A genetically encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling

被引:37
作者
Prole, David L. [1 ]
Taylor, Colin W. [1 ]
机构
[1] Univ Cambridge, Dept Pharmacol, Tennis Court Rd, Cambridge CB2 1PD, England
基金
英国生物技术与生命科学研究理事会; 英国惠康基金;
关键词
Cell signalling; Endoplasmic reticulum; Fluorescence microscopy; Fluorescent protein; GFP; Intrabody; Membrane contact site; Mitochondria; Nanobody; RFP; CALCIUM INDICATOR; CONTACT SITES; GENE-PRODUCTS; CA2+ STORE; MITOCHONDRIA; DYNAMICS; LOCALIZATION; LIBRARY; INACTIVATION; RECEPTOR;
D O I
10.1186/s12915-019-0662-4
中图分类号
Q [生物科学];
学科分类号
07 ; 0710 ; 09 ;
摘要
BackgroundIntrabodies enable targeting of proteins in live cells, but generating specific intrabodies against the thousands of proteins in a proteome poses a challenge. We leverage the widespread availability of fluorescently labelled proteins to visualize and manipulate intracellular signalling pathways in live cells by using nanobodies targeting fluorescent protein tags.ResultsWe generated a toolkit of plasmids encoding nanobodies against red and green fluorescent proteins (RFP and GFP variants), fused to functional modules. These include fluorescent sensors for visualization of Ca2+, H+ and ATP/ADP dynamics; oligomerising or heterodimerising modules that allow recruitment or sequestration of proteins and identification of membrane contact sites between organelles; SNAP tags that allow labelling with fluorescent dyes and targeted chromophore-assisted light inactivation; and nanobodies targeted to lumenal sub-compartments of the secretory pathway. We also developed two methods for crosslinking tagged proteins: a dimeric nanobody, and RFP-targeting and GFP-targeting nanobodies fused to complementary hetero-dimerizing domains. We show various applications of the toolkit and demonstrate, for example, that IP3 receptors deliver Ca2+ to the outer membrane of only a subset of mitochondria and that only one or two sites on a mitochondrion form membrane contacts with the plasma membrane.ConclusionsThis toolkit greatly expands the utility of intrabodies and will enable a range of approaches for studying and manipulating cell signalling in live cells.
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页数:24
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