为52。该协议中使用的GAL4系还表达mCherry标记的肌球蛋白调节轻链Sqh（意大利面南瓜）37 和GFP标记的E-钙粘蛋白53。来自光遗传系的苍蝇可能仍含有FM7和TM6平衡染色体。来自母体GAL4驱动系的苍蝇可能仍含有CyO和TM3平衡染色体。</li>
	<li>~10天后，选择具有以下基因型的F1雌性苍蝇：UASp-CIBNpm/+;67 平方/小时樱桃/+;15 E-钙粘蛋白-GFP/UASp-CRY2-Rho1DN-mCherry。与雄性苍蝇一起设置一个鸡蛋收集杯。<ol><li>确保具有正确基因型的雌性果蝇不包含任何平衡染色体（即，它们不应包含卷曲的翅膀[Cy]，短刚毛[Sb]，条形或肾形眼睛[B]或额外的肱骨刚毛[Hu]），这些染色体分别是用于产生种群的CyO，TM3，FM7和TM6平衡器的标记。用苹果汁盘盖住杯子，表面有一丝新鲜的酵母酱。 注意：在F1女性中，光遗传学成分以母系表达。因此，用于设置杯子的F1女性不需要是处女。为方便起见，建议使用来自同一F1种群的雄性苍蝇作为杯子。</li>
	</ol></li>
	<li>将杯子放在覆盖有铝箔的纸板箱中，以避免任何可能的光线从环境中泄漏。对于保存在室温（~21-23°C）下的杯子，每天更换苹果汁盘，或每两天更换一次保存在18°C的杯子。<ol><li>在更换盘子之前，将杯子敲在平坦的表面上（例如，工作台），以保持苍蝇在杯子底部并防止它们逃逸。在黑暗的房间里更换苹果汁盘，并使用带红光的前照灯进行照明（见 材料表）。 注意：为了增加受母体微管蛋白-GAL4和UASp控制的构建体的表达，建议在胚胎收集前将杯子保持在18°C至少3天。这个孵化期还允许苍蝇适应杯子并很好地喂食酵母酱，以实现最佳的产蛋量。</li>
	</ol></li>
</ol>2. 在所需阶段收集胚胎并为光遗传学刺激做好准备
注意：所有样品收集和制备步骤都需要在暗室中进行，使用“安全灯”（例如红灯）进行照明。光遗传学成分对环境光非常敏感。即使是最轻微的环境光照射也会导致标本过早受到刺激。通常，绿红色范围（>532 nm）的光不会引起不必要的刺激。
<ol><li>为了在早期胚胎发生时收集胚胎，在胚胎收集前8至16小时（在18°C）将新的苹果汁板放在杯子上。</li>
	<li>在胚胎收集时，更换苹果汁盘。标记从杯子上取下的板，并用一层薄薄的卤烃油27覆盖板的表面（见 材料表）。等待 30-60 秒，让蛋壳变得透明。</li>
	<li>将橙红色塑料防护罩（见 材料表）放在直立立体镜的舞台上。橙红色屏蔽的位置通过阻挡透射光的蓝绿色波长来防止样品照明期间的不良刺激。 注意：在该协议中，直立立体镜用于胚胎收集（见 材料表）。</li>
	<li>将苹果汁盘放在橙红色盾牌的顶部。打开体视显微镜的透射光以照亮样品。使用一把镊子在适当的阶段从苹果汁盘中收集5-15个胚胎。不要用镊子挤压胚胎。 注意：在此协议中，适当的胚胎应处于早期 - 中期细胞化阶段。这个阶段的胚胎应该有一个深色不透明的蛋黄，周围环绕着一个清晰、均匀的周质层，其中包含正在形成的胚层细胞。裂解沟的前缘（“细胞化前沿”）显示为平行于胚胎表面的连续线，不应通过周质深度的一半。</li>
	<li>用一张纸巾（~1.5厘米×1.5厘米）轻轻吸干胚胎，用镊子去除胚胎中多余的油脂。</li>
	<li>使用塑料移液管将几滴新鲜制备的40%漂白剂（~3%次氯酸钠;见 材料表）加入新的小方形纸巾（~1.5厘米×1.5厘米）中，使纸巾上覆盖一层薄薄的漂白剂。使用镊子将胚胎从干纸巾转移到漂白剂浸泡的纸巾上，并确保胚胎浸泡在漂白剂中。等待2-4分钟，让胚胎去皮。</li>
	<li>去皮后，用镊子将方形纸巾吸干在一大张薄纸上，以去除多余的漂白剂。确保胚胎的一侧朝上。</li>
	<li>要冲洗胚胎，请使用镊子将方形纸巾轻轻浸泡在一滴去离子水中，然后快速将其吸干在一大张薄纸上。重复此过程八次，以确保去除任何残留的漂白剂。</li>
	<li>使用睫毛工具将胚胎从纸巾转移到35毫米的玻璃底培养皿中（见 材料表）。在培养皿中加入去离子水以完全覆盖胚胎。使用睫毛工具微调胚胎的位置和方向。 注意：去皮后，胚胎倾向于粘在玻璃表面并且不动而不会受到干扰，因此无需进行额外的处理（例如胶水）将胚胎固定在玻璃底培养皿上。</li>
	<li>将装有胚胎的35毫米玻璃底培养皿放入防光黑匣子（见 材料表）中，以保护样品在转移过程中免受光照。将盒子带到带有多光子显微镜的房间。</li>
</ol>3. 光遗传学刺激、激光消融和胚胎成像
注意：本实验中使用的多光子系统（见 材料表）能够同时进行双波长成像。它还包含一个带有 458 nm 激光器的光刺激单元和一个单独的振镜扫描仪，允许在定义的感兴趣区域 （ROI） 内进行光激活/刺激。值得注意的是，用于激发绿黄色荧光蛋白的920 nm激光将刺激Opto-Rho1DN，尽管与蓝色激光介导的刺激相比更慢。
<ol><li>用防光黑布覆盖多光子显微镜（见 材料表），以避免在样品设置和成像过程中对胚胎造成不必要的刺激。 注意：在常规多光子成像过程中使用相同的方法来保护显微镜上配备的光敏探测器。</li>
	<li>关闭房间灯和电脑屏幕。通过单击软件中“触摸屏控制器背光”下的“关闭” 关闭 触摸面板控制器。确保房间内没有其他环境光。<ol><li>打开显微镜上黑布盖的正面。从黑匣子中取出35毫米玻璃底培养皿并将其放在显微镜载物台上。</li>
	</ol></li>
	<li>用绿光照亮胚胎，绿灯通常用作落射荧光的激发光。为此，请打开荧光照明单元，在软件中的“目镜”面板下选择目镜，然后将“立方体转盘”更改为4：TRITC。使用目镜识别感兴趣的胚胎并将其聚焦。 注意：绿光可视化是通过允许荧光照明单元产生的白光通过内置的标准TRITC滤光片立方体来实现的，该滤光片立方体包含一个528-553 nm激发滤光片，一个565 nm分束器和一个590-650 nm发射滤光片。其他非蓝光也应该适用于样品照明。在这一步中没有必要佩戴护目镜，因为激发光强度低，不会对准目镜。由于自发荧光，胚胎将呈现均匀的红色。</li>
	<li>关闭黑布盖，使样品完全避光。打开计算机屏幕以访问控制显微镜的软件。在软件中将“目镜”更改为 LSM 以进行图像采集。</li>
	<li>使用25倍水浸物镜在对照中执行激光消融，未受刺激的胚胎。<ol><li>从软件的“工具窗口”中单击 亮 Z、 序列管理器和 LSM 刺激 。将扫描仪类型设置为 Galvano ，将扫描大小设置为 512 × 512。在“PMT 设置”面板下打开 CH1 和 CH3 以允许使用 1,040 nm 激光，然后单击 Live × 4 以可视化胚胎。</li>
		<li>使用软件中的旋转功能 旋转 胚胎，使胚胎的前后轴垂直定向。将缩放比例设置为 3。使用“扫描设置”下的形状工具绘制感兴趣区域（ROI），然后在“参考”面板中设置ROI的大小。将 ROI 设置为宽度为 512 像素，高度为 100 像素（171 × 33 μm2）。</li>
		<li>设置烧蚀前 Z 堆栈的采集参数。<ol><li>在“Z 部分”下将胚胎表面注册为 0 。将开始设置为 0 ，结束设置为 100 μm。将步长设置为 2 μm。通过选中“系列”选项卡下的Z来激活 Z 采集模式。</li>
			<li>使用 Bright Z 功能将 1,040 nm 激光强度设置为从 3% 线性增加到 7%。</li>
