Imaging Trans-Cellular Neurexin-Neuroligin Interactions by Enzymatic Probe Ligation

Liu, Daniel S.; Loh, Ken H.; Lam, Stephanie S; White, Katharine A.; Ting, Alice Y.

doi:10.1371/journal.pone.0052823

Cited by 37 publications

(47 citation statements)

References 38 publications

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“…PRIME with resorufin ligase can be used to study specific cellular proteins in an optimal spectral window while minimizing steric perturbation to that protein. Thus, PRIME can be an attractive alternative to red fluorescent proteins such as mCherry, which is 15 times larger, in cases where bulky tags are not tolerated [such as for β-actin shown in this work and neuroligin-1 shown elsewhere (38,39)]. Our study shows that despite the posttranslational nature of our labeling the tagging specificity is exceptionally specific, comparable to that obtained by direct genetic fusion to fluorescent proteins.…”

Section: Discussionmentioning

confidence: 50%

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Liu¹,

Nivón²,

Richter

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.fluorescence microscopy | enzyme redesign | LplA | PRIME | chemical probe targeting F luorescent proteins are used ubiquitously in imaging, but their dim fluorescence, rapid photobleaching, and large size limit their utility. At ∼27 kDa (∼240 aa), fluorescent proteins can disrupt protein folding and trafficking or impair protein function (1, 2). Chemical fluorophores, in comparison, are typically less than 1 kDa in size, and are brighter and more photostable. These properties allow chemical fluorophores to perform better than fluorescent proteins in advanced imaging modalities such as singlemolecule tracking and superresolution microscopies (3, 4).Site-specific labeling of proteins with chemical fluorophores inside living cells is challenging because these fluorophores are not genetically encodable and therefore must be posttranslationally targeted inside a complex cellular milieu. Existing methods to achieve this targeting either require large fusion tags [such as HaloTag (5), the SNAP tag (6), and the DHFR tag (7)] or have insufficient specificity [such as biarsenical dye targeting (8) and amber codon suppression (9)]. To achieve a labeling specificity comparable to fluorescent proteins, we developed PRIME (PRobe Incorporation Mediated by Enzymes), which uses Escherichia coli lipoic acid ligase to attach small molecules to a 13-aa peptide tag (Fig. 1A) (10). To make PRIME more useful for cellular protein imaging, we sought to engineer the system for the targeting of bright chemical fluorophores. The challenge, though, is that lipoic acid ligase (LplA) has a small and fully enclosed substrate-binding pocket that even with extensive structure-guided mutagenesis has not until this point been able to accommodate large chemical structures. ResultsSynthesis of Resorufin Derivatives for PRIME. We considered fluorophores for PRIME based on the steric constraints of LplA. Far-red emitters such as Cy5 and Atto 647N, although photophysically desirable, are so bulky that binding inside LplA would require...

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Section: Discussionmentioning

confidence: 50%

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Liu¹,

Nivón²,

Richter

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…However, the transsynaptic interaction is strong and may not be reversible (Chen et al 2014), thus precluding dynamic studies. STaR, on the other hand, relies on colocalization of pre-and postsynaptic labels without a direct trans-synaptic interaction (Chen et al 2014).To develop a methodology that involves a trans-synaptic enzymatic reaction, we adapted Figure 1A) (Liu et al 2013). The in vivo BLINC method, which we term iBLINC, seems to capture the directionality of synaptic connections and permits the quantification of synaptic patterns in a population of animals.…”

mentioning

confidence: 99%

“…To develop a methodology that involves a trans-synaptic enzymatic reaction, we adapted Figure 1A) (Liu et al 2013). The in vivo BLINC method, which we term iBLINC, seems to capture the directionality of synaptic connections and permits the quantification of synaptic patterns in a population of animals.…”

