Cell-Type-Specific Intracellular Protein Delivery with Inactivated Botulinum Neurotoxin

The ability to deliver proteins and peptides across the plasma membrane into the cytosol of living mammalian cells would be highly impactful for both basic science and medicine. Natural cell-penetrating protein toxins have shown promise as protein delivery platforms, but existing approaches are limited by immunogenicity, lack of cell-type-specificity, or their multi-component nature. Here we explore inactivated botulinum neurotoxin (BoNT) as a protein delivery platform. Using split luciferase reconstitution in the cytosol as a readout for endosomal escape and cytosolic delivery, we showed that BoNT chimeras with nanobodies replacing their natural receptor binding domain can be selectively targeted to cells expressing nanobody-matched surface markers. We used chimeric BoNTs to deliver a range of cargo from 1.3 to 55 kDa in size, and demonstrated selective delivery of orthogonal cargoes to distinct cell populations within a mixed culture. These explorations suggest that BoNT may be a versatile platform for targeted protein and peptide delivery into mammalian cells

Pyrenebutyrate Leads to Cellular Binding, Not Intracellular Delivery, of Polyarginine Quantum Dots

The intracellular, cytosolic, delivery of quantum dots is an important goal for cellular imaging. Recently, a hydrophobic anion, pyrenebutyrate, has been proposed to serve as a delivery agent for cationic quantum dots as characterized by confocal microscopy. Using an extracellular quantum dot quencher, QSY-21, as an alternative to confocal microscopy, we demonstrate that quantum dots remain on the cell surface and do not cross the plasma membrane following pyrenebutyrate treatment, a result that is confirmed with transmission electron microscopy. Pyrenebutyrate leads to increased cellular binding of quantum dots rather than intracellular delivery. These results characterize the use of QSY-21 as a quantum dot quencher and highlight the importance of the use of complementary techniques when using confocal microscopy

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

We present a methodology for targeting quantum dots to specific proteins on living cells in two steps. In the first step, Escherichia coli lipoic acid ligase (LplA) site-specifically attaches 10-bromodecanoic acid onto a 13 amino acid recognition sequence that is genetically fused to a protein of interest. In the second step, quantum dots derivatized with HaloTag, a modified haloalkane dehalogenase, react with the ligated bromodecanoic acid to form a covalent adduct. We found this targeting method to be specific, fast, and fully orthogonal to a previously reported and analogous quantum dot targeting method using E. coli biotin ligase and streptavidin. We used these two methods in combination for two-color quantum dot visualization of different proteins expressed on the same cell or on neighboring cells. Both methods were also used to track single molecules of neurexin, a synaptic adhesion protein, to measure its lateral diffusion in the presence of neuroligin, its trans-synaptic adhesion partner

ID-PRIME for imaging neurexin-neuroligin interactions in HEK cells.

<p>(<b>A</b>) ID-PRIME with lipoic acid readout (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052823#pone-0052823-g001" target="_blank">Figure 1B</a>). HEK cells were separately transfected with *LplA₃₆-NRX3β plus a membrane-localized tdTomato marker (shown in blue), or 3xLAP-NLG1 plus a Venus marker. After mixing and replating, cells were labeled with 50 µM lipoic acid +500 µM ATP for 15 minutes. Ligated lipoic acid was detected with an anti-lipoic acid antibody followed by a secondary antibody-AF647 conjugate (shown in red) for 5 minutes each. For row 1, a magnified view representing the boxed region, and a more contrasted view of the transfection markers are shown on the right. Controls were performed with lipoic acid omitted (row 2), the acceptor peptide for BirA substituted for LAP (row 3), and the interaction-deficient NRX mutant (row 4). (<b>B</b>) ID-PRIME with picolyl azide readout (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052823#pone-0052823-g001" target="_blank">Figure 1C</a>). HEK cells were transfected as in (A), and labeling was performed with 100 µM picolyl azide +500 µM ATP for 15 minutes, followed by detection with copper-catalyzed click chemistry, using 50 µM copper and 20 µM alkyne-AF647. Color schemes and controls are the same as for (A). All scale bars, 10 µm.</p

Scheme showing BLINC and ID-PRIME methods for imaging trans-cellular protein-protein interactions.