			<li>通过单击“序列管理器”中的 LSM ，将当前映像设置保存为管道的第一个任务。 注意：获得预消融Z堆栈以确认胚胎的阶段。在这个实验中，CRY2-Rho1DN-mCherry和Sqh-mCherry将被1,040纳米激光激发。在顶端收缩期间，Sqh-mCherry信号在腹侧中胚层细胞的中尖区域增强，而CRY2-Rho1DN-mCherry在刺激前是胞质的。用于刺激前和刺激后成像的激光强度是根据最佳信噪比和避免光漂白之间的平衡根据经验决定的。</li>
		</ol></li>
		<li>设置预消融视频的采集参数。<ol><li>删除任何现有的ROI，以允许对胚胎腹面附近的整个512×512像素区域（171×171μm 2,3倍变焦）进行成像，如步骤3.5.2中所述。将 1,040 nm 激光强度设置为 3%。</li>
			<li>检查 时间 并取消选中“系列”面板下的 Z 。将“间隔”保留为“延时摄影”面板下的 FreeRun 。将周期设置为 10。</li>
			<li>通过单击“序列管理器”中的 LSM ，将当前设置保存为管道的下一个任务。 注意：此任务的目的是使用1,040 nm激光器获得10帧单Z平面预烧蚀视频以进行图像采集。图像采集速度约为每帧1秒。</li>
		</ol></li>
		<li>设置激光烧蚀的参数。<ol><li>定义从卵黄膜正下方到~20μm深的3D区域以进行激光烧蚀（图1A）。将Z堆栈的起点设置为紧邻玻璃膜下方的平面，并将终点设置为比起始平面深20μm。将步长设置为 1.5 μm。 注意：消融区域的ROI宽度为~30像素，高度为~10像素（沿内侧轴~10μm，沿A-P轴~3.3μm）。在多个Z平面上烧蚀样品的目的是确保腹侧细胞的顶端表面被烧蚀。这对于受刺激的胚胎尤其重要，因为腹侧细胞在Rho1抑制后会经历快速的顶端松弛。</li>
			<li>在“PMT 设置”面板下打开 CH2 和 CH4 ，以允许使用 920 nm 激光器。 将 920 nm 激光的强度设置为 30%。在步骤3.5.5.1中定义的用于激光烧蚀的3D区域内，使用920 nm激光器为单个Z堆栈设置图像采集。 注意：在此步骤中选择的激光强度足以消融组织（如激光治疗后立即组织后坐力所示），但同时不会明显损坏质膜（如细胞膜上没有烧伤痕迹所示）（图1B，C）。</li>
			<li>通过单击“序列管理器”中的 LSM ，将当前设置保存为管道的下一个任务。</li>
		</ol></li>
		<li>设置消融后视频的采集参数。<ol><li>使用 1,040 nm 和 920 nm 激光器为 100 帧单 Z 平面烧蚀后电影设置图像采集。将 1,040 nm 激光和 920 nm 激光的强度分别设置为 3% 和 0.3%。步骤3.5.4中指定的区域将以相同的图像采集速度进行成像。</li>
			<li>通过单击“序列管理器”中的 LSM，将当前设置保存为管道的下一个任务。 注意：在实际采集之前，将步骤3.5.3-3.5.6中描述的参数作为顺序任务存储在“序列管理器”中的目的是确保捕获激光烧蚀后的即时组织反应。</li>
		</ol></li>
		<li>选择“获取”下的 序列 。根据需要更改数据保存路径和文件名。单击 就绪 并等待软件初始化管道。然后单击“ 启动 ”以执行管道。</li>
	</ol></li>
	<li>在受刺激的胚胎中进行激光消融。<ol><li>设置烧蚀前Z堆栈的采集参数，如步骤3.5.1-3.5.3中所述。通过单击“序列管理器”中的 LSM，将当前设置另存为管道的第一个任务。</li>
		<li>在定义的ROI内设置光遗传学刺激的参数。<ol><li>将缩放更改为 1 并选择覆盖胚胎腹面的 ROI（~512 × 300 μm2）。关闭 CH1-CH4 探测器。</li>
			<li>单击“LSM 刺激”。取消选中持续时间内的连续并键入 12 秒。检查 458 nm 的激光强度为 0.3%。</li>
			<li>通过单击“序列管理器”中的 “刺激 ”，将当前设置保存为管道的下一个任务。 注意：通过增加458 nm激光强度可以实现更快速的刺激。然而，当使用更高的激光强度时，散射的激光可能会刺激与ROI相邻的区域，如果需要空间受限的刺激，这是不理想的。</li>
		</ol></li>
		<li>在刺激后设置3分钟的等待时间，以确保肌球蛋白完全失活和顶端F-肌动蛋白的拆卸，并在激光消融前实现静态组织形态。这是通过单击“序列管理器”下的“等待 /暂停”并为“等待 ”设置所需时间来实现的。 注意：刺激后10-15分钟，由单轮刺激（0.3%458nm激光持续12秒）引起的CRY2-Rho1DN-mCherry募集到膜上是可以清楚地检测到的。这与公布的解离半衰期~9分47一致。</li>
		<li>设置单个Z平面预烧蚀电影的采集参数，如步骤3.5.4中所述，但1,040 nm和920 nm激光器均用于图像采集。打开 CH1-CH4 探测器。将 1,040 nm 激光和 920 nm 激光的强度分别设置为 3% 和 0.3%。通过单击“序列管理器”中的 LSM ，将当前设置保存为管道的下一个任务。</li>
		<li>设置激光烧蚀的参数，如步骤3.5.5中所述。通过单击“序列管理器”中的 LSM ，将当前设置保存为管道的下一个任务。</li>
		<li>设置单个Z平面烧蚀后电影的采集参数，如步骤3.5.6中所述。通过单击“序列管理器”中的 LSM ，将当前设置保存为管道的下一个任务。</li>
		<li>选择“获取”下的 序列 。根据需要更改数据保存路径和文件名。单击 就绪 并等待软件初始化管道。然后，单击“启动”以执行管道。</li>
	</ol></li>
</ol>4. 量化激光消融后组织后坐力率
<ol><li>在 ImageJ 中打开消融后影片。</li>
	<li>使用 矩形选择 工具沿覆盖消融区域的腹侧中线绘制一个小的 ROI。ROI 的宽度为 9 个像素。ROI的高度设置为足够大，以覆盖激光消融后的所有组织后坐力。<ol><li>点击“图片”标签下的复制。在弹出窗口中，选中复制堆栈，然后单击确定以在所选ROI内复制堆栈。</li>
	</ol></li>
	<li>通过图像>堆栈从复制的堆栈生成蒙太 奇>蒙太奇。在弹出窗口中，将行号设置为 1 ，将列号设置为堆栈中的帧总数（在本例中为 100 ）。</li>
	<li>从生成的蒙太奇中测量烧蚀区域的 A-P 宽度随时间的变化。<ol><li>使用 ImageJ 中的多点工具标记蒙太奇上每个时间 点 的消融区域的 A-P 边界。</li>
		<li>使用 “文件>另存为XY坐标”保存标记点>坐标。</li>
		<li>将测量的 XY 坐标导入 MATLAB。通过从下边界的 Y 坐标中减去上边界的 Y 坐标来计算每个时间点的烧蚀区域的 A-P 宽度。</li>
	</ol></li>
	<li>通过使用 MATLAB 中的“polyfit”函数将“随时间变化的宽度”曲线的前 20 秒拟合成一条线来确定组织后坐力率。将拟合线的斜率报告为组织后坐力。</li>
	<li>执行统计测试以比较受刺激和非受刺激样品之间的组织反冲率。 注意：建议每种情况下至少从五个胚胎中获取数据。双侧威尔科克森秩和检验和双侧学生 t 检验都可用于统计比较。</li>
</ol>

