| A Bioimaging Pipeline to Show Membrane Trafficking Regulators Localized to the Golgi Apparatus and Other Organelles in Plant Cells
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Author:
Date:
2015-09-05
[Abstract] The plant Golgi apparatus is composed of numerous stacks of cisterna, designated as cis, medial, and trans Golgi cisternae; these stacks move within the cytoplasm along the actin cytoskeleton. Cis cisternae receive secretory products from endoplasmic reticulum (ER) and they subsequently progress through the stack to the trans cisternae, where they are sorted to other destinations, including cell wall, plasma membrane (PM), vacuoles, and chloroplasts. In addition, the plant Golgi apparatus plays a role of glycosylating proteins as well as synthesizing cell wall polysaccharides, such as hemicelluloses and pectins. This protocol describes procedures for imaging fluorescently-tagged proteins localized to the plant Golgi apparatus of Arabidopsis ...
[摘要] 植物高尔基体包括许多叠状体，称为顺，内侧和反式高尔基这些堆栈在细胞质内沿着肌动蛋白细胞骨架移动。 Ctern 池从内质网（ER）接收分泌产物，并且随后通过堆叠进入反转池，在那里它们被分选到其他目的地，包括细胞壁，血浆膜（PM），液泡和叶绿体。此外，植物高尔基体起到糖基化蛋白质以及合成细胞壁多糖如半纤维素和果胶的作用。该协议描述了使用共聚焦激光显微镜（CLSM），全内反射荧光显微镜（TIRF）和高压的免疫金标记标记定位于拟南芥幼苗的植物高尔基体的荧光标记的蛋白质的程序冷冻/冷冻取代的样品通过透射电子显微镜（TEM）。我们特别关注长期时间推移成像和蛋白质本地化在高尔基内的子域。该方案还可以用于其他细胞器，组织和植物物种。
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| Differentiation of Naturally Produced Extracellular Membrane Vesicles from Lipid Aggregation by Glucuronoxylomannan Immunogold Transmission Electron Microscopy in Bacillus subtilis
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Author:
Date:
2015-03-05
[Abstract] Recently, membrane vesicle (MV) production was described in Gram-positive bacteria, which harbor a variety of components such as toxins, antibiotic resistance proteins, proteases, DNA, and immune modulators. Free lipids have the ability to form micelles, thus it is important to rule out spontaneous association of lipids into vesicle-like structures and rather, that MVs are produced naturally by a metabolically active cell. Here, we describe a protocol utilizing the polysaccharide, glucuronoxylomannan (GXM) from Cryptococcus neoformans (C. neoformans) as a marker to differentiate naturally produced MVs from vesicles that form spontaneously in the Gram-positive model organism, Bacillus subtilis (B. subtilis). MVs are purified from bacterial cultures grown ...
[摘要] 最近，在革兰氏阳性细菌中描述了膜囊泡（MV）产生，其含有多种组分，例如毒素，抗生素抗性蛋白，蛋白酶，DNA和免疫调节剂。游离脂质具有形成胶束的能力，因此排除脂质自发缔合成囊泡样结构是重要的，并且更确切地说，MV由代谢活性细胞天然产生。在这里，我们描述了利用来自新隐孢子虫的多糖葡萄糖醛酸甘露聚糖（GXM）（ C。新型隐球菌）作为标记物来区分天然产生的MV与自发形成的囊泡革兰氏阳性模式生物，枯草芽孢杆菌（<枯草芽孢杆菌）。从在GXM存在下生长的细菌培养物中纯化MV;由细胞天然产生的MV在管腔中不含有GXM，而在培养基中形成的囊泡结构可包封GXM，并且这可通过免疫金属透射电子显微镜可视化。
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