<p>(<b>A</b>) In <u>B</u>iotin <u>L</u>abeling of <u>IN</u>tercellular <u>C</u>ontacts (BLINC), protein A is genetically tagged with the 35 kDa <i>E. coli</i> biotin ligase (BirA) on the extracellular side. Protein B is genetically tagged with a 15-amino acid acceptor peptide (AP) for BirA. When proteins A and B interact, BirA ligates biotin onto protein B, which can be detected using a monovalent streptavidin-fluorophore conjugate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052823#pone.0052823-Howarth2" target="_blank">[23]</a>. (<b>B</b>) In <u>I</u>nteraction-<u>D</u>ependent <u>PR</u>obe <u>I</u>ncorporation <u>M</u>ediated by <u>E</u>nzymes (ID-PRIME), protein A is genetically tagged with a 38 kDa mutant of <i>E. coli</i> lipoic acid ligase (*LplA = W37A, T57I, F147L, H267R mutant of LplA) on its extracellular side. Protein B is genetically tagged with a 13-amino acid ligase acceptor peptide (LAP) for LplA. When proteins A and B interact, *LplA ligates lipoic acid onto protein B, which can be detected using an antibody-fluorophore conjugate. <b>(C)</b> Alternative ID-PRIME detection using picolyl azide ligation onto protein B. Ligated azide can be detected by copper-catalyzed click chemistry with alkyne-fluorophore conjugates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052823#pone.0052823-Uttamapinant1" target="_blank">[7]</a>.</p

ID-PRIME for imaging neurexin-neuroligin interactions in neuron cultures and in HEK-neuron mixed cultures.

<p>(<b>A</b>) Lipoic acid ID-PRIME labeling of pure neuron cultures. Dissociated rat hippocampal neurons were separately nucleofected at DIV0 with either 1xLAP-NLG1 plus a Venus transfection marker (shown in green), or *LplA₃₆-NRX3β plus a membrane-localized tdTomato transfection marker (shown in blue). The two pools of neurons were mixed and plated. At DIV5, neurons were labeled with lipoic acid and anti-lipoic acid antibody as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052823#pone-0052823-g004" target="_blank">Figure 4A</a>. ID-PRIME signal was detected in 22 out of 23 fields of view, and was localized to contact sites (arrow heads, row 1). Negative controls with lipoic acid omitted (row 2), AP-NLG1 in place of LAP-NLG (row 3), or with an interaction deficient mutant of NRX (row 4) are also shown. Asterisks in row 1 and 2 indicate sites where the over-expression of ID-PRIME constructs caused neuronal processes to “zip up”. (<b>B</b>) Lipoic acid ID-PRIME labeling of mixed HEK-neuron cultures. HEK cells expressing *LplA₃₆-NRX3β and a membrane-localized tdTomato marker (shown in blue) were plated on top of neurons, transfected with lipofectamine at DIV 7 with 3xLAP-NLG1 plus a Venus marker (shown in green). Labeling was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052823#pone-0052823-g004" target="_blank">Figure 4A</a>, at DIV8. ID-PRIME signal was detected in 9 out of 11 fields of view in which Venus-expressing neurons contacted Tomato-expressing HEK cells. The bottom row shows a control with a D137A mutation in NRX; no ID-PRIME signal was observed in any field of view. All scale bars, 10 µm.</p

BLINC for imaging neurexin-neuroligin interactions in neuron cultures and in HEK-neuron mixed cultures.

<p>(<b>A</b>) BLINC labeling of pure neuron cultures. Two pools of hippocampal neurons were separately nucleofected at DIV0 with BirA-NRX plus a membrane tdTomato marker (shown in blue), or 3xAP-NLG1 plus a Venus marker (shown in green). For the top row, the BirA₃₆-NRX3β construct was used, and for the bottom row the BirA₂₇₂-NRX3β construct was used. All constructs had CAG promoters. Labeling was performed at DIV5 with biotin+ATP for 15 minutes, followed by monovalent streptavidin-AF647 detection for 5 minutes. Confocal images of live neurons showed no detectable BLINC signal for the BirA₃₆-NRX3β fusion across 10 fields of view in which Venus- and Tomato-expressing neurons were observed to be crossing. For the BirA₂₇₂-NRX3β fusion (bottom row), BLINC signal was detected in 5 out of 10 such fields of view. (<b>B</b>) BLINC labeling of mixed HEK-neuron cultures. HEK cells expressing BirA₂₇₂-NRX3β and a dsRed marker (shown in blue) were plated on top of rat hippocampal neurons transfected with lipofectamine at DIV10 with 3xAP-NLG1 plus a Venus marker (shown in green). Labeling was performed at DIV11 as in (A). BLINC signal could be detected in 22 out of 30 fields of view, and was localized to contact sites (arrow heads). The bottom row shows a control with a D137A mutation in NRX3β; BLINC signal was not observed in any field of view. All scale bars, 10 µm.</p