果蝇中 Rho1 介导的肌动球蛋白收缩性的光遗传学抑制

作者声明，这项研究是在没有任何商业或财务关系的情况下进行的，这些关系可以解释为潜在的利益冲突。

该协议描述了光遗传学和激光消融的组合使用，以在肌动肌蛋白收缩性失活后立即探测组织张力的变化。这里描述的光遗传学工具利用Rho1（Rho1DN）的主要阴性形式来急性抑制内源性Rho1和Rho1依赖性肌动肌素收缩力。先前在果蝇腹侧沟形成背景下对Opto-Rho1DN的表征表明，该工具通过同时肌球蛋白失活和肌动蛋白拆卸来介导顶端肌动肌蛋白收缩力的快速失活非常有效41。特别是...

肌动蛋白收缩力是非肌肉肌球蛋白II（以下简称“肌球蛋白”）对F-肌动蛋白网络施加的收缩力，是改变细胞形状和驱动组织水平形态发生的最重要力量之一1,2。例如，上皮细胞顶端结构域肌动蛋白收缩性的激活导致顶端收缩，这促进了各种形态发生过程，包括上皮折叠、细胞挤出、分层和伤口愈合 3,4,5,6,7.肌球蛋白的活化需要其调节轻链的磷酸化。这种修饰减轻了肌球蛋白分子的抑制构象，使它们能够形成两端具有多个头部结构域的双极肌球蛋白丝束。双极肌球蛋白丝驱动肌动蛋白丝的反平行运动并产生收缩力1,8,9。
进化上保守的Rho家族小GTP酶RhoA（果蝇中的Rho1）在各种细胞环境中的肌动肌蛋白收缩力的激活中起核心作用10,11。Rho1通过结合GTP（活性形式）或GDP（非活性形式）作为双分子开关起作用12。GTP 或 GDP 结合的 Rho1 之间的循环由其 GTP 酶激活蛋白 （GAP） 和鸟嘌呤核苷酸交换因子 （GEF） 调节13。全球环境基金的作用是促进国内生产总值换取全球通用技术伙伴关系，从而激活Rho1活动。另一方面，GAPs增强了Rho1的GTP酶活性，从而使Rho1失活。活化的Rho1通过与下游效应物Rho相关激酶（Rok）和Diaphanous14相互作用并激活肌动肌肽收缩力。Rok通过磷酸化肌球蛋白15的调节轻链来诱导肌球蛋白活化和肌动球蛋白收缩力。此外，Rok还抑制肌球蛋白调节轻链磷酸酶，从而进一步促进肌球蛋白丝组装16。Rok还可以磷酸化LIM激酶，当激活时，通过磷酸化和抑制肌动蛋白解聚因子cofilin17,18来防止肌动蛋白分解。Diaphanous是一种福尔马林家族肌动蛋白成核剂，可促进肌动蛋白聚合，为肌球蛋白与19,20,21相互作用提供基础。
虽然激活肌动肌蛋白收缩力的细胞机制已经得到很好的阐明，但我们对其在调节动态组织重塑中的功能的理解仍然不完整。填补这一知识空白需要能够快速灭活体内特定组织区域的肌动球蛋白并记录对组织行为和性质的直接影响的方法。该协议描述了使用光遗传学方法在果蝇中胚层内陷期间急性抑制肌动肌蛋白收缩力，然后使用激光消融测量上皮张力。在果蝇原肠胚形成过程中，腹侧定位的中胚层前体细胞通过形成前后定向的沟22,23，经历顶端收缩并从胚胎表面内陷。腹沟的形成长期以来一直被用作研究上皮折叠机制的模型。腹侧沟的形成由果蝇24,25,26,27的背腹模式系统施用。位于胚胎腹侧的两种转录因子Twist和Snail的表达控制腹侧沟的形成并指定中胚层细胞命运28。扭曲和蜗牛通过G蛋白偶联受体途径和RhoGEF2衔接蛋白T48 29,30,31,32,33激活Rho1 GEF RhoGEF2募集到中胚层前体细胞的顶点。接下来，RhoGEF2通过Rho-Rho激酶途径34,35,36,37,38,39激活潜在中胚层的整个顶端表面的肌球蛋白。活化的肌球蛋白在整个中胚层原基的顶端表面形成细胞上肌动球蛋白网络，其收缩驱动顶端收缩并导致顶端组织张力迅速增加14,37,40。
本协议中描述的光遗传学工具Opto-Rho1DN通过蓝光依赖性质膜募集主要阴性形式的Rho1（Rho1DN）41来抑制肌动肌蛋白收缩力。Rho1DN中的T19N突变消除了突变蛋白将GDP交换为GTP的能力，从而使蛋白质永久失活34。随后的Rho1DN突变C189Y消除了其幼稚膜靶向信号42,43。当 Rho1DN 注入质膜时，它会结合并储存 Rho1 GEF，从而阻断 Rho1 的活化以及 Rho1 介导的肌球蛋白和肌动蛋白34,44 的活化。Rho1DN的质膜募集是通过衍生自Cryptochrome 2及其结合伴侣CIB1的光依赖性二聚化模块实现的。隐花色素2是拟南芥45中的蓝光激活隐色素感光器。隐花色素2仅以光激发态45与CIB1（一种碱性的螺旋-环-螺旋蛋白）结合。后来发现，来自隐花色素2（CRY2 PHR，以下简称CRY2）的保守N端，光解酶同源区（PHR）和CIB1（以下简称CIBN）的N端结构域（aa 1-170）对于光诱导二聚化很重要46。Opto-Rho1DN包含两个组件。第一个组成部分是与CAAX锚融合的CIBN蛋白，它将蛋白质定位到质膜47。第二个组件是mCherry标记的CRY2与Rho1DN41融合。在没有蓝光的情况下，CRY2-Rho1DN保留在细胞质中。在蓝光刺激下，CRY2-Rho1DN通过膜锚定CIBN和激发的CRY2之间的相互作用靶向质膜。Opto-Rho1DN可以通过紫外A（UVA）光和蓝光（400-500nm，450-488nm处的峰值激活）激活，或者在进行双光子刺激41,46,47,48时通过830-980nm脉冲激光激活。因此，Opto-Rho1DN受到通常用于激发GFP的波长的刺激（单光子成像为488 nm，双光子成像为920 nm）。相比之下，通常用于激发mCherry的波长（单光子成像为561 nm，双光子成像为1,040 nm）不会刺激光遗传模块，因此可用于刺激前成像。该协议描述了用于最小化样品操作过程中不需要的刺激风险的方法。
激光消融已被广泛用于检测和测量细胞和组织中的张力49。先前的研究表明，当激光强度得到适当控制时，使用飞秒近红外激光的双光子激光烧蚀可以物理损害某些亚细胞结构（例如，皮质肌动肌蛋白网络），而不会引起质膜狂喜50,51。如果组织处于张力下，激光消融组织内感兴趣的区域会导致与烧蚀区域相邻的细胞立即向外反冲。反冲速度是承受后坐力的结构周围的介质（细胞质）的张力大小和粘度的函数49。由于近红外激光具有优越的穿透深度和实现良好局限聚焦消融的能力，双光子激光消融对于检测体内组织张力特别有用。如该协议所示，该方法可以很容易地与Opto-Rho1DN介导的肌动球蛋白收缩力失活相结合，以研究动态组织重塑过程中Rho1依赖性细胞收缩力对组织力学的直接影响。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm glass-bottom dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-10-C</td>