mentioning

confidence: 99%

“…To develop a methodology that involves a trans-synaptic enzymatic reaction, we adapted Biotin Labeling of INtercellular Contacts (BLINC), a technique established in cell culture (Liu et al 2013) and developed it for transgenic use in C. elegans. BLINC is based on enzymatic transfer of biotin onto a 15-amino-acid peptide (the acceptor peptide, or AP) by the Escherichia coli biotin ligase BirA ( Figure 1A) (Liu et al 2013).…”

mentioning

confidence: 99%

“…BLINC is based on enzymatic transfer of biotin onto a 15-amino-acid peptide (the acceptor peptide, or AP) by the Escherichia coli biotin ligase BirA ( Figure 1A) (Liu et al 2013). The in vivo BLINC method, which we term iBLINC, seems to capture the directionality of synaptic connections and permits the quantification of synaptic patterns in a population of animals.…”

mentioning

confidence: 99%

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Directional Trans-Synaptic Labeling of Specific Neuronal Connections in Live Animals

et al. 2015

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Understanding animal behavior and development requires visualization and analysis of their synaptic connectivity, but existing methods are laborious or may not depend on trans-synaptic interactions. Here we describe a transgenic approach for in vivo labeling of specific connections in Caenorhabditis elegans, which we term iBLINC. The method is based on BLINC (Biotin Labeling of INtercellular Contacts) and involves trans-synaptic enzymatic transfer of biotin by the Escherichia coli biotin ligase BirA onto an acceptor peptide. A BirA fusion with the presynaptic cell adhesion molecule NRX-1/neurexin is expressed presynaptically, whereas a fusion between the acceptor peptide and the postsynaptic protein NLG-1/neuroligin is expressed postsynaptically. The biotinylated acceptor peptide::NLG-1/neuroligin fusion is detected by a monomeric streptavidin::fluorescent protein fusion transgenically secreted into the extracellular space. Physical contact between neurons is insufficient to create a fluorescent signal, suggesting that synapse formation is required. The labeling approach appears to capture the directionality of synaptic connections, and quantitative analyses of synapse patterns display excellent concordance with electron micrograph reconstructions. Experiments using photoconvertible fluorescent proteins suggest that the method can be utilized for studies of protein dynamics at the synapse. Applying this technique, we find connectivity patterns of defined connections to vary across a population of wild-type animals. In aging animals, specific segments of synaptic connections are more susceptible to decline than others, consistent with dedicated mechanisms of synaptic maintenance. Collectively, we have developed an enzyme-based, trans-synaptic labeling method that allows high-resolution analyses of synaptic connectivity as well as protein dynamics at specific synapses of live animals. KEYWORDS biotin; circuit; connectome; labeling; synapse U NDERSTANDING animal behavior requires the determination and analysis of their precise neural connectivity, i.e., their connectome. Historically, this has been accomplished through reconstruction of serial sections of electron micrographs (White et al. 1986;Jarrell et al. 2012). Pioneered in the nematode Caenorhabditis elegans with its defined and invariant nervous system, partial circuits from Drosophila and the mouse retina have now also been reconstructed (Helmstaedter et al. 2013;Takemura et al. 2013). However, both the experimental effort and the static nature of the connectome at the time of analysis render investigations into the variability of synaptic connections between individuals, during development, or in different genotypes laborious at best. Live-imaging techniques to visualize synaptic connectivity have been developed such as GFP Reconstitution Across Synaptic Partners (GRASP) in C. elegans and, more recently, Synaptic Tagging with Recombination (STaR) in flies (Feinberg et al. 2008;Chen et al. 2014). GRASP takes advantage of the strong molecular intera...

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Bioorthogonal Labeling of Cellular Proteins by Enzymatic and Related Mechanisms

Walper

Turner

Medintz

2017

Chemoselective and Bioorthogonal Ligation Reactions

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Imaging Trans-Cellular Neurexin-Neuroligin Interactions by Enzymatic Probe Ligation

Cited by 37 publications

References 38 publications

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Directional Trans-Synaptic Labeling of Specific Neuronal Connections in Live Animals

Bioorthogonal Labeling of Cellular Proteins by Enzymatic and Related Mechanisms

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