我们感谢 Dr. r. 伯格, 在唐纳德丹佛斯植物科学中心的综合显微镜设施主任, 为他的指导和帮助与共焦显微镜。作者没有利益冲突的声明。

1. 用荧光标记防御
<ol>
	<li>选择一种对抗菌性能影响最小的荧光, 以及对活细胞内防御的吸收和定位。 注意: 选择最佳荧光取决于特定的实验目标。还应考虑荧光的光谱和化学性质、光、大小和电荷。</li>
	<li>标签防御与选定的荧光使用适当的肽标签套件可从商业供应商。另外, 化学合成荧光标记的防御。在这项研究中, 标签 MtDef5 防御与 DyLight550 使用商业标签套件根据制造商的协议和四甲基罗丹明 (TAMRA) 标记 MtDef4 防御是化学合成商业。 注: 建议使用化学合成的荧光标记的多肽 (特别是50氨基酸或更少的短肽), 因为荧光可以附着在肽的 n-或 C 端残留物上, 以确保对其的最小影响抗菌活性。合成具有三以上二硫化键的长肽具有挑战性。在这种情况下, 建议使用适当的商业可用的肽标记试剂盒, 将肽标记为最佳荧光。</li>
	<li>测试体外标记防御的抗菌活性, 并测定对真菌的最小抑菌浓度 (MIC)。 注意: 在本研究中, DyLight550-MtDef5 和 TMR-MtDef4 的 MIC 是针对孢菌和镰刀菌菌确定的。</li>
</ol>
2. 真菌培养和生长培养基
<ol>
	<li>将孢子从N. 菌砧木培养物转移到含有伏戈尔琼脂培养基21的斜管上, 在室温下孵育5天。</li>
	<li>区域性F. 菌PH-1 在包含完整介质 (CM)22的板上的应变为 28 c 5 天。为了生产孢子, 接种 4 (10 毫米直径) 5 天的老文化的插头F. 菌PH-1 为50毫升羧甲基纤维素 (CMC) 培养基 (15 克羧甲基纤维素, 1 克酵母提取物, 0.5 g MgSO4. 7 H2O, 1 g NH4没有3和 1 g 的2PO4) 和文化4-7 天在 28 c 在旋转振动筛 180 rpm。</li>
</ol>
3. 准备的孢子悬挂
<ol>
	<li>F. 菌孢子悬挂
	<ol>
		<li>涡流的F. 菌液体培养和收集孢子通过过滤1.5 毫升真菌培养通过两层过滤材料 (如 Miracloth) 到2毫升离心管。离心孢子悬浮在 13226 x g 速度在一个离心为2分钟。</li>
		<li>丢弃上清液, 加入1毫升的无菌水清洗颗粒。</li>
		<li>离心机孢子悬浮在 13226 x g 速度在一个离心 2 min. 丢弃上清和重在1毫升的2X 可持续森林 (合成真菌培养基)23中的颗粒。</li>
		<li>在光显微镜下用例计数孢子。</li>
		<li>将孢子悬浮液调整为 105生孢子/毫升, 2X 可持续使用。</li>
	</ol>
	</li>
	<li>N. 菌孢子悬浮
	<ol>
		<li>在N. 菌中使用一个接种回路将少量的生长培养物 (5 天老) 转移到含有2毫升液体介质的离心管上。</li>
		<li>涡流的孢子悬浮和过滤通过过滤材料进入一个新的离心管。</li>
		<li>将孢子悬浮液以最大速度离心离心2分钟, 在1毫升液体介质中丢弃上清和重孢子颗粒。</li>
		<li>在光显微镜下用例计数孢子。</li>