			<td>Used for sample preparation</td>
		</tr>
		<tr>
			<td>60 mm &#215; 15 mm Petri dish with lid</td>
			<td>Falcon</td>
			<td>351007</td>
			<td>Used for sample preparation</td>
		</tr>
		<tr>
			<td>Black cloth for covering the microscope</td>
			<td>Online</td>
			<td>NA</td>
			<td>Used to avoid unwanted light stimulation</td>
		</tr>
		<tr>
			<td>Clorox Ultra Germicadal Bleach (8.25% sodium hypochlorite)</td>
			<td>VWR</td>
			<td>10028-048</td>
			<td>Used for embryo dechorination</td>
		</tr>
		<tr>
			<td>CO2 pad</td>
			<td>Genesee Scientific</td>
			<td>59-114</td>
			<td>Used for cross set-up</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>NA</td>
			<td>NA</td>
			<td>Used for sample preparation</td>
		</tr>
		<tr>
			<td>Dumont Style 5 tweezers</td>
			<td>VWR</td>
			<td>102091-654</td>
			<td>Used for sample preparation</td>
		</tr>
		<tr>
			<td>Eyelash tool (made from pure red sable round brush #2)</td>
			<td>VWR</td>
			<td>22940-834</td>
			<td>Used for sample preparation</td>
		</tr>
		<tr>
			<td>FluoView (Software)</td>
			<td>Olympus</td>
			<td>NA</td>
			<td>Used for image acquisition and optogenetic stimulation</td>
		</tr>
		<tr>
			<td>Halocarbon oil 27</td>
			<td>Sigma Aldrich</td>
			<td>H8773-100ML</td>
			<td>Used for embryo stage visualization</td>
		</tr>
		<tr>
			<td>ImageJ/FIJI</td>
			<td>NIH</td>
			<td>NA</td>
			<td>Used for image analysis</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>NA</td>
			<td>Used for image analysis</td>
		</tr>
		<tr>
			<td>Nikon SMZ-745 stereoscope</td>
			<td>Nikon</td>
			<td>NA</td>
			<td>Used for sample preparation</td>
		</tr>
		<tr>
			<td>Olympus FVMPE-RS multiphoton microscope with InSight DS Dual-line Ultrafast Lasers for simultaneous dual-wavelength multiphoton imaging, , a 25x/NA1.05 water immersion objective (XLPLN25XWMP2), and an IR/VIS stimulation unit for photo-activation/stimulation. This system is also equipped with a TRITC filter (39005-BX3; AT-TRICT-REDSHFT 540/25x, 565BS, 620/60M), and a fluorescence illumination unit that emits white light.</td>
			<td>Olympus</td>
			<td>NA</td>
			<td>Used for image acquisition and optogenetic stimulation</td>
		</tr>
		<tr>
			<td>SP Bel-Art 100-place polypropylene freezer storage box (Black, light-proof box for sample transfer)</td>
			<td>VWR</td>
			<td>30621-392</td>
			<td>Used to avoid unwanted light stimulation</td>
		</tr>
		<tr>
			<td>UV Filter Shield for FM1403 Fluores (Orange-red plastic shield)</td>
			<td>Bolioptics</td>
			<td>FM14036151</td>
			<td>Used to avoid unwanted light stimulation</td>
		</tr>
		<tr>
			<td>VITCHELO V800 Headlamp with White and Red LED Lights</td>
			<td>Amazon</td>
			<td>NA</td>
			<td>Used to avoid unwanted light stimulation</td>
		</tr>
	</tbody>
</table>

