REGULATION OF SUBCELLULAR ORGANIZATION IN SKELETAL MUSCLE
骨骼肌亚细胞组织的调节
基本信息
- 批准号:7732813
- 负责人:
- 金额:$ 124.56万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:AcuteAddressAffectAntibodiesAreaBiological AssayBiopsyCardiacCell Differentiation processCell LineCell NucleusCell membraneCellsCellular biologyCentrosomeChronicComplementary DNAConditionCultured CellsCytoskeletonDefectDesminDestinationsDiseaseDisruptionDominant-Negative MutationDrug usageElectronsElementsEndoplasmic ReticulumEnzymesEventFailureFiberFilamentGenerationsGlycogen Synthase Kinase 3Glycogen Synthase KinasesGlycogen storage disease type IIGoalsGolgi ApparatusHumanImageImmunofluorescence ImmunologicInfantIntermediate Filament ProteinsIntermediate FilamentsKnockout MiceKnowledgeLateralLifeLinkLysosomesMammalian CellManualsManuscriptsMembraneMembrane ProteinsMicroscopyMicrotubule BundleMicrotubule StabilizationMicrotubule-Associated ProteinsMicrotubule-Organizing CenterMicrotubulesModalityModelingMolecularMusMuscleMuscle CellsMuscle DevelopmentMuscle FibersMuscle functionMuscular DystrophiesMutateMyoblastsMyofibrilsMyogeninMyopathyMyosin ATPaseNocodazoleOrganellesOsteoclastsPaclitaxelPaste substancePathologyPathway interactionsPharmaceutical PreparationsPhotonsPhysiologicalPhysiologyPlasticsPlayPositioning AttributePost-Translational Protein ProcessingPrevalenceProteinsPublishingRecyclingRegulationRoleSiteSkeletal MuscleSourceStagingStructureSystemTechniquesTissuesUndifferentiatedViral ProteinsWorkWritingYeastsbasehuman subjectimpressioninhibitor/antagonistinterestkinase inhibitorknock-downlight microscopymouse modelmyogenesispericentrinpreventresearch studysatellite cellsecond harmonicsizetooltraffickingtranscription factor
项目摘要
The Golgi complex is an essential organelle involved in the post-translational modifications of proteins and in the targeting of membrane and secreted proteins to their destination in the cell. It is easily identified in electron micrographs as a stack of flattened cisternae, each representing a different functional compartment through which secreted and trans-membrane proteins transit sequentially. Yet, there is plasticity in its organization. The muscle Golgi complex is constituted of many small stacks, whereas most mammalian cells have a single Golgi complex. Its positioning near the nucleus is due to microtubules, and different microtubule organizations seem responsible for different Golgi complex organizations. We have published the only comprehensive model, so far, for the reorganization of the Golgi complex during muscle differentiation (Lu et al., 2001). During muscle differentiation, both microtubules and Golgi complex are coordinately redistributed as muscle cells elongate and fuse to form large multinucleated cells. We demonstrated that reorganization of the Golgi complex involves its fragmentation in small elements. We proposed that this reorganization depends on the constant recycling of Golgi complex proteins through the endoplasmic reticulum (ER), on the reorganization of microtubule organizing centers (MTOC, or centrosome), and on redistribution of the ER exit sites. Our current goal is to refine this model and, particularly, to find a hierarchy in the several changes that appear to be taking place simultaneously.
We have started by asking whether reorganization of the Golgi complex can take place in the absence of a normal microtubule network. Working with the mouse muscle cell line C2, we have established concentrations of different drugs that chronically affect microtubules while allowing muscle differentiation (expression of the transcription factor myogenin). We have used the drugs nocodazole, which prevents microtubule nucleation and elongation, taxol, which instead forces massive stabilization and bundling of microtubules, and an organometallic specific inhibitor of glycogen synthase kinase 3, expected to affect microtubule orientation and stabilization.
As we had hypothesized, differentiation in presence of these drugs affects different aspects of the cellular reorganization distinctly. Reorganization of the MTOC is the least affected, with more than 75% normal reorganization in all conditions. In contrast, Golgi complex reorganization fails or is incomplete in a large fraction of myogenin-positive cells and is never observed unless MTOC reorganization is normal. A small percentage of cells seem to have a normal reorganization of the Golgi complex without ER exit sites redistribution but in >90% of the cells the two reorganize coordinately. We therefore propose that the primary event during differentiation is the reorganization of the MTOC which then leads to the coordinate reorganization of Golgi complex and ER exit sites. Kristien Zaal is presently writing up this work.