		<li>将孢子悬浮液调整到 105的生孢子/毫升, 并使用伏地尔液体介质。</li>
	</ol>
	</li>
</ol>
4. 样品制备和共聚焦显微镜
<ol>
	<li>MtDef4 防御的亚细胞定位
	<ol>
		<li>吸管50µL 每孢子悬浮液 (105生孢子/毫升) 入10毫米微35毫米玻璃底部微菜。为观察分生孢子, 直接进行下一步, 或为 germlings 准备, 吸管50µL 的N. 菌和F. 菌分生孢子进入 35 mm 培养皿, 并让发芽3-6 小时室温。</li>
		<li>添加50µL 荧光标记防御在麦克风每微菜和孵化2.5 小时。此处使用的荧光标记防御的麦克风 (步骤 1.3) 为N 菌和F菌的3µM, TMR-MtDef4 菌和f菌的麦克风分别为1µM 和12µM。</li>
		<li>在培养皿中加入2µL 的膜选择性染料 FM4-64 (最终浓度: 5 µM), 并在共聚焦显微镜下孵育30分钟, 立即装入成像。</li>
		<li>选择白光激光器 (环路)。使用两个激光源 488 nm 和 550 nm 分别激发 TMR-MtDef4 和 FM4-64 染料。检测荧光 TMR-MtDef4 在 580-700 nm 和检测 FM4-64 染料在 690-800 nm。 注: 在暗室中进行共聚焦显微镜。</li>
	</ol>
	</li>
	<li>MtDef5 防御内部化的时间推移成像
	<ol>
		<li>吸管50µL 的N. 菌和F. 菌孢子悬浮液 (105生孢子/毫升) 成 10 mm 微的 35 mm 玻璃底微菜。10毫米微无涂层, 并在底部有一个 No. 1.5 盖玻璃。对于分生孢子的观察, 直接进行下一步。为观察 germlings, 在室温下孵育孢子3-6 小时, 然后再进行显微镜检查。</li>
		<li>打开。选择 550 nm 的激光线, 以激发荧光标记的防御 (步骤 1.3) 和 FM4-64, 并使用1.00% 强度并激活相应的探测器。在这里使用的荧光标记的防御 (步骤 1.3) 在 560-600 nm 检测到, FM4-64dye 在 690-800 nm 检测到。 注: 使用低激光强度的活细胞成像作为高激光强度可能会造成对活菌细胞的损害。</li>
		<li>在显微镜上安装微盘。首先找到使用 10 x 目标的单元格, 然后切换到 100 x/1.44 油目标, 以获得更高的放大倍数。 注意: 将扫描模式设置为 xyzt (z 堆栈和延时模式的组合)、z 位置、缩放、图像捕获频率等。</li></ol></li></ol>在继续下一步之前。
		<li>添加50µL 的荧光标记防御使用这里 (步骤 1.3) 的麦克风3µM 和2µL 膜选择性染料 FM4-64 (最终浓度5µM) 每微菜, 并开始延时成像。将小块湿滤纸放在微盘中, 以防蒸发。三十年代的图像采集频率为3分钟, 总周期为2小时30分钟。 注: 在显微镜下安装微盘后添加防御和荧光可以使感兴趣的区域散焦。因此, 在将防御和荧光添加到微后, 需要重新调整感兴趣区域, 这会导致图像获取的延迟。</li>
		<li>使用共聚焦显微镜进行所有共聚焦成像, 并在暗室的室温下进行显微镜观察。</li>