optogenetic inhibition of rho1-mediated actomyosin contractility coupled with measurement of epithelial tension in drosophila embryos

肌动蛋白和非肌球蛋白II产生的收缩力（“肌动肌蛋白收缩力”）对于细胞和组织在多个长度尺度上的形态变化至关重要，例如细胞分裂，细胞迁移，上皮折叠和分支形态发生。深入了解肌动球蛋白收缩性在形态发生中的作用需要允许快速灭活肌动球蛋白的方法，这是使用传统的遗传学或药理学方法难以实现的。所提出的协议展示了使用基于CRY2-CIBN的光遗传学二聚化系统Opto-Rho1DN通过精确的时间和空间控制来抑制 果蝇 胚胎中的肌动球蛋白收缩力。在该系统中，CRY2融合到Rho1（Rho1DN）的主要负形式，而CIBN锚定在质膜上。蓝光介导的CRY2和CIBN二聚化导致Rho1DN从细胞质快速转移到质膜，在那里它通过抑制内源性Rho1使肌动球蛋白失活。此外，本文还提出了将Opto-Rho1DN介导的肌动球蛋白失活与激光消融偶联的详细方案，以研究肌动肌蛋白在 果蝇 腹侧沟形成过程中产生上皮张力中的作用。该协议可以应用于许多其他形态过程，这些过程涉及 果蝇 胚胎中的肌动肌收缩性，只需最少的修改。总体而言，这种光遗传学工具是剖析动态组织重塑过程中肌动肌蛋白收缩力在控制组织力学中的功能的有力方法。