BLINC for imaging neurexin-neuroligin interactions in HEK cells.

<p>(<b>A</b>) Scheme showing the BLINC experimental protocol. Two pools of HEK cells were separately transfected with BirA₆₄-NRX1β plus YFP, or AP-NLG1 plus BFP. The pools were then mixed and allowed to form contacts over 24 hours. BLINC labeling was performed with 10 µM biotin-AMP for 2 minutes (note that biotin+ATP was used instead for neuron cultures in other figures, for reasons explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052823#pone.0052823.s006" target="_blank">Figure S6</a>). Biotinylated AP sites were detected by live-cell staining with streptavidin-AF568 for 5 minutes. (<b>B</b>) BLINC imaging results. Controls are shown with a D137A mutation in BirA-NRX to abolish its interaction with NLG (rows 2 and 4), and 1 µM exogenous BirA added during the biotin-AMP step to label total cell surface AP-NLG1 (rows 3 and 4). When a NLG-expressing cell apposes a NRX-expressing cell, BLINC signal is localized at contact sites (thin arrow heads, row 1). The same phenomenon was observed when exogenous BirA was added to label the total NLG pool (thick arrow heads, row 3). All scale bars, 10 µm.</p

Diels–Alder Cycloaddition for Fluorophore Targeting to Specific Proteins inside Living Cells

The inverse-electron-demand Diels–Alder cycloaddition between <i>trans</i>-cyclooctenes and tetrazines is biocompatible and exceptionally fast. We utilized this chemistry for site-specific fluorescence labeling of proteins on the cell surface and inside living mammalian cells by a two-step protocol. <i>Escherichia coli</i> lipoic acid ligase site-specifically ligates a <i>trans</i>-cyclooctene derivative onto a protein of interest in the first step, followed by chemoselective derivatization with a tetrazine–fluorophore conjugate in the second step. On the cell surface, this labeling was fluorogenic and highly sensitive. Inside the cell, we achieved specific labeling of cytoskeletal proteins with green and red fluorophores. By incorporating the Diels–Alder cycloaddition, we have broadened the panel of fluorophores that can be targeted by lipoic acid ligase

Fluorophore Targeting to Cellular Proteins via Enzyme-Mediated Azide Ligation and Strain-Promoted Cycloaddition

Methods for targeting of small molecules to cellular proteins can allow imaging with fluorophores that are smaller, brighter, and more photostable than fluorescent proteins. Previously, we reported targeting of the blue fluorophore coumarin to cellular proteins fused to a 13-amino acid recognition sequence (LAP), catalyzed by a mutant of the Escherichia coli enzyme lipoic acid ligase (LplA). Here, we extend LplA-based labeling to green- and red-emitting fluorophores by employing a two-step targeting scheme. First, we found that the W37I mutant of LplA catalyzes site-specific ligation of 10-azidodecanoic acid to LAP in cells, in nearly quantitative yield after 30 min. Second, we evaluated a panel of five different cyclooctyne structures and found that fluorophore conjugates to aza-dibenzocyclooctyne (ADIBO) gave the highest and most specific derivatization of azide-conjugated LAP in cells. However, for targeting of hydrophobic fluorophores such as ATTO 647N, the hydrophobicity of ADIBO was detrimental, and superior targeting was achieved by conjugation to the less hydrophobic monofluorinated cyclooctyne (MOFO). Our optimized two-step enzymatic/chemical labeling scheme was used to tag and image a variety of LAP fusion proteins in multiple mammalian cell lines with diverse fluorophores including fluorescein, rhodamine, Alexa Fluor 568, ATTO 647N, and ATTO 655