Subsynaptic organization of the major NMDAR subtypes
主要 NMDAR 亚型的突触亚组织
基本信息
- 批准号:10620768
- 负责人:
- 金额:$ 4.36万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2021
- 资助国家:美国
- 起止时间:2021-04-01 至 2024-03-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
Glutamatergic signaling via NMDA receptors (NMDARs) is critical in synaptic plasticity, excitotoxicity, and
numerous degenerative and cognitive disorders. Each NMDAR comprises four subunits: two obligatory GluN1
subunits and two GluN2A-D subunits. Notably, NMDARs with different subunit compositions display different
channel kinetics and protein interactions, supporting the widely held notion that subunit composition dictates
NMDAR function. The subsynaptic organization of NMDARs influences the probability of their activation during
synaptic transmission, and extrasynaptic receptors are thought to play roles in cell death, plasticity and gene
expression. However, despite evidence for subunit-specific NMDAR nanoclustering, to date there is almost no
information about the nanoscale distribution of specific NMDAR subunits within synapses or with respect to sites
of vesicular release. Thus, to elucidate the organization of synaptic and extrasynaptic NMDARs with respect to
key postsynaptic molecules and presynaptic release sites, in the first aim of my project, I will use a combination
of CRISPR-mediated gene tagging and nanobody labeling with DNA-Exchange PAINT super-resolution imaging.
These measures will provide a comprehensive map of major NMDAR subunits and provide insight into their roles
in neurons. Next, though many NMDARs contain GluN1 and two identical GluN2 subunits, in many brain regions,
NMDARs commonly are “triheteromeric” receptors containing both GluN2A and GluN2B with GluN1. However,
despite their deduced importance, there are no methods to unambiguously distinguish triheteromeric NMDARs
from other subtypes in neurons, and thus their distribution and subcellular trafficking remain mysterious. To
overcome this, I have designed a method for direct visualization of triheteromeric NMDARs using bimolecular
fluorescent complementation (BiFC). I tagged GluN2A and GluN2B with two parts of a modified fluorescent
protein that complement to produce fluorescence only when an NMDAR is assembled containing both the
GluN2A and GluN2B subunits (split-tagged NMDARs). In the second aim of my proposal, following a series of
validation experiments, I will use these split-tagged NMDARs to address two straightforward but longstanding
questions surrounding NMDARs. First, though GluN2A and GluN2B traffic with different dynamics and target
subsynaptic regions in part through differing interactions with scaffolding molecules, it is unknown which subunit
dominates synaptic integration of triheteromeric receptors. Using my new probes, I will provide the first direct
measure of triheteromeric synaptic enrichment. Second, the rate of NMDAR turnover in synapses is critical for
determining synaptic strength and plasticity, yet the exchange rate and mobile fraction of triheteromeric receptors
have not previously been possible to measure. Using FRAP, I will time determine if triheteromeric NMDARs
display unique turnover compared to other NMDARs. Completion of this work will provide a training plan that
combines deep and diverse training in methodologies and professional development that will leave me well-
positioned to excel in my career as an academic researcher at a biomedical research-oriented university.
NMDA受体(NMDAR)的谷氨酸能信号传导在突触可塑性,兴奋性毒性和
许多退化和认知障碍。每个NMDAR都包含四个亚基:两个强制性的Glun1
亚基和两个glun2a-d亚基。值得注意的是,具有不同亚基组成的NMDAR显示不同
通道动力学和蛋白质相互作用,支持亚基组成的广泛持有的观念
NMDAR功能。 NMDAR的次突触组织会影响其激活期间激活的可能性
突触传播和突触外受体被认为在细胞死亡,可塑性和基因中起着作用
表达。但是,dospite证据表明亚基特异性NMDAR纳米群集,迄今为止几乎没有
有关突触中特定NMDAR亚基的纳米级分布的信息
囊泡释放。这是为了阐明有关突触和突触外NMDAR的组织
关键的突触后分子和突触前释放点,在我的项目的第一个目的中,我将使用一个组合
用DNA交换涂料超分辨率成像的CRISPR介导的基因标记和纳米型标记。
这些措施将为主要的NMDAR亚基提供全面的地图,并洞悉其角色
在神经元中。接下来,尽管许多NMDAR都包含Glun1和两个相同的Glun2亚基,但在许多大脑区域中,但
NMDAR通常是包含glun2a和glun2b的“三分球”受体。然而,
尽管它们的重要性被推论出来,但没有任何方法可以明确区分三个月室NMDAR
从神经元中的其他亚型中,它们的分布和亚细胞贩运仍然神秘。到
克服了这一点,我设计了一种使用双分子直接可视化三分态NMDAR的方法
荧光完成(BIFC)。我用修改的荧光灯标记了Glun2a和Glun2b
仅当NMDAR组装在一起时,蛋白质才能产生荧光
glun2a和glun2b亚基(分开标记的NMDAR)。在我的提议的第二个目标中,跟随一系列
验证实验,我将使用这些拆分标签的NMDAR来解决两个直接但长期存在的
围绕NMDAR的问题。首先,尽管具有不同动态和目标的Glun2a和Glun2b流量
突触区域部分通过区分与脚手架分子的相互作用,是未知的亚基
主导三位异构体受体的突触整合。使用我的新问题,我将提供第一个直接
三分机突触富集的度量。其次,突触中的NMDAR周转率对于
确定突触强度和可塑性,但汇率和移动率的分数
以前没有可能测量。使用FRAP,我会时间确定三分态NMDARS是否
与其他NMDAR相比,显示独特的营业额。这项工作的完成将提供一个培训计划
结合了方法和专业发展的深入而多样的培训,这将使我变得很好
在我在生物医学研究的大学中担任学术研究员的职业生涯中,他的职业生涯脱颖而出。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)

暂无数据
数据更新时间:2024-06-01
Michael C. Anderson其他文献
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The Role of Inhibition in Learning
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Synthesis of new ferecrystals (SnSe)y(TSe2) where T = V and Ta
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Forgetting our facts: the role of inhibitory processes in the loss of propositional knowledge.
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- DOI:10.1037/0096-3445.130.3.54410.1037/0096-3445.130.3.544
- 发表时间:20012001
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- 作者:Michael C. Anderson;Theodore A. BellMichael C. Anderson;Theodore A. Bell
- 通讯作者:Theodore A. BellTheodore A. Bell
Suppression-induced forgetting of motor sequences
抑制引起的运动序列遗忘
- DOI:
- 发表时间:20222022
- 期刊:
- 影响因子:3.4
- 作者:Markus Schmidt;Michael C. Anderson;Tobias TempelMarkus Schmidt;Michael C. Anderson;Tobias Tempel
- 通讯作者:Tobias TempelTobias Tempel
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Michael C. Anderso...的其他基金
Subsynaptic organization of the major NMDAR subtypes
主要 NMDAR 亚型的突触亚组织
- 批准号:1040867610408676
- 财政年份:2021
- 资助金额:$ 4.36万$ 4.36万
- 项目类别:
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