| Strategy of Isolating ‘Primed’ Tumor Initiating Cells Based on Mitochondrial Transmembrane Potential
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Author:
Date:
2021-03-05
[Abstract] Various stem cells have been found to be dependent on mitochondrial energetics. The role of mitochondria in regulating the self-renewal of normal stem cells and stem-like tumor initiating cells (TICs) is increasingly being appreciated. We proposed that TIC populations have a sub population of cells that are “primed” by mitochondria for self-renewal. Using ovarian cancer model, we have developed a protocol to identify and isolate these “primed” cells using Fluorescence-Assisted Cell Sorting (FACS). We combined live cell stains for a functional marker of TICs and for mitochondrial transmembrane potential to enrich TICs with higher mitochondrial potential that form in vitro spheroids 10-fold more than the other TICs with lower mitochondrial potential. This protocol ...
[摘要] [摘要]已经发现各种干细胞都依赖于线粒体的能量学。线粒体在调节正常干细胞和干细胞样肿瘤起始细胞（TICs）自我更新中的作用日益受到人们的重视。我们提出，TIC种群具有由线粒体“引发”自我更新的亚细胞群。使用卵巢癌模型，我们开发了一种协议，可以使用荧光辅助细胞分选（FACS）识别和分离这些“初免”细胞。我们结合活细胞染色剂作为TIC的功能标记物和线粒体跨膜电位，以富集具有更高线粒体电位的TIC（在体外形成）球状体比线粒体电位较低的其他TIC高10倍。该协议可以直接使用或修改以用于各种小区类型。因此，预期该方案对于干细胞群体中线粒体和能量异质性的基本理解是不可侵犯的，并且在再生医学和癌症生物学的转化研究中也可能被证明是有价值的。
背景技术肿瘤中的细胞异质性对癌症治疗提出了严峻的挑战（Magee等，2012）。当某些肿瘤通过自身更新和分化维持肿瘤时，其与肿瘤抗性的肿瘤起始细胞（也称为癌症干细胞）分层地排列在细胞层次的底部（Magee et ...
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| In vitro AMPylation/Adenylylation of Alpha-synuclein by HYPE/FICD
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Author:
Date:
2020-09-20
[Abstract] One of the major histopathological hallmarks of Parkinson’s disease are Lewy bodies (LBs) –cytoplasmic inclusions, enriched with fibrillar forms of the presynaptic protein alpha-synuclein (α-syn). Progressive deposition of α-syn into LBs is enabled by its propensity to fibrillize into insoluble aggregates. We recently described a marked reduction in α-syn fibrillation in vitro upon posttranslational modification (PTM) by the Fic (Filamentation induced by cAMP) family adenylyltransferase HYPE/FICD (Huntingtin yeast-interacting protein E/FICD). Specifically, HYPE utilizes ATP to covalently decorate key threonine residues in α-syn’s N-terminal and NAC (non-amyloid-β component) regions with AMP (adenosine monophosphate), in a PTM termed AMPylation or adenylylation. Status quo in ...
[摘要] [摘要 ] 帕金森氏病的主要组织病理学标志之一是路易体（LB） –细胞质内含物，富含纤维状形式的突触前蛋白α-突触核蛋白（α-syn）。由于α-syn易于原纤维化成不溶性聚集体，因此可以逐步沉积到LBs中。我们最近描述了Fic（由cAMP诱导的细丝化）家族腺苷酸转移酶HYPE / FICD（与亨廷顿酵母相互作用的蛋白E / FICD）在翻译后修饰（PTM）后体外α-syn纤颤的明显减少。具体而言，HYPE利用ATP在称为AMPylation 或adenylylation 的PTM中，以AMP（单磷酸腺苷）共价修饰α-syn's N末端和NAC（非淀粉样β成分）区域中的关键苏氨酸残基。HYPE底物（例如α-syn）的体外AMPyl化反应现状使用多种ATP类似物，包括放射性标记的α - 32 P-ATP 或α - 33 P-ATP，荧光ATP类似物，生物素化ATP类似物（N6- [6-六甲基] -ATP-生物素），以及基于点击化学的烷基-ATP方法检测基于凝胶的AMPylation 。当前描述HYPE介导AMPylation 的分步方案的文献依赖于α - 33 P-ATP核苷酸，而不是更常见的α - 32 P-ATP。尽管有效，但前一种方法需要长时间且危险的DMSO-PPO（二甲基亚砜- 聚苯基恶唑）沉淀。因此，我们提供了基于α - 33 ...
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