Quantum Dot Targeting with Lipoic Acid Ligase and HaloTag for Single-Molecule Imaging on Living Cells

Liu, Daniel S.; Phipps, William S.; Loh, Ken H.; Howarth, Mark; Ting, Alice Y.

doi:10.1021/nn304793z

Cited by 65 publications

(47 citation statements)

References 34 publications

(63 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.

Self Cite

View full text Add to dashboard Cite

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...

show abstract

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.

Self Cite

View full text Add to dashboard Cite

show abstract

“…More recently, a fluorophore ligase was generated, enabling direct incorporation of 7-hydroxycoumarin, 7-aminocoumarin and Pacific Blue (Fig. 2e) 45,46 . Steric collisions within the active site of lipoic acid ligase seem to necessitate small fluorophores, traditionally limiting imaging to the near-UV.…”

Section: Bio-orthogonal Methods For Protein Labelingmentioning

confidence: 99%

Advances in fluorescence labeling strategies for dynamic cellular imaging

2014

View full text Add to dashboard Cite

Synergistic advances in optical physics, probe design, molecular biology, labeling techniques and computational analysis have propelled fluorescence imaging into new realms of spatiotemporal resolution and sensitivity. This review aims to discuss advances in fluorescent probes and live-cell labeling strategies, two areas that remain pivotal for future advances in imaging technology. Fluorescent protein– and bio-orthogonal–based methods for protein and RNA imaging are discussed as well as emerging bioengineering techniques that enable their expression at specific genomic loci (for example, CRISPR and TALENs). Important attributes that contribute to the success of each technique are emphasized, providing a guideline for future advances in dynamic live-cell imaging.

show abstract

“…Biotin naturally present in the culture medium is covalently added to a central lysine residue in the AP-tag through the enzymatic action of the biotin ligase, and AP-Nrx1β reaches the plasma membrane in a biotinylated form. 25 The biotinylated tag is then detected by incubation with monomeric streptavidin (mSA) conjugated to an organic dye (Alexa647), compatible with dSTORM. In DIV 15 neurons, we observed an excellent colocalization of the Alexa647 mSA conjugate with VGlut1-SEP [ Fig.…”

Section: Nanoscale Organization Of Nrx1β At Presynapsesmentioning

confidence: 99%

Nanoscale organization of synaptic adhesion proteins revealed by single-molecule localization microscopy

et al. 2016

View full text Add to dashboard Cite

Abstract. The advent of superresolution imaging has created a strong need for both optimized labeling strategies and analysis methods to probe the nanoscale organization of complex biological structures. We present a thorough description of the distribution of synaptic adhesion proteins at the nanoscopic scale, namely presynaptic neurexin-1β (Nrx1β), and its two postsynaptic binding partners neuroligin-1 (Nlg1) and leucine-richrepeat transmembrane protein 2 (LRRTM2). We monitored these proteins in the membrane of neurons by direct stochastic optical reconstruction microscopy, after live surface labeling with Alexa647-conjugated monomeric streptavidin. The small probe (∼3 nm) efficiently penetrates into crowded synaptic junctions and reduces the distance to target. We quantified the organization of the single-molecule localization data using a tesselation-based analysis technique. We show that Nlg1 exhibits a fairly disperse organization within dendritic spines, while LRRTM2 is organized in compact domains, and Nrx1β in presynaptic terminals displays a dual-organization pattern intermediate between that of Nlg1 and LRRTM2. These results suggest that part of Nrx1β interacts transsynaptically with Nlg1 and the other part with LRRTM2.

show abstract

Quantum Dot Targeting with Lipoic Acid Ligase and HaloTag for Single-Molecule Imaging on Living Cells

Cited by 65 publications

References 34 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

Advances in fluorescence labeling strategies for dynamic cellular imaging

Nanoscale organization of synaptic adhesion proteins revealed by single-molecule localization microscopy

Contact Info

Product

Resources

About