Actomyosin contractility, the contractile force exerted by non-muscle myosin II (hereafter &#39;myosin&#39;) on the F-actin network, is one of the most important forces in changing cell shape and driving tissue-level morphogenesis1,2. For example, the activation of actomyosin contractility at the apical domain of the epithelial cells results in apical constriction, which facilitates a variety of morphogenetic processes, including epithelial folding, cell extrusion, delamination, and wound healing3,4,5,6,7. The activation of myosin requires phosphorylation of its regulatory light chain. This modification alleviates the inhibitory conformation of the myosin molecules, allowing them to form bipolar myosin filament bundles with multiple head domains on both ends. The bipolar myosin filaments drive the anti-parallel movement of actin filaments and result in the generation of contractile force1,8,9.
The evolutionarily conserved Rho family&#160;small GTPase RhoA (Rho1 in Drosophila)&#160;plays a central role in the activation of actomyosin contractility in various cellular contexts10,11. Rho1 functions as a bimolecular switch by binding either GTP (active form) or GDP (inactive form)12. The cycling between GTP- or GDP-bound Rho1 is regulated by its GTPase-activating proteins (GAPs) and guanine nucleotide-exchange factors (GEFs)13. GEFs function to facilitate the exchange of GDP for GTP and thus activate Rho1 activity. GAPs, on the other hand, enhance the GTPase activity of Rho1 and thus deactivate Rho1. Activated Rho1 promotes actomyosin contractility through interacting with and activating its downstream effectors, Rho-associated kinase (Rok) and Diaphanous14. Rok induces myosin activation and actomyosin contractility by phosphorylating the regulatory light chain of myosin15. In addition, Rok also inhibits the myosin regulatory light chain phosphatase, and hence further promotes myosin filament assembly16. Rok can also phosphorylate LIM kinases, which, when activated, prevent actin disassembly by phosphorylating and inhibiting the actin-depolymerization factor cofilin17,18. Diaphanous is a formin family actin nucleator that promotes actin polymerization, providing a base for myosin to interact with19,20,21.
While the cellular mechanisms that activate actomyosin contractility have been well elucidated, our understanding of its function in regulating dynamic tissue remodeling remains incomplete. Filling this knowledge gap requires approaches that can rapidly inactivate actomyosin at specific tissue regions in vivo and record the immediate impact on tissue behavior and properties. This protocol describes the use of an optogenetic approach to acutely inhibit actomyosin contractility during Drosophila mesoderm invagination, followed by measurement of epithelial tension using laser ablation. During Drosophila gastrulation, the ventrally localized mesoderm precursor cells undergo apical constriction and invaginate from the surface of the embryo by forming an anterior-posteriorly oriented furrow22,23. The formation of ventral furrows has long been used as a model for studying the mechanism of epithelial folding. Ventral furrow formation is administered by the dorsal-ventral patterning system in Drosophila24,25,26,27. The expression of two transcription factors, Twist and Snail, located at the ventral side of the embryo, controls ventral furrow formation and specifies mesodermal cell fate28. Twist and Snail activate the recruitment of the Rho1 GEF RhoGEF2 to the apex of the mesoderm precursor cells via a G-protein coupled receptor pathway and a RhoGEF2 adaptor protein, T4829,30,31,32,33. Next, RhoGEF2 activates myosin throughout the apical surface of the potential mesoderm through the Rho-Rho kinase pathway34,35,36,37,38,39. Activated myosin forms a supracellular actomyosin network throughout the apical surface of the mesoderm primordium, the contractions of which drive apical constriction and result in a rapid increase in apical tissue tension14,37,40.
The optogenetic tool described in this protocol, Opto-Rho1DN, inhibits actomyosin contractility through blue-light dependent plasma membrane recruitment of a dominant negative form of Rho1 (Rho1DN)41. A T19N mutation in Rho1DN eliminates the ability of the mutant protein to exchange GDP for GTP and thus renders the protein perpetually inactive34. A subsequent mutation in Rho1DN, C189Y, eliminates its naive membrane targeting signal42,43. When Rho1DN is infused to the plasma membrane, it binds to and impounds Rho1 GEFs, thereby blocking the activation of Rho1 as well as Rho1-mediated activation of myosin and actin34,44. The plasma membrane recruitment of Rho1DN is achieved through a light-dependent dimerization module derived from Cryptochrome 2 and its binding partner CIB1. Cryptochrome 2 is a blue-light activated Cryptochrome photoreceptor in Arabidopsis thaliana45. Cryptochrome 2 binds to CIB1, a basic helix-loop-helix protein, only in its photoexcited state45. It was later found that the conserved N-terminal, photolyase homology region (PHR), from Cryptochrome 2 (CRY2PHR, hereafter referred to as CRY2) and the N-terminal domain (aa 1-170) of CIB1 (hereafter CIBN) are important for light-induced dimerization46. Opto-Rho1DN contains two components. The first component is the CIBN protein fused with a CAAX anchor, which localizes the protein to the plasma membrane47. The second component is mCherry-tagged CRY2 fused with Rho1DN41. In the absence of blue light, CRY2-Rho1DN remains in the cytoplasm. Upon blue light stimulation, CRY2-Rho1DN is targeted to the plasma membrane through the interaction between membrane-anchored CIBN and excited CRY2. Opto-Rho1DN can be activated by ultraviolet A (UVA) light and blue light (400-500 nm, peak activation at 450-488 nm), or by an 830-980 nm pulsed laser when performing two-photon stimulation41,46,47,48. Therefore, Opto-Rho1DN is stimulated by wavelengths normally used for exciting GFP (488 nm for single photon imaging and 920 nm for two-photon imaging). In contrast, wavelengths commonly used for exciting mCherry (561 nm for single photon imaging and 1,040 nm for two-photon imaging) do not stimulate the optogenetic module and therefore can be used for pre-stimulation imaging. The protocol describes the approaches used to minimize the risk of unwanted stimulation during sample manipulation.
Laser ablation has been extensively employed to detect and measure tension in cells and tissues49. Previous studies have shown that, when laser intensity is appropriately controlled, two-photon laser ablation employing a femtosecond near-infrared laser can physically impair some subcellular structures (e.g., cortical actomyosin networks) without causing plasma membrane rapture50,51. If the tissue is under tension, laser ablation of a region of interest within the tissue results in an immediate outward recoil of the cells adjacent to the ablated region. The recoil velocity is a function of the magnitude of the tension and the viscosity of the media (cytoplasm) surrounding the structures undergoing recoil49. Because of the superior penetration depth of the near-infrared lasers and the ability to achieve well-confined focal ablation, two-photon laser ablation is particularly useful for detecting tissue tension in vivo. As demonstrated in this protocol, this method can be easily combined with Opto-Rho1DN-mediated inactivation of actomyosin contractility to investigate the direct impact of Rho1-dependent cellular contractility on tissue mechanics during dynamic tissue remodeling.

作者聚焦： Rho1 介导的肌动球蛋白收缩力的光遗传学抑制与果蝇胚胎上皮张力的测量相结合  

Rho1介导的肌动肌蛋白收缩力的光遗传学抑制与 果蝇 胚胎上皮张力的测量

Our research studies tissue morphogenesis, the formation of complex three-dimensional tissue structures in development. We are interested in the genes and the molecules that regulate morphogenesis and seek to understand the physical principles underlying morphogenesis. For example, how mechanical forces are generated and how they drive tissue rehabilitation.Contractile forces generated by filamentous actin and non-muscle myosin II, also known as actomyosin contractility, is one of the most important forces that drive tissue morphogenesis. Our current research addresses how actomyosin contractility mediates the folding of blood epithelial cell sheets, a fundamental tissue construction mechanism in development. An in-depth understanding of the role of actomyosin contractility in epithelial folding and other morphogenetic processes requires approaches that can quickly inactivate actomyosin at designated time and location, and records the immediate impact of tissue behavior and properties.However, this is difficult to achieve using conventional genetic approaches. The approach described in this protocol is designed to address how cells and tissues respond to sudden changes in mechanical forces that normally drive tissue remodeling. This is an important question but has been difficult to tackle due to limited approaches that can quickly alter mechanical forces in intact tissues.In this study, we developed an optogenetic tool to inactivate actomyosin at specific tissue regions in Drosophila embryos quickly. We found that combining these two with laser ablation to investigate the direct impact of actomyosin contractility on tissue mechanics in epithelial folding. Our findings provide new insights into the mechanical mechanism apical constriction media to epithelial folding.With manual modifications, the protocol can be easily adapted to study the function of actomyosin contractility in a wide range of morphogenetic processes.