Although failure to reorganize completely may have physiological consequences if carried over to a mature muscle fiber, this is not necessarily the case in cultured cells. The Golgi complex may be plastic enough to function in several geometries. To assess its functionality, we have used a well-known assay, in which trafficking of a fluorescently tagged viral protein (VSV-G-GFP) is followed from the endoplasmic reticulum to the plasma membrane, through the Golgi complex. We have demonstrated that trafficking of VSV-G appears normal in cells treated with a glycogen synthase kinase inhibitor, despite the abnormal distribution of the Golgi complex elements.
Information about the MTOC organization in muscle cells is sought through experiments carried out by Tan Zhang. He has been investigating the role of the microtubule-associated protein EB1 in muscle differentiation. In other mammalian cells, EB1 is necessary for microtubule stabilization. We hypothesized that EB1 may play a similar role during muscle development. Indeed, we found that dominant-negative constructs of EB1 affect myoblast elongation. Knocking down EB1 permanently prevents microtubule stabilization and, unexpectedly, prevents differentiation of the muscle cells (this work is described in a recently submitted manuscript). In addition to its role at the plus end tips of microtubules, EB1 is also present in the centrosome. We confirmed that EB1 knock-down cells have defects in microtubule nucleation and also found that pericentrin, one of the best known proteins of the MTOC, is partially displaced to the Golgi complex in EB1 knock-down cells. We are now ready to use cDNA constructs which affect EB1 selectively at the centrosome in order to determine which of its localizations is related to its role in cell differentiation. These results point to a link between the microtubule cytoskeleton organization and the capacity for muscle cell differentiation.
Microtubules play an important role in the reorganization of the Golgi complex during myogenesis but they may not be the only cytoskeletal system involved in the distribution of the Golgi complex in muscle fibers and others as well as our own results suggested intermediate filament involvement. Since desmin is the main intermediate filament protein of skeletal muscle, we have used desmin-null mice. We have observed that in slow muscle fibers from desmin-null mice, Golgi complex elements and ER exit sites are abnormal and heterogeneous in size compared to those in control mice. In the past year, John Sheridan has prepared satellite cell cultures from single muscle fibers of specific muscles from control and desmin-null mice. The Golgi complex reorganization/ fragmentation in desmin-null myotubes is indeed incomplete, suggesting that desmin does plays a role in the reorganization of the Golgi complex at the early stages of differentiation.
The best tool to obtain the information we require is immunofluorescence of single fibers. The manual teasing of the fibers is labor-intensive, yet is necessary because antibodies do not penetrate bundles of fibers and sections provide a view that is too limited. There are cases, however, where handling single fibers is close to impossible. We have been collaborating with Drs. Plotz and Raben in NIAMS who study Pompe Disease, a lysosomal storage disorder in which cardiac and skeletal muscles are the source of the pathology. Our work has emphasized the presence of large areas of autophagic debris in muscles of the mouse model and of Pompe Disease human subjects. If such buildup is linked to the pathology, it is very important to be able to assess its prevalence in biopsies, particularly in those of infants who suffer the most devastating form of the disease. Single infant fibers, however, are thin and frail, and extremely difficult to handle. To address this problem, we have used a new imaging modality which provides structural information from unstained tissue at the molecular level, the 2-photon microscopy technique called Second Harmonic Generation (SHG). SHG detects arrays of molecules arranged in a semi-crystalline order. Myosin filaments are a good source of SHG and the technique can be applied to large bundles of fibers. We have found that SHG is capable of detecting all the inclusions (enlarged lysosomes and autophagic buildup areas) found in the mouse model and in human biopsies of Pompe disease subjects (Ralston et al., 2008). This was the first published application of SHG to assess muscle disease. SHG quantitation of muscle defects should be also of interest in other pathological conditions in which structural defects of muscles may be more subtle and not as well documented.
