Department of Pharmacology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, CO, 80045, USA.
Nat Commun. 2024 Nov 29;15(1):10374. doi: 10.1038/s41467-024-54530-5.
Tools for visualizing and manipulating protein dynamics in living cells are critical for understanding cellular function. Here we leverage recently available monoclonal antibody sequences to generate a set of affinity tags for labeling and manipulating AMPA-type glutamate receptors (AMPARs), which mediate nearly all excitatory neurotransmission in the central nervous system. These antibodies can be produced from heterologous cells for exogenous labeling applications or directly expressed in living neurons as intrabodies, where they bind their epitopes in the endoplasmic reticulum and co-traffic to the cell surface for visualization with cell impermeant fluorescent dyes. We show these reagents do not perturb AMPAR trafficking, function, mobility, or synaptic recruitment during plasticity and therefore can be used as probes for monitoring endogenous receptors in living neurons. We also adapt these reagents to deplete AMPARs from the cell surface by trapping them in the endoplasmic reticulum, providing a simple approach for loss of excitatory neurotransmission. The strategies outlined here serve as a template for generating similar reagents targeting diverse proteins as more antibody sequences become available.
用于可视化和操作活细胞中蛋白质动力学的工具对于理解细胞功能至关重要。在这里，我们利用最近可用的单克隆抗体序列生成了一组用于标记和操作 AMPA 型谷氨酸受体 (AMPAR) 的亲和标签，AMPAR 介导中枢神经系统中几乎所有的兴奋性神经递质传递。这些抗体可以从异源细胞中产生，用于外源标记应用，也可以直接在活神经元中作为内抗体表达，在那里它们结合内质网中的表位，并共同运输到细胞表面，以便用细胞不可渗透的荧光染料进行可视化。我们表明，这些试剂不会在可塑性过程中干扰 AMPAR 的运输、功能、迁移或突触募集，因此可以作为监测活神经元中内源性受体的探针。我们还通过将它们困在内质网中来耗尽细胞表面的 AMPAR，从而提供了一种简单的方法来抑制兴奋性神经递质传递。这里概述的策略为生成针对不同蛋白质的类似试剂提供了模板，因为更多的抗体序列变得可用。