在经历顶端收缩的未刺激胚胎中，Sqh-mCherry在腹侧中胚层细胞的中尖区域富集，而CRY2-Rho1DN-mCherry是胞质的（图1A）。收缩域内的激光消融导致沿A-P轴的快速组织反冲（图1B，C）。在受刺激的胚胎中，CRY2-Rho1DN-mCherry信号变得质膜定位，而Sqh-mCherry的中等神经质地信号完全消失（图1A）。受刺激胚胎中的激光消融不会导致?...

Watch this Scientific Journal Video about Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in Drosophila Embryos at JoVE.com

肌动肌蛋白收缩力在细胞和组织形态发生中起重要作用。然而，在 体内 急性操纵肌动肌蛋白收缩力具有挑战性。该协议描述了一种光遗传学系统，该系统可快速抑制 果蝇 胚胎中Rho1介导的肌动肌蛋白收缩力，揭示了 体内肌动球蛋白失活后上皮张力的立即丧失。

Contractile forces generated by actin and non-muscle myosin II ("actomyosin contractility") are critical for morphological changes of cells and tissues at ...

我们的研究研究组织形态发生，发育中复杂三维组织结构的形成。我们对调节形态发生的基因和分子感兴趣，并试图了解形态发生背后的物理原理。例如，机械力是如何产生的以及它们如何驱动组织康复。丝状肌动蛋白和非肌球蛋白II产生的收缩力，也称为肌动肌蛋白收缩力，是驱动组织形态发生的最重要力量之一。我们目前的研究解决了肌动肌素收缩性如何介导血上皮细胞片的折叠，这是发育中的基本组织构建机制。深入了解肌动肌蛋白收缩性在上皮折叠和其他形态发生过程中的作用需要能够在指定时间和地点快速灭活肌动球蛋白的方法，并记录组织行为和性质的直接影响。然而，使用传统的遗传方法很难做到这一点。本协议中描述的方法旨在解决细胞和组织如何响应通常驱动组织重塑的机械力的突然变化。这是一个重要的问题，但由于可以快速改变完整组织中机械力的方法有限，因此很难解决。在这项研究中，我们开发了一种光遗传学工具，可以快速灭活果蝇胚胎中特定组织区域的肌动球蛋白。我们发现将这两者与激光消融相结合，以研究肌动肌蛋白收缩性对上皮折叠中组织力学的直接影响。我们的研究结果为上皮折叠的根尖收缩介质的机械机制提供了新的见解。通过手动修改，该方案可以很容易地适应研究肌动球蛋白收缩性在各种形态发生过程中的功能。

optogenetic-inhibition-rho1-mediated-actomyosin-contractility-coupled

Research

JoVE Journal

Developmental Biology

Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in Drosophila Embryos

1.0K 次观看.  Dartmouth College. 我们的研究研究组织形态发生，发育中复杂三维组织结构的形成。我们对调节形态发生的基因和分子感兴趣，并试图了解形态发生背后的物理原理。例如，机械力是如何产生的以及它们如何驱动组织康复。丝状肌动蛋白和非肌球蛋白II产生的收缩力，也称为肌动肌蛋白收缩力，是驱动组织形态发生的最重要力量之一。我们目前的研究解决了肌动肌素收缩性如何介导血上?...

视频: 作者聚焦： Rho1 介导的肌动球蛋白收缩力的光遗传学抑制与果蝇胚胎上皮张力的测量相结合  

Contractile forces generated by actin and non-muscle myosin II ("actomyosin contractility") are critical for morphological changes of cells and tissues at multiple length scales, such as cell division, cell migration, epithelial folding, and branching morphogenesis. An in-depth understanding of the role of actomyosin contractility in morphogenesis requires approaches that allow the rapid inactivation of actomyosin, which is difficult to achieve using conventional genetic or pharmacological approaches. The presented protocol demonstrates the use of a CRY2-CIBN based optogenetic dimerization system, Opto-Rho1DN, to inhibit actomyosin contractility in Drosophila embryos with precise temporal and spatial controls. In this system, CRY2 is fused to the dominant negative form of Rho1 (Rho1DN), whereas CIBN is anchored to the plasma membrane. Blue light-mediated dimerization of CRY2 and CIBN results in rapid translocation of Rho1DN from the cytoplasm to the plasma membrane, where it inactivates actomyosin by inhibiting endogenous Rho1. In addition, this article presents a detailed protocol for coupling Opto-Rho1DN-mediated inactivation of actomyosin with laser ablation to investigate the role of actomyosin in generating epithelial tension during Drosophila ventral furrow formation. This protocol can be applied to many other morphological processes that involve actomyosin contractility in Drosophila embryos with minimal modifications. Overall, this optogenetic tool is a powerful approach to dissect the function of actomyosin contractility in controlling tissue mechanics during dynamic tissue remodeling.

Actomyosin contractility plays an important role in cell and tissue morphogenesis. However, it is challenging to manipulate actomyosin contractility in vivo acutely. This protocol describes an optogenetic system that rapidly inhibits Rho1-mediated actomyosin contractility in Drosophila embryos, revealing the immediate loss of epithelial tension after the inactivation of actomyosin in vivo.

