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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 35 mm glass-bottom dish | MatTek | P35G-1.5-10-C | Used for sample preparation |
| 60 mm × 15 mm Petri dish with lid | Falcon | 351007 | Used for sample preparation |
| Black cloth for covering the microscope | Online | NA | Used to avoid unwanted light stimulation |
| Clorox Ultra Germicadal Bleach (8.25% sodium hypochlorite) | VWR | 10028-048 | Used for embryo dechorination |
| CO2 pad | Genesee Scientific | 59-114 | Used for cross set-up |
| ddH2O | NA | NA | Used for sample preparation |
| Dumont Style 5 tweezers | VWR | 102091-654 | Used for sample preparation |
| Eyelash tool (made from pure red sable round brush #2) | VWR | 22940-834 | Used for sample preparation |
| FluoView (Software) | Olympus | NA | Used for image acquisition and optogenetic stimulation |
| Halocarbon oil 27 | Sigma Aldrich | H8773-100ML | Used for embryo stage visualization |
| ImageJ/FIJI | NIH | NA | Used for image analysis |
| MATLAB | MathWorks | NA | Used for image analysis |
| Nikon SMZ-745 stereoscope | Nikon | NA | Used for sample preparation |
| 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. | Olympus | NA | Used for image acquisition and optogenetic stimulation |
| SP Bel-Art 100-place polypropylene freezer storage box (Black, light-proof box for sample transfer) | VWR | 30621-392 | Used to avoid unwanted light stimulation |
| UV Filter Shield for FM1403 Fluores (Orange-red plastic shield) | Bolioptics | FM14036151 | Used to avoid unwanted light stimulation |
| VITCHELO V800 Headlamp with White and Red LED Lights | Amazon | NA | Used to avoid unwanted light stimulation |
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