高尔基复合体是一种重要的细胞器,参与蛋白质的翻译后修饰以及将膜和分泌蛋白靶向至细胞中的目的地。在电子显微照片中很容易将其识别为一堆扁平的池,每个池代表一个不同的功能区室,分泌蛋白和跨膜蛋白依次通过该区室。然而,其组织具有可塑性。肌肉高尔基复合体由许多小堆组成,而大多数哺乳动物细胞只有一个高尔基复合体。它位于细胞核附近是由于微管,不同的微管组织似乎负责不同的高尔基复合体组织。迄今为止,我们已经发表了唯一一个关于肌肉分化过程中高尔基复合体重组的综合模型(Lu et al., 2001)。在肌肉分化过程中,随着肌肉细胞伸长并融合形成大的多核细胞,微管和高尔基复合体协调地重新分布。我们证明高尔基复合体的重组涉及其小元素的碎片化。我们提出,这种重组取决于高尔基复合体蛋白通过内质网(ER)的不断循环、微管组织中心(MTOC或中心体)的重组以及ER出口位点的重新分配。我们当前的目标是完善这个模型,特别是在似乎同时发生的几个变化中找到一个层次结构。
我们首先询问高尔基复合体的重组是否可以在没有正常微管网络的情况下发生。通过使用小鼠肌肉细胞系 C2,我们确定了长期影响微管同时允许肌肉分化(转录因子肌生成素表达)的不同药物的浓度。我们使用了药物诺考达唑(可防止微管成核和伸长)、紫杉醇(可强制微管大量稳定和成束)以及糖原合成酶激酶 3 的有机金属特异性抑制剂(预计会影响微管定向和稳定)。
正如我们所假设的,这些药物存在下的分化会明显影响细胞重组的不同方面。 MTOC 的重组受到的影响最小,在所有条件下重组率均超过 75%。相反,在大部分肌生成素阳性细胞中,高尔基复合体重组失败或不完整,并且除非 MTOC 重组正常,否则从未观察到。一小部分细胞似乎具有正常的高尔基复合体重组,没有 ER 出口位点重新分布,但在 > 90% 的细胞中,两者协调重组。因此,我们认为分化过程中的主要事件是 MTOC 的重组,然后导致高尔基复合体和 ER 出口位点的协调重组。克里斯蒂安·扎尔 (Kristien Zaal) 目前正在撰写这部作品。
尽管如果转移到成熟的肌纤维上,完全重组失败可能会产生生理后果,但在培养细胞中情况不一定如此。高尔基复合体可能具有足够的可塑性,能够在多种几何形状中发挥作用。为了评估其功能,我们使用了一种众所周知的测定法,其中荧光标记的病毒蛋白(VSV-G-GFP)通过高尔基复合体从内质网运输到质膜。我们已经证明,尽管高尔基复合体元件分布异常,但在用糖原合酶激酶抑制剂处理的细胞中,VSV-G 的运输似乎正常。
张谈通过实验寻找有关肌肉细胞中 MTOC 组织的信息。他一直在研究微管相关蛋白 EB1 在肌肉分化中的作用。在其他哺乳动物细胞中,EB1 对于微管稳定是必需的。我们假设 EB1 可能在肌肉发育过程中发挥类似的作用。事实上,我们发现 EB1 的显性失活结构影响成肌细胞伸长。敲除 EB1 永久地阻止微管稳定,并且出乎意料地阻止肌肉细胞的分化(这项工作在最近提交的手稿中进行了描述)。除了在微管正端发挥作用外,EB1 还存在于中心体中。我们证实 EB1 敲低细胞在微管成核方面存在缺陷,并且还发现 MTOC 中最著名的蛋白质之一周中心蛋白在 EB1 敲低细胞中部分移位至高尔基复合体。我们现在准备使用在中心体选择性影响 EB1 的 cDNA 构建体,以确定其哪个定位与其在细胞分化中的作用相关。这些结果表明微管细胞骨架组织与肌细胞分化能力之间存在联系。
微管在肌发生过程中高尔基复合体的重组中发挥着重要作用,但它们可能不是参与高尔基复合体在肌纤维和其他纤维中分布的唯一细胞骨架系统,我们自己的结果表明中间丝也参与其中。由于结蛋白是骨骼肌的主要中间丝蛋白,因此我们使用了结蛋白缺失小鼠。我们观察到,与对照小鼠相比,结蛋白无效小鼠的慢肌纤维中,高尔基复合体元件和内质网出口位点的大小异常且不均匀。去年,约翰·谢里丹(John Sheridan)利用对照小鼠和结蛋白无效小鼠的特定肌肉的单肌纤维制备了卫星细胞培养物。无结蛋白的肌管中的高尔基复合体重组/断裂确实是不完整的,这表明结蛋白确实在分化早期阶段的高尔基复合体重组中发挥作用。