科研

发育生物学

Collection of Drosophila Embryos and Preparing Them for Optogenetic Stimulation

Collection of Drosophila Embryos and Preparing Them for Optogenetic Stimulation  

To begin collecting the drosophila embryos, change the apple juice plate from the cup and label the plate. Cover the surface of the plate with a thin layer of halocarbon oil 27. Next position, an orange red plastic shield on the stage of an upright stereoscope.Place the apple juice plate on the orange red shield. Turn on the transmitted light of the stereo microscope to illuminate the sample. Collect 5 to 15 embryos from the apple juice plate using a pair of tweezers.Gently blot the embryos on a paper towel, sized approximately 1.5 by 1.5 centimeters. Then add several drops of freshly prepared 40%bleach to a new paper towel using plastic transfer pipette to cover the paper towel with a thin layer of bleach. Transfer the embryo from the dry paper towel to the bleach soaked paper towel and ensure the embryos are soaked in the bleach.Wait two minutes for the embryo to become dechorionated. After dechorionation, using tweezers, blot the paper towel on a large piece of tissue paper to remove the excess bleach. Rinse the embryos with deionized water eight times to remove residue bleach.Using an eyelash tool, transfer the embryo from the paper towel to a 35 millimeter glass bottom dish. Then add deionized water to the dish to cover the embryos completely. Finally, fine tune the position and orientation of the embryos using the eyelash tool.Place the dish with the embryos inside a light proof black box to protect the sample from light exposure during the transfer process.

收集 果蝇 胚胎并为光遗传学刺激做好准备

Optogenetic Stimulation, Laser Ablation, and Imaging of Drosophila Embryos

Optogenetic Stimulation, Laser Ablation, and Imaging of Drosophila Embryos  

To begin, turn off the room light. Select Ocular under the ocular panel in the FlowView software. Change the cube turret to four TRITC, and turn off the touch panel controller by clicking off under backlight of touch panel controller in the software.Then turn off the computer screen. Open the front side of the black cloth cover on the microscope. Take the 35 millimeter glass-bottomed dish containing the embryos from the black box and place it on the microscope stage.Then turn on the fluorescence illumination unit, and use the eyepiece to identify the embryo of interest. Bring it into focus. Close the black cloth cover to protect the sample from light.Turn on the computer screen to access the software that controls the microscope. Change the ocular to LSM in the software for image acquisition. Perform laser ablation in control unstimulated embryos using a 25 times magnification water immersion objective.Click Bright Z, Sequence Manager, and LSM stimulation from the tool window. Set the scanner type as Galvano, and the scan size as 512 by 512. Turn on channel one and channel three under the PMT setting panel to allow the use of the 1040 nanometer laser, and click live four times speed to visualize the embryo.Rotate the embryo using the rotation function to make the anterior posterior axis vertically oriented, and set the zoom to three. Draw a region of interest or ROI using the shape tool under scan settings, and set the ROI size in the reference panel. Next, set the ROI as 512 pixels in width and 100 pixels in height.To set the acquisition parameters for the pre-ablation Z stack, register the embryo's surface as zero under the Z section. Set the start as zero and the end as 100 micrometers. Set the step size as two micrometers, and activate the Z acquisition mode by checking Z under the series tab.Using the bright Z function, set the 1040 nanometer laser intensity to increase linearly from 3%to 7%Save the current imaging setting as the first task of the pipeline by clicking LSM in Sequence Manager. To set acquisition parameters for the pre-ablation movie, set an ROI of 512 by 512 pixels near the embryo's ventral surface as previously demonstrated. Set the 1, 040 nanometer laser intensity to 3%Check time, and uncheck Z under the series panel.Keep the interval as free run under the time lapse panel, and set the cycle as 10. Save the current setting as the next task of the pipeline by clicking LSM in Sequence Manager. To set the parameters for laser ablation, define a 3D region immediately below the vitelline membrane.Set the start of the Z stack as the plane, and the end as 20 micrometers deeper. Set the step size as 1.5 micrometers. Turn on channel two and channel four under the PMT setting panel to allow the use of the 920 nanometer laser.Set the intensity of the laser to 30%and set image acquisition with the laser for a single Z stack within the defined 3D region. Save the current setting as the next task of the pipeline by clicking LSM in Sequence Manager. To set acquisition parameters for the post ablation movie, set image acquisition for a 100 frame single Z plane post ablation movie using the 1, 040 and 920 nanometer lasers.Set the intensity of the lasers to 3%and 0.3%Save the current setting as the next task of the pipeline by clicking LSM in Sequence Manager. Select sequence under acquire. Change the data saving path and file name as needed.Click ready, and wait for the software to initialize the pipeline, then click start to execute the pipeline. to perform laser ablation in stimulated embryos, Set acquisition parameters for the pre-ablation Z stack as demonstrated for the unstimulated embryos. Save the current setting as the first task of the pipeline by clicking LSM in Sequence Manager.To set parameters for optogenetic stimulation within a defined ROI, change the zoom to one, and select an ROI that covers the embryo's ventral surface. Turn off the channel one to channel four detectors, click LSM stimulation, uncheck continuous within duration, and type 12 seconds. Save the current setting as the next task of the pipeline by clicking stimulation in Sequence Manager.Set a three minute wait time after stimulation to ensure total inactivation of myosin and apical F-actin disassembly and achieve a static tissue morphology before laser ablation. Next, set acquisition parameters for the single Z plane pre-ablation movie as demonstrated for the unstimulated embryos, except that the 1, 040 and 920 nanometer lasers are used for image acquisition. Turn on the channel one to channel four detectors.Set the intensity of the lasers to 3%and 0.3%Save the current setting as the next task of the pipeline by clicking LSM in Sequence Manager. Set parameters for laser ablation as demonstrated for the unstimulated embryos. Save the current setting as the next task of the pipeline by clicking LSM in Sequence Manager.Set acquisition parameters for the single Z plane post-ablation movie as demonstrated for the unstimulated embryos. Save the current setting as the next task of the pipeline by clicking LSM in Sequence Manager. Select sequence under acquire.Change the data saving path and file name as needed. Click ready and wait for the software to initialize the pipeline, then click start to execute the pipeline. In the unstimulated embryos undergoing apical constriction, spaghetti squash mCherry became enriched at the medial apical region, whereas CRY2Rho one dominant negative mCherry was cytosolic.In the stimulated embryos, the CRY2Rho one dominant negative mCherry signal became plasma membrane localized, whereas the medial apical signal of spaghetti squash mCherry completely disappeared. Laser ablation of the unstimulated embryos within the constriction domain led to a rapid tissue recoil along the anterior posterior axis, whereas laser ablation in the stimulated embryos did not result in noticeable tissue recoil. The ablation was quantified and shown here.