枯草芽孢杆菌中细菌体和内膜的可视化
Summary
萌发细胞受体蛋白聚集在枯草芽孢杆菌内膜的 “生殖细胞” 中。我们描述了一个协议, 使用超级分辨率显微镜和荧光报告蛋白可视化生殖细胞。该协议还确定了优先与 fm4-64 的膜染料染色的孢子内膜域。
Abstract
孢子体积小, 萌发蛋白丰度相对较低, 因此难以使用荧光显微镜进行显微分析。超分辨率三维结构化照明显微镜 (3D-SIM) 是克服这一障碍并揭示枯草芽孢杆菌 (b. 枯草芽孢杆菌) 萌发过程分子细节的一个很有前途的工具。在这里, 我们描述了改进的 SIMcheck (ImageJ) 辅助三维成像过程和荧光报告蛋白在b. 枯草孢子的萌发体、萌发蛋白簇的 sim 显微镜中的应用。我们还提出了一个 (standard)3D-sim 成像程序 FM4-64 染色 b. 枯草孢子膜。通过这些方法, 我们获得了无与伦比的萌发细胞定位分辨率, 并表明在定义的最小 MOPS 培养基上培养后获得的 b . 枯草kgb80 休眠孢子有一个或两个 Gerd-gfp 和 GerKB-mCherry 的 gt;80%灶。在 FM4-64 染色孢子的3D-SIM 图像中也观察到明亮的病灶, 表明可能存在不同流动性的内膜脂质域。进一步的研究, 使用双重标记程序与膜染料和生殖细胞报告蛋白, 以评估共同定位, 从而得到一个最佳的概述, 在内孢子膜芽孢杆菌萌发蛋白的组织可能。
Introduction
Bacillales 和 Clostridiales 的孢子在代谢上处于休眠状态, 对苛刻的去污状态非常耐受, 但除非它们发芽, 否则不能对人类造成有害影响 1.在营养萌发引发枯草芽孢杆菌(枯草芽孢杆菌) 孢子萌发时, 萌发事件与位于孢子内膜 (im) 中的萌发受体 (gr) 结合。随后, Gs 将信号传递到也位于 IM 中的 SpoVA 通道蛋白。这导致孢子核心吡啶-2, 6-二羧酸 (二甲二胺酸) 的交换开始;政治部;由20% 的孢子芯干重) 组成, 用于通过 SpoVA 通道的水。随后, dpa 释放触发皮层肽多糖水解的激活, 额外的吸水跟随 2,3,4。这些事件导致涂层层的机械应力, 其随后破裂, 开始生长, 最后, 植物生长。然而, 萌发过程的确切分子细节仍远未解决。
孢子萌发的一个主要问题涉及 im 萌发蛋白周围脂质的生物物理特性以及 IM SpoVA 通道蛋白。这种基本不动的 im 脂质双层是许多小分子的主要渗透屏障, 包括有毒化学防腐剂, 其中一些在孢子核或营养细胞细胞质5,6中发挥作用。IM 脂质双层可能处于凝胶状态, 尽管在 IM5中有相当一部分的移动脂质。孢子的 IM 也有显著扩张的潜力5。因此, im 的表面积在不进行额外膜合成的情况下在萌发时增加 1.6倍, 并伴随着这种膜的低渗透性和脂质不动5,6的丧失。
虽然萌发蛋白活化和在孢子中组织 im 脂质的分子细节是一个很有吸引力的研究课题,但枯草芽孢杆菌的体积小, 萌发蛋白的丰度相对较低, 构成了一个挑战微观分析。Griffiths 等人在荧光显微镜下使用荧光记者融合到萌发蛋白的证据表明, 在b. 枯草孢子中, 支架蛋白 Gerd 为 Gda、B 和 k grs 组织三个 gr 亚基 (A、B 和 c),在一个集群7。他们为这一萌发蛋白群创造了 “萌发细胞” 一词, 并将其描述为 ~ 300 纳米大 IM 蛋白焦点8。在孢子萌发开始后, 荧光萌发病灶最终变成更大的分散荧光模式, 在 l-valine8萌发1小时的孢子中, 有 gt;75% 的孢子群表现出这种模式。请注意, 上面提到的论文使用了数十张连续荧光图像的平均图像, 以获得统计能力, 并克服成像过程中观察到的低荧光信号的障碍。这些结构在细菌孢子中的这种可视化是在技术上是可行的, 与经典的微观工具, 既不是一个单一的孢子中的病灶的数量的评估, 也不是他们更详细的亚细胞定位这种方法的可能性。
在这里, 我们演示了使用结构照明显微镜 (SIM), 以获得详细的可视化和定量的萌发体 (s) 在孢子, 以及他们的 im 脂域9。该协议还包含由 SIMcheck (v1.0, imageJ 插件) 以及 ImageJ10、11、12进行运动、幻灯片准备和图像分析的说明。
Protocol
Representative Results
Discussion
所介绍的协议包含一个标准的3D-SIM 程序, 用于分析 FM4-64 染色b.枯草孢子, 包括孢子, 幻灯片制备和成像过程。此外, 该协议还描述了一种改进的 SIMcheck (ImageJ) 辅助三维成像过程, 用于用荧光记者标记的b.枯草孢子萌发体的 sim 显微镜。后一个过程使我们能够观察到这个暗淡的子结构与增强对比度。通过将两个成像过程耦合起来, 可以用相同的 SIM 显微镜对同一孢子中的离散子结构进行可?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢克里斯蒂安·泽伦伯格在 SIM 成像过程中提供的帮助。JW 感谢中国奖学金委员会获得博士奖学金, 并感谢艾琳·斯泰林沃夫在成像的最初阶段提供的帮助。
Materials
| Air dried glass slides | Menzel Gläser | 630-2870 | |
| APO TIRF N20R8 100× oil objective (NA=1.49) | |||
| B. subtilis KGB80 (PS4150 gerKA gerKC gerKB-mCherry cat, gerD-gfp kan) | |||
| B. subtilis PS4150 (PS832 ΔgerE::spc, ΔcotE::tet) | |||
| Erlenmeyer flasks 1 L | Sigma-Aldrich | Z567868 | |
| Erlenmeyer flasks 250 mL | Sigma-Aldrich | Z723088 | |
| FluoSpheres carboxylate-modified microspheres | Invitrogen, 0.1 μm | F8803 | |
| FM4-64 | Thermo Fisher Scientific | F34653 | |
| Histodenz nonionic density gradient medium | Sigma-Aldrich | D2158 | |
| Image J | |||
| iXON3 DU-897 X-6515 CCD camera | Andor Technology | https://imagej.net/Welcome | |
| LB Agar | Sigma-Aldrich | L2897 | |
| Microfuge tubes 1.5 mL | Thermo Fisher Scientific | 3451PK | |
| Microscope imaging software | Nikon, Japan | NIS-Element AR 4.51.01 | |
| MilliQ Ultrapure Deminerilzed Water | Millipore | Milli-Q IQ 7003 | |
| Nikon Eclipse Ti microscope | |||
| Polypropylene Screw Cap Bottle 180 mL | Thermo Fisher Scientific | 75003800 | |
| Precision Coverslips | Paul Marienfeld | 117650 | |
| Round Bottom tubes 15 mL | Thermo Fisher Scientific | Nunc TM | |
| Screw cap tubes 50 mL | Thermo Fisher Scientific | Nunc TM |
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