TEM TOMOGRAPHY OF A DROSOPHILA ATP6 MUTATION

果蝇 ATP6 突变的 TEM 断层扫描

• 批准号: 7721714
• 负责人: Michael John Palladino
• 金额: $ 1.11万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721714
• 关键词: Address; Catalysis; Complement; Computer Retrieval of Information on Scientific Projects Database; Couples; Crista ampullaris; Disease; Drosophila genus; F1F0-ATP synthase; Frequencies; Funding; Genes; Genotype; Grant; Human; Impairment; Inherited; Institution; Ions; Journals; Longevity; Membrane; Mitochondria; Mitochondrial Encephalomyopathies; Modeling; Morphology; Mutation; Nerve Degeneration; Orthologous Gene; Outer Mitochondrial Membrane; Paper; Peripheral; Production; Protocols documentation; Publishing; Radial; Reporting; Research; Research Personnel; Resolution; Resources; Source; Surface; Techniques; Testing; Tube; Tubular formation; United States National Institutes of Health; Vesicle; abstracting; aging brain; animation; density; improved; movie; mutant; reconstruction; tomography

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. ABSTRACT Our studies of mutations that cause shortened lifespan and neurodegeneration have led us to identify a maternally inherited mutation in the mitochondrial gene ATP6 (ortholog of ATPa). This gene encodes the H+ channel component of the F1F0 ATP synthase, which couples ion gradient dissipation with ATP production via rotary catalysis. In humans, impairment of ATP6 generally manifests in diseases when the mutation is nearly homoplastic. For example, humans bearing the best-characterized mutation in the mitochondrial ATP6 gene (L156R) have MILS, NARP, or are normal depending upon the heteroplasmy of the mutation (>~85%, ~70-85% or <~70% mutant, respectively). In our mutant, ATP6[1] the mutation in nearly homoplastic (98¿4 % mutant). TEM analysis revealed aberrant cristae in mitochondria from ATP6[1] mutants (Figure 1). We have acquired the micrographs using stereological protocols and have discovered the density of mitochondria in ATP6[1] (31 ¿ 3 per 100mM2) does not appear to be significantly different than wild type (35 ¿ 2 per 100mM2, p=0.4). We would like to better understand this morphological aberration to complement our ongoing functional studies of these mitochondria. Specifically, is the internal mitochondiral membrane vesiciular (as it appears in 2D TEM) or is it tubular? Does the internal membrane remain separate from the outer mitochondrial membrane? TEM tomography with a 5nm resolution is extremely well-suited to directly address both of these questions, which have profound consequences on the function of mitochondria. The quality of our 2D TEM and the high frequency with which we see aberrant mitochondria in our mutants suggests the proposed project will not be overly burdensome and will yield instructive results. Figure 1: TEM analysis of aged brains reveals aberrant mitochondrial morphology in ATP6[1] mutants (B). N > 3 each genotype. Quantification demonstrates a high frequency of abnormal mitochondria (>60% of total mitochondria, C). Comparison to wild type (* p<0.01, T-test). In the previous reporting periods, blocks from wild-type and ATP6 mutant were received. A double-tilt reconstruction made from each. Dr. Renken collected an additional single-tilt reconstruction from the mutant, using the Tecnai F20. Surface-rendered models, using both tracing and thresholding techniques, were made. In mitochondria from the mutant, there appears to be a radial progression from peripheral tubes to central vesicles. Surface models were improved, and animation movies were made. One of the surface models was used on the journal cover when the paper was published: + Celotto, A.M., Frank, A.C., McGrath, S.W., Fergestad, T., Van Voorhies, W.A. Buttle, K.F., Mannella, C.A., Palladino, M.J. (2006) Mitochondrial encephalomyopathy in Drosophila. J. Neurosci. 26:810-820.

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。 抽象的 我们对导致寿命缩短和神经退行性变的突变的研究使我们发现了线粒体基因 ATP6（ATPa 的直系同源物）中的母系遗传突变。该基因编码 F1F0 ATP 合酶的 H+ 通道组件，该组件将离子梯度耗散与 ATP 耦合。在人类中，当突变接近同源性时，ATP6 的损伤通常会出现在疾病中，例如，人类携带最典型的突变。线粒体 ATP6 基因 (L156R) 具有 MILS、NARP 或正常，具体取决于突变的异质性（在我们的突变体 ATP6 中分别为 >~85%、~70-85% 或 <~70% 突变体）[1]。几乎同质的突变（98¿4 % 突变体）显示 ATP6[1] 突变体的线粒体中存在异常嵴（图 1）。使用立体学协议并发现 ATP6[1] 中线粒体的密度（31 ¿ 3 每 100mM2）似乎与野生型（35 ¿ 2 每 100mM2，p=0.4）。我们希望更好地了解这种形态畸变，以补充我们正在进行的这些线粒体的功能研究。具体来说，线粒体内部膜是囊泡状的（如 2D TEM 中所示）还是管状的？内膜与线粒体外膜保持分离吗？具有 5 nm 分辨率的 TEM 断层扫描非常适合直接解决这两个问题，这会产生深远的影响我们的 2D TEM 的质量以及我们在突变体中看到异常线粒体的高频率表明，拟议的项目不会过于繁琐，并且会产生有启发性的结果。 图 1：衰老大脑的 TEM 分析揭示了 ATP6[1] 突变体的异常线粒体形态 (B)，每种基因型的定量显示异常线粒体的频率较高（> 总线粒体的 60%，C）。类型（* p<0.01，T 检验）。 在之前的报告期间，Renken 博士使用 Tecnai 表面渲染模型收集了来自野生型和 ATP6 突变体的块，并从突变体中收集了额外的单倾斜重建。使用追踪和阈值技术，在突变体的线粒体中，似乎存在从外围管到中央囊泡的径向进展，并且制作了动画电影。论文发表时用于期刊封面的： + Celotto, A.M.、Frank, A.C.、McGrath, S.W.、Fergestad, T.、Van Voorhies、W.A. Buttle, K.F.、Mannella, C.A.、Palladino, M.J. (2006) 果蝇线粒体脑肌病 26:810-820。 。