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作者没有什么可透露的。

在这项研究中, 一个可靠的活细胞成像方法, 使用荧光标记的抗真菌防御被描述为研究的动力学, 这些肽内化的真菌细胞和确定其亚型靶点。这种方法是一个强大的工具, 以可视化的动态的互动之间的防御和真菌细胞的时空。
采用多种方法研究了真菌细胞内植物防御的内部化和细胞定位。在这些方法中, 防御处理的细胞通常是固定的, 然后处理的 immunolocalization, 电子显微镜, 或 X ...

植物进化了一个复杂的先天免疫系统, 以抵御微生物植物病原体1。他们表达了许多基因编码的宿主防御肽与假定的抗菌活性2。事实上, 这些肽中的许多都是在体外3中显示抗菌活性的。防御包括植物王国中最大的寄主防御肽组之一4。这些富含半胱氨酸的阳离子肽在 micromolar 浓度下表现出对真菌和卵病原体的有效生长抑制作用, 并且是抵御这些病原体的第一条防线5,6。由于其强大的抗真菌活性, 防御可以被利用在 agribiotechnological 的应用, 以产生抗病作物。几种植物防御的本构表达提高了温室的抗病性和转基因作物的田间试验6。重要的是要解开这些抗真菌肽的作用机制 (MOA), 以便充分利用其潜力作为有效的作物保护工具。然而, 对这些植物防御的 MOA 了解得很差。目前的证据表明, 他们表现出不同的 MOA5,6,7,8。一些防御行为 extracellularly 真菌和靶向特定细胞壁/细胞膜驻留鞘, 扰乱膜完整性和激活细胞毒性通路9,10,11。最近, 然而, 抗真菌防御, 转移到真菌细胞已被发现12,13,14。这些防御结合到膜驻留生物活性磷脂, 形成聚复合物和 permeabilize 等离子膜15,16,17。从而阐明了植物防御的某些方面。然而, 植物防御的 MOA 可能涉及一系列复杂的事件, 它们尚未被识别并集成到一个综合模型中。特别是, 在我们对这些肽的细胞靶点的理解上仍然存在很大的差距。
近年来, 随着显微镜技术的发展和新的荧光探针的开发, 活体细胞成像技术已被广泛应用于抗菌肽 (安培) 的研究。这些技术补充了广泛使用的方法, 如 immunolocalization, 电子显微镜, 原子力显微镜或 X 射线断层摄影术18, 主要用于分析抗真菌肽对形态学的影响和真菌细胞的生长, 包括细胞壁完整性的研究, 细胞生长/分支模式的改变, 以及血浆膜性和杀戮。然而, 这些研究仅限于在治疗后的某个时间点的影像细胞, 而不是在同一个细胞上进行延时成像, 以监测它们对防御挑战的动态变化。近年来, 使用荧光标记多肽与活细胞成像结合使用共焦显微镜, 使 real-time 可视化动力学的 AMP–microbe 相互作用。天然纯化和化学合成的抗真菌肽可以标记为荧光标签 (如, DyLight, 罗丹明, BODIPY, 或 Alexa 的染料), 并直接观察在他们与细胞的互动时, 延时活细胞成像。这些标记多肽的使用大大增加了我们对其 MOA 的不同方面的理解, 包括进入模式、亚细胞定位、胞内贩运以及活真菌细胞中的抗真菌作用部位18。
最近, 有几项研究表明, 包括植物防御在内的各种抗真菌肽都是由活真菌细胞内化的12,14,19,20。这些防御的 MOA 可能涉及与细胞内目标的相互作用。我们最近报告了植物防御 MtDef4 在两个囊真菌的抗真菌作用,孢菌和镰刀菌菌。MtDef4 被证明使用不同的途径真菌细胞进入和亚细胞定位这些真菌14。本研究采用化学合成的四甲基罗丹明 (TAMRA) 标记 MtDef4 与生命荧光染料 (膜选择染料, FM4-64; 膜 permeant 染料, SYTOX 绿色; 细胞死亡记者染料, 碘碘化物) 和代谢抑制剂。这些分析显示了 MtDef4 内化的动力学, 其胞内转运机制和亚细胞靶向14。
本文提出了一种利用共焦显微镜进行活细胞成像的协议。该协议利用荧光标记多肽与生命荧光染料结合, 研究植物防御真菌相互作用, 特别是真菌细胞的易位途径和胞内防御的细胞靶点。

<table><tbody><tr><td>FM4-64 Dye</td><td>Life Technologies</td><td>T13320</td><td></td></tr><tr><td>DyLight 550 Antibody Labeling Kit</td><td>Thermo Scientific</td><td>84530</td><td></td></tr><tr><td>Glass Bottom Microwell Dishes</td><td>Mat TeK</td><td>P35G-1.5-10-C</td><td></td></tr><tr><td>Mira cloth</td><td>EMD Millipore Corp</td><td>475855-1R</td><td></td></tr><tr><td>SP8-X confocal microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>ImageJ software</td><td>FiJi</td><td></td><td>For Image analysis</td></tr><tr><td>Imaris software</td><td>Bitplane</td><td></td><td>For Image analysis</td></tr></tbody></table>

live-cell imaging of fungal cells to investigate modes of entry and subcellular localization of antifungal plant defensins

小的半胱氨酸丰富的防御是在所有植物中存在的最大的寄主防御肽群之一。许多植物防御展示了强有力的在体外抗真菌活性的 broad-spectrum 霉菌病原体, 因此有潜力被用作抗真菌剂在转基因作物。为了充分发挥植物防御对疾病控制的潜力, 阐明其作用机制是至关重要的。随着先进的显微技术的问世, 活细胞成像已经成为了解植物防御抗真菌的动力学的有力工具。在这里, 用两个荧光标记植物防御 (MtDef4 和 MtDef5) 结合生命荧光染料描述了一种基于共聚焦显微镜的活体细胞成像方法。这一技术使 real-time 可视化和分析的动态事件的 MtDef4 和 MtDef5 内化为真菌细胞。重要的是, 这种分析产生了丰富的信息, 包括内化动力学, 进入方式和亚细胞定位这些肽。这些方法与其他细胞生物工具一起, 为这些肽的动态和复杂性提供了重要的洞察力。这些工具也可以用来比较这些肽对不同真菌的 MOA。

