Intracellular Protein-Labeling Probes for Multicolor Single-Molecule

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Intracellular protein-labeling probes for multicolor singlemolecule imaging of immune receptor-adaptor molecular dynamics Ryota Sato, Jun Kozuka, Masahiro Ueda, Reiko Mishima, Yutaro Kumagai, Akimasa Yoshimura, Masafumi Minoshima, Shin Mizukami, and Kazuya Kikuchi J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b08262 • Publication Date (Web): 09 Nov 2017 Downloaded from http://pubs.acs.org on November 9, 2017

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Journal of the American Chemical Society

Intracellular protein-labeling probes for multicolor singlemolecule imaging of immune receptor-adaptor molecular dynamics Ryota Sato1‡, Jun Kozuka2‡, Masahiro Ueda2*, Reiko Mishima3, Yutaro Kumagai3*, Akimasa Yoshimura1, Masafumi Minoshima1, Shin Mizukami4 & Kazuya Kikuchi1,5* 1

Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan 2 RIKEN Quantitative Biology, Suita, Osaka 565-0874, Japan 3 Quantitative Immunology Research Unit, WPI-Immunology Frontier Research Center, Osaka University, Suita, Osaka 5650871, Japan 4 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi, 980-8577, Japan 5 Chemical Imaging Techniques, WPI-Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan KEYWORDS: fluorescent probes, single-molecule imaging, protein-labeling tag, protein dynamics, innate immune system.

ABSTRACT: Single-molecule imaging (SMI) has been widely utilized to investigate biomolecular dynamics and protein-protein interactions in living cells. However, multicolor SMI of intracellular proteins is challenging because of high background signals and other limitations of current fluorescence labeling approaches. To achieve reproducible intracellular SMI, a labeling probe ensuring both efficient membrane permeability and minimal non-specific binding to cell components is essential. We developed near-infrared (NIR) fluorescent probes for protein labeling, which specifically binds to a mutant β-lactamase tag. By structural fine-tuning of cell permeability and minimized non-specific binding, SiRcB4 enabled multicolor SMI in combination with a HaloTag-based red-fluorescent probe. Upon addition of both chemical probes at sub-nanomolar concentrations, single-molecule imaging revealed the dynamics of TLR4 and its adaptor protein, TIRAP, which are involved in the innate immune system. Statistical analysis of the quantitative properties and time-lapse changes in dynamics revealed a protein-protein interaction in response to ligand stimulation.

Introduction Multicolor imaging is widely used in cell biology to study the localization of specific biomolecules and track their dynamics. In particular, visualization of ligandreceptor-adaptor interactions in response to ligand stimulation is essential for investigating signal transduction in living cells. Recently, analysis of signaling pathways (e.g., that of epidermal growth factor receptor (EGFR)1, 2) by multicolor imaging revealed the receptor dynamics in response to ligand stimulation at singlemolecule resolution3. Single-molecule imaging (SMI) enables tracking of hundreds or thousands of individual molecules over a period of several seconds, and provides direct information about molecular dynamics and interactions in the cellular context, and consequently, about dynamic and kinetic parameters of biological reactions in living cells4–9. Multicolor SMI in particular, would be a useful method for visualizing intermolecular interactions or analyzing molecular dynamics in living cells.

Genetically encoded fluorescent proteins (FPs) are widely used as fluorescence reporters. However, the ability to use FPs for SMI and other photon-intensive imaging applications is limited because of their poor photostability10. Moreover, FPs have common limitations in strictly controlling their expression levels. For SMI, the labeling density of fluorescent molecules in regions of interest must be controlled to observe individual fluorophores. It is challenging to optimize the experimental conditions to observe an appropriate number of fluorescent molecules. Dual-color SMI using two FPs is particularly challenging, as it requires optimization of the expression levels of both FPs to determine conditions under which FPs display similar fluorescence intensities within a cell. Over the past decade, protein-labeling approaches with organic dyes have been developed. Particularly, enzyme-based ‘self-labeling tags’ (e.g., HaloTag11, SNAP-tag12, and ACP-tag13) have been extensively stud-

