IMAGING APPROACHES FOR DIATOM SILICA CELL WALL SYNTHESIS

硅藻土细胞壁合成的成像方法

• 批准号: 7601091
• 负责人: MARK HILDEBRAND
• 金额: $ 0.22万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601091
• 关键词: Algae; Cell Count; Cell Wall; Cells; Computer Retrieval of Information on Scientific Projects Database; Culture Techniques; Diatoms; Dyes; Electron Microscopy; Electrons; Event; Fluorescence; Fluorescence Microscopy; Fluorescent Dyes; Funding; Genes; Goals; Grant; Green Fluorescent Proteins; Image; Imaging Techniques; Institution; Investigation; Label; Localized; Location; Maps; Membrane; Monitor; Photobleaching; Phytoplankton; Pilot Projects; Process; Proteins; Research; Research Personnel; Resolution; Resources; Scanning; Set protein; Silicon Dioxide; Source; Staging; Staining method; Stains; Structure; Subcellular structure; Techniques; Three-Dimensional Image; Three-Dimensional Imaging; Time; USA Georgia; United States National Institutes of Health; Western Asia Georgia; Work; electron tomography; interest; intracellular protein transport; nanostructured; protein localization location; two-photon

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are investigating the process of cell wall synthesis in diatoms, unicellular eukaryotic phytoplankton that make cell walls of nanostructured silica. Electron and fluorescence microscopy is being applied to monitor subcellular structures and their dynamics during cell wall synthesis. In addition, the intracellular location of specific proteins in relation to silica structures is being determined, with a goal of correlating formation of specific structures with the proteins involved. We are interested in applying advanced imaging techniques to understand how diatoms, unicellular eukaryotic algae, make their cell walls out of silica. We discussed with Mark Ellisman, Guido Gaietta, and Tom Deerinck at NCMIR three pilot scale projects and one longer term investigation. One pilot project is to generate high-resolution 3D images of the diatom Thalassiosira pseudonana during cell wall synthesis using electron tomography. We have developed a synchronized culture technique that enables enrichment of cells forming particular silica structures, which facilitates this approach. Because we can incorporate fluorescent dyes into diatom silica, examination of these cells using two-photon confocal scanning fluorescence microscopy could also provide valuable information. A second pilot project is to attempt real-time confocal imaging of cell wall formation, monitoring silica incorporation and membrane dynamics with specific fluorescent dyes. By analyzing a reasonable number of cells, we should be able to identify those in specific stages in cell wall synthesis. We propose to initially look at Nitzschia alba, a non-photosynthetic diatom on which we have done preliminary work in this regard, but would like to extend this to T. pseudonana. Examination of cytoskeletal dynamics during this process would also be valuable. A third pilot level project is to investigate protein localization approaches using tags that enable sequential fluorescence and electron microscopy to fine map proteins during formation of specific structures. Our collaborators, Nicole Poulsen and Nils Kr¿ger at Georgia Tech, have developed transformation and GFP tagging techniques for T. pseudonana, and have shown that one protein isolated from the silica cell wall is specifically localized to a particular silica substructure. We propose to generate a fusion construct with this gene, GFP, and a tetracysteine tag that would enable biarsenical dye labeling of the localized protein. After evaluating fluorescence localization, we would then photobleach the dye to facilitate staining for TEM to more precisely map location. The longer term investigation would involve localizing, in relationship to silica structure and larger scale dynamic events, sets of proteins we have identified using microarrays that are suspected to be involved in cell wall synthesis. These approaches have not been previously applied to diatoms, and are likely to change our fundamental understanding of the components and dynamics involved in silica structure formation.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 我们正在研究硅藻、单细胞真核浮游植物的细胞壁合成过程，利用电子和荧光显微镜来监测细胞壁合成过程中的亚细胞结构及其动态。与二氧化硅结构相关的蛋白质正在被确定，目标是将特定结构的形成与所涉及的蛋白质相关联。 我们有兴趣应用先进的成像技术来了解硅藻（单细胞真核藻类）如何用二氧化硅制造细胞壁。我们与 NCMIR 的 Mark Ellisman、Guido Gaietta 和 Tom Deerinck 讨论了三个试点项目和一项长期研究。试点项目是使用电子断层扫描技术在细胞壁合成过程中生成硅藻假海藻的高分辨率 3D 图像。能够富集形成特定二氧化硅结构的细胞，这有利于这种方法，因为我们可以将荧光染料掺入硅藻二氧化硅中，所以使用双光子共焦扫描荧光显微镜检查这些细胞也可以提供有价值的信息。细胞壁形成的实时共聚焦成像，用特定荧光染料监测二氧化硅的掺入和膜动力学。通过分析合理数量的细胞，我们应该能够识别细胞壁合成中特定阶段的细胞。首先看一下 Nitzschia alba，一种非光合作用硅藻，我们已经在这方面进行了初步工作，但希望将其扩展到 T.pseudonana，在此过程中对细胞骨架动力学的检查也很有价值。我们的合作者 Nicole Poulsen 和 Nils Kr¿ 旨在研究使用标签的蛋白质定位方法，这些标签使顺序荧光和电子显微镜能够在特定结构的形成过程中精细绘制蛋白质图谱。佐治亚理工学院的 ger 开发了 T.pseudonana 的转化和 GFP 标记技术，并表明从二氧化硅细胞壁中分离出的一种蛋白质特异性定位于特定的二氧化硅亚结构，我们建议用该基因 GFP 生成融合构建体。和四半胱氨酸标签，可以对定位蛋白质进行双砷染料标记。在评估荧光定位后，我们将对染料进行光漂白，以方便 TEM 染色以更精确地绘制位置图。 长期研究将涉及与二氧化硅结构和更大规模的动态事件相关的定位，我们使用微阵列鉴定出怀疑参与细胞壁合成的蛋白质组。这些方法以前尚未应用于硅藻，但目前尚未应用于硅藻。可能会改变我们对二氧化硅结构形成所涉及的成分和动力学的基本理解。