从苜蓿蒺藜中进行了活细胞成像, 以追踪和比较两个防御、MtDef4 和 MtDef5 的内化和亚单位定位;在真菌细胞中。TMR-MtDef4 是化学合成的, 而 MtDef5 被标记为 Dylight550 (Dylight550-MtDef5)。用防御与膜选择性染料 FM4-64 联合培养孢子。图 1显示, 与F. 菌相比, TMR-MtDef4 在N. 菌中具有不同的贩运路径。在N. 菌中, FM4-64 不 co-localize 与防御, 而?...

Watch this Scientific Journal Video about Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins at JoVE.com

Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins

植物防御在植物防御病原体中起着重要的作用。为了有效地使用这些抗真菌肽作为抗真菌剂, 了解其作用方式 (MOA) 是至关重要的。在这里, 描述了一个活细胞成像方法, 以研究这些肽的 MOA 的关键方面。

真菌细胞的活细胞成像研究抗真菌植物防御的进入和亚单位定位

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro ...

live-cell-imaging-fungal-cells-to-investigate-modes-entry-subcellular

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

6.6K 次观看.  Donald Danforth Plant Science Center. 植物防御在植物防御病原体中起着重要的作用。为了有效地使用这些抗真菌肽作为抗真菌剂, 了解其作用方式 (MOA) 是至关重要的。在这里, 描述了一个活细胞成像方法, 以研究这些肽的 MOA 的关键方面。

视频: Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

活细胞视频显微镜病病原菌的吞噬作用

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

科研

免疫与感染

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

生物学

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

为了应对抑菌活性的实时成像人力初级中性粒细胞和单核细胞<em&gt;一种。曲霉</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

二维和三维活细胞成像的DNA损伤反应蛋白

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

用于在细胞水平上探测真菌 - 微生物相互作用的微流体工具

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

生物工程

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

分离绿色荧光蛋白系统可视化细菌在感染过程中传递的效应

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

膜和细胞壁选择性荧光染料在丝状真菌活细胞成像中的应用

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...

玉米鞘和真菌之间细胞相互作用的可视化

Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one originally applied to rice leaf sheaths and allows direct microscopic observation of fungal growth and development in living plant cells. Leaf sheaths collected from maize seedlings with two fully emerged leaf collars are inoculated with 20 &#181;L drops of 5 x 105 spores/mL fungal spore suspensions and incubated in humidity chambers at 23 &#176;C under continuous fluorescent light. After 24-72 h, excess tissue is removed with a razor blade to leave a single layer of epidermal cells, an optically clear sample that can be imaged directly without the necessity for chemical fixation or clearing. Plant and fungal cells remain alive for the duration of the experiment and interactions can be visualized in real-time. Sheaths can be stained or subjected to plasmolysis to study the developmental cytology and viability of host and pathogen cells during infection and colonization. Fungal strains transformed to express fluorescent proteins can be inoculated or co-inoculated on the sheaths for increased resolution and to facilitate the evaluation of competitive or synergistic interactions. Fungal strains expressing fluorescent fusion proteins can be used to track and quantify the production and targeting of these individual proteins in planta. Inoculated sheath tissues can be extracted to characterize nucleic acids, proteins, or metabolites. The use of these sheath assays has greatly advanced the detailed studies of the mechanisms of fungal pathogenicity in maize and also of fungal protein effectors and secondary metabolites contributing to pathogenicity.

作者聚焦:研究玉米中的真菌致病机制 

This manuscript details an optimized inoculation protocol that uses detached maize leaf sheaths for reproducible cytological, physiological, and molecular studies of maize interactions with fungal plant pathogens. The leaf sheaths facilitate real-time observation of cellular interactions between the living plant and fungus in unfixed tissues.

用于真菌叶面玉米病原体感染的活细胞成像的分离玉米鞘

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one ...

真菌细胞的活细胞成像研究抗真菌植物防御的进入和亚单位定位

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