丝状蓝藻延时显微镜分析的垂直固定方法
Summary
我们提出了一种简单易用的XY平面丝状蓝藻可视化方法。使用低熔点琼脂糖基质，允许以垂直方向获取参与分裂的蛋白质的图像。因此，该方法可以应用于任何丝状生物和不同种类的蛋白质。
Abstract
细菌细胞分裂的主要事件是分离过程，其中蛋白质FtsZ是关键元素。FtsZ在细胞中间聚合形成环状结构（Z环），作为其他分裂蛋白的支架。细菌模型 大肠杆菌 和 枯草芽孢杆菌 的超分辨率显微镜显示Z环是不连续的，而活细胞成像研究表明FtsZ通过一种称为跑步机的机制沿着环移动。为了研究FtsZ在 体内的动力学，需要在垂直位置放置特殊的细胞，以便在XY平面中对环的完整结构进行成像。在多细胞蓝藻（例如 Anabaena sp. PCC7120）中进行FtsZ成像的情况下，由于细胞的大小和细丝的长度，将细丝保持在垂直位置具有挑战性。在本文中，我们描述了一种方法，该方法允许使用低熔点琼脂糖和注射器垂直固定 Anabaena sp. PCC 7120细丝，以记录表达FtsZ-sfGFP融合蛋白的突变体中的Z环。该方法是一种使用共聚焦显微镜在分裂位点记录蛋白质动力学的快速且廉价的方法。
Introduction
细菌细胞分裂是母细胞产生两个子细胞的过程，在大多数情况下是通过称为二元裂变的机制。分离过程中最早的事件之一是FtsZ在细胞1中间的定位。这种蛋白质在结构上与微管蛋白2 同源，在大多数细菌中是保守且广泛分布的，其聚合产生称为 Z 环3 的收缩结构。这个环充当其他分裂蛋白的支架，它们一起形成一种称为分裂体的分子机制。多项研究表明，Z 环是高度动态的，FtsZ 原丝通过跑步 4,5,6 移动。为了在延时实验中研究Z环，建议在XY平面中记录分裂位点，以获得更好的分辨率和快速采样。为了实现这一目标，有必要开发垂直细胞固定方法，该方法通常包括微孔细胞陷阱和复杂微流体装置的纳米制造7。
蓝藻是光合微生物，根据其细胞形态被归类为革兰氏阴性菌。然而，在系统发育上，它们更接近革兰氏阳性细菌8。这些生物体具有在革兰氏阳性和革兰氏阴性细菌中常见的细胞分裂基因，但它们的分裂体也含有独特的元素9。Anabaena sp. PCC 7120（以下简称Anabaena sp.）是一种具有一个分裂平面的丝状蓝藻，是研究多细胞蓝藻细胞分裂的模型。在该菌株中，已经能够确定FtsZ在细胞10中间的位置。然而，没有研究表明FtsZ在该模型中的体内动力学。在我们的实验室中，通过三亲交配和同源重组，我们获得了Anabaena sp.的完全分离突变体，该突变体表达与sfGFP融合的FtsZ蛋白，取代了完整的内源性ftsZ基因。我们开发了一种快速简单的细胞固定方法，该方法有利于突变菌丝的垂直方向，用于延时实验，以可视化丝状蓝藻中的分裂蛋白。这种方法不需要昂贵且难以开发的微流体装置。例如，我们使用该协议通过共聚焦显微镜可视化FtsZ-sfGFP突变体中的Z环。
Protocol
Representative Results
Discussion
对分裂体蛋白质动力学的研究无疑是一个挑战。特别是在丝状蓝藻中，挑战之一是水平面Z环的可视化，细胞必须垂直定向。我们在这里描述的方法允许在XY平面中定位Z环以执行不同的分析。这是丝状蓝藻的第一种简单方法，可以完全可视化Z环。
尽管该方案简单快速，但在琼脂糖基质的样品制备步骤中必须特别小心，因为低熔点琼脂糖必须处于合适的温度，以使流体足以与?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢国家博士奖学金（ANID 21211333; 21191389）的资助。格兰特·丰德西特1161232。
这项工作得到了de Advanced Microscopy Facility UMA UC的支持。
Materials
| 1 ml syringe | Qingdao Agna Medical Technology Co., Ltd. | – | The disposable medical plastic syringe with 1 ml needle generally used to pump liquid or injection liquid, in this experiment it is used to suck the sample and make it polymerize. |
| Attofluor Cell Chamber | ThermoFischer Scientific | A7816 | The Attofluor Cell Camera is a durable and practical coverslip holder designed for viewing live cell samples in upright or inverted microscopes. |
| Axygen MaxyGene II Thermal Cycler with 96 well block | CORNING | THERM-1001 | The new MaxyGene II Thermal Cycler increased speed and advanced features, providing the premium performance you have come to expect from Axygen brand products. Unique flexible programming. Rapid run times. Improved workflow over traditional gradient cyclers. Ramping rates up to 5°C/sec. Adjustable heated lid accommodates strips, tubes and microplates. |
| Fisherbrand Cover Glasses: Circles | ThermoFischer Scientific | 12-546-2P | Made of finest optical borosilicate glass, with uniform thickness and size. Circular shape. Corrosion-resistant. |
| Low Melting Point agarose | Promega | V3841 | Agarose, Low Melting Point, Analytical Grade, is ideal for applications that require recovery of intact DNA fragments after gel electrophoresis. |
| LSM 880 microscope from Zeiss Airyscan | Zeiss | – | The Zeiss Airyscan LSM 880 microscope is a laser scanning focal microscope that can acquire images under the resolution limit (lateral resolution ~ 120nm and axial resolution ~ 350nm). The detectors are highly sensitive making it possible to acquire super-resolution images at high speed. |
| Microcentrífuga Fresco 17 | ThermoFischer Scientific | 75002402 | Speed up routine sample preparation processes up to 17,000 × g with our standard microcentrifuge, available with refrigeration. These microcentrifuges offer productivity, versatility, safety and convenience in an easy-to-use, compact design laboratory instrument. |
| Nikon Timelapse Microscope | Nikon | – | The Nikon C2 laser scanning confocal microscope is an ideal microscope for long-term timelapse, because thanks to its incubation chamber it is possible to keep samples in optimal conditions for more than 8 hours. |
| Thin razor blades Schick Super Chromium | Farmazon | SKU: 401146 | The thin razor blades is used to cut the agarose matrix, allowing us to obtain the disks with the sample while maintaining the integrity of the matrix. |
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