获得我们所需信息的最佳工具是单纤维的免疫荧光。手动梳理纤维是劳动密集型的,但也是必要的,因为抗体不会穿透纤维束,并且切片提供的视图过于有限。然而,在某些情况下,处理单根光纤几乎是不可能的。我们一直在与博士合作。 NIAMS 的 Plotz 和 Raben 研究庞贝病,这是一种溶酶体贮积症,心肌和骨骼肌是其病理根源。我们的工作强调了小鼠模型和庞贝病人类受试者的肌肉中存在大面积的自噬碎片。如果这种积聚与病理学有关,那么能够评估其在活检中的患病率就非常重要,特别是在患有该疾病最具破坏性的婴儿中。然而,单根婴儿纤维又薄又脆弱,并且极难处理。为了解决这个问题,我们使用了一种新的成像方式,即称为二次谐波产生(SHG)的双光子显微镜技术,它可以在分子水平上提供未染色组织的结构信息。 SHG 检测以半晶体顺序排列的分子阵列。肌球蛋白丝是二次谐波的良好来源,该技术可应用于大束纤维。我们发现 SHG 能够检测在小鼠模型和庞贝病受试者的人体活检中发现的所有内含物(扩大的溶酶体和自噬堆积区域)(Ralston 等人,2008)。这是首次发表的 SHG 评估肌肉疾病的应用。肌肉缺陷的 SHG 定量也应该对其他病理状况感兴趣,在这些病理状况中,肌肉的结构缺陷可能更微妙且没有很好的记录。
项目成果
期刊论文数量(3)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Clearing skeletal muscle with CLARITY for light microscopy imaging.
- DOI:10.1002/cbin.10578
- 发表时间:2016-04
- 期刊:
- 影响因子:3.9
- 作者:Milgroom A;Ralston E
- 通讯作者:Ralston E
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Evelyn Ralston其他文献
Evelyn Ralston的其他文献
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{{ truncateString('Evelyn Ralston', 18)}}的其他基金
Regulation of Subcellular Organization in Skeletal Muscle
骨骼肌亚细胞组织的调节
- 批准号:
8939419 - 财政年份:
- 资助金额:
$ 124.56万 - 项目类别:
REGULATION OF SUBCELLULAR ORGANIZATION IN SKELETAL MUSCL
骨骼肌亚细胞组织的调节
- 批准号:
6690255 - 财政年份:
- 资助金额:
$ 124.56万 - 项目类别:
REGULATION OF SUBCELLULAR ORGANIZATION IN SKELETAL MUSCLE
骨骼肌亚细胞组织的调节
- 批准号:
7969925 - 财政年份:
- 资助金额:
$ 124.56万 - 项目类别:
Regulation of Subcellular Organization in Skeletal Muscle
骨骼肌亚细胞组织的调节
- 批准号:
10006383 - 财政年份:
- 资助金额:
$ 124.56万 - 项目类别:
REGULATION OF SUBCELLULAR ORGANIZATION OF EXCITABLE CELLS
兴奋细胞亚细胞组织的调节
- 批准号:
6290649 - 财政年份:
- 资助金额:
$ 124.56万 - 项目类别:
REGULATION OF SUBCELLULAR ORGANIZATION IN SKELETAL MUSCL
骨骼肌亚细胞组织的调节
- 批准号:
6823119 - 财政年份:
- 资助金额:
$ 124.56万 - 项目类别:
REGULATION OF SUBCELLULAR ORGANIZATION OF EXCITABLE CELLS
兴奋细胞亚细胞组织的调节
- 批准号:
6111884 - 财政年份:
- 资助金额:
$ 124.56万 - 项目类别:
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