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ied and applied in advanced imaging experiments, such as super-resolution microscopy14. Recently, we developed a protein-labeling method named as the BL-tag system, which is based on a mutant TEM-1 β-lactamase. This system enables covalent modification of β-lactam derivatives with a functional molecule, such as a fluorophore15–20. One advantage of the BL-tag is that membrane-permeable probes can be synthesized19, 20. These protein-tagging approaches are superior to FPs because the density of dye-labeled proteins can be simply controlled by adjusting the probe concentration and are useful for biomolecular tracking of membrane proteins at single-molecule resolution in living cells21–26. To further expand the SMI applications, development of cell-permeable protein-labeling probes with longerwavelength excitation are required. Near-infrared (NIR) fluorophores have many advantages in biological applications such as their low phototoxicity, low spectral overlap with cellular autofluorescence, and good tissue penetration27, 28. NIR-emitting protein-labeling probes can be used in combination with red-fluorescent probes for multicolor SMI. Although various synthetic NIRfluorophores with exceptional photostability and brightness have been reported, they tend to show non-specific adsorption to cellular components because of their high lipophilicity29. Thus, protein-labeling probes with NIR dyes show off-target fluorescent spots in SMI applications and significantly interfere with the analysis of protein dynamics, despite the use of the protein tag system like a SNAP-tag25. Introduction of a negatively charged sulfonate group can increase dye hydrophilicity and decrease non-specific adsorption30, but results in membrane impermeability31, making it impossible to label intracellular proteins. Recently, photostable Sirhodamine (SiR) fluorophores were developed and applied in live-cell and super-resolution imaging29, 30, 32–37. However, reducing the high hydrophobicity of these probes was still necessary to enhance the signal-tobackground (S/B) ratio for SMI in living cells. Thus, analysis of protein-protein interactions and simultaneous monitoring of the dynamics of two different proteins in living cells remains challenging. Here, we developed NIR fluorescent probes for intracellular protein labeling and applied to multicolor SMI. Control of the hydrophilicity by introducing a noncharged oligoethylenoxy linker remarkably enhanced the labeling S/B ratio to achieve SMI of intracellular proteins. As results, our probe design enabled dual color SMI of intracellular proteins in living cells without requiring optimization of the expression levels of two different proteins. Using our multicolor SMI platform, we performed quantitative time-lapse analysis of intracellular protein dynamics at single-molecule resolution in the context of innate immune system receptor-adaptor interactions.

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Results Design and synthesis of SiR-based labeling probes. To develop NIR-fluorescent probes for cell imaging based on the BL-tag, we selected SiR-Me and SiRcarboxyl as the NIR fluorophores, which possess excellent brightness, photostability, and cell permeability29, 30, 33 , and bacampicillin as the BL-tag ligand for intracellular protein labeling19. The membrane-permeable SiRB and SiRcB probes were synthesized by conjugating bacampicillin with SiR-Me and SiR-carboxyl (Figure 1a, Schemes S1, S6). To evaluate labeling selectivity, fluorescence imaging with SiRB and SiRcB was performed on HEK293T cells expressing BL-NLS, which is a fusion construct between the BL-tag and nuclear localization signal (NLS). BL-NLS was specifically labeled with SiRcB (Figure S1a), whereas strong NIR-fluorescence signals from SiRB were detected in both the nucleus and the cytosol (Figure S3a), as also reported in a previous paper29. This result indicates that SiRcB has sufficient cell permeability and limited non-specific intracellular accumulation for imaging of intracellular proteins. To evaluate its capabilities for intracellular SMI, we used SiRcB for SMI of the BL-tag anchored to the inner leaflet of the plasma membrane. A fusion construct of the BL-tag and the Lyn N-terminus sequence (Lyn11-BL) was expressed in HEK293T cells, and SMI was performed by total internal reflection fluorescence microscopy (TIRFM). However, following cell incubation with SiRcB and washing, many bright spots were observed in both Lyn11-BL expressing and non-transfected cells (Figure S5a, b). The spots were blinking (Movie S1), probably because the carboxyl group of the dye was in equilibrium with the non-fluorescent spirolactone form. Therefore, although SiRcB could specifically label the target proteins, it was inadequate for SMI without further modifications. Improved design and synthesis of SiRc-based labeling probes for intracellular SMI. Fluorescence signals from SiRcB were specifically found in the cell nucleus, where BL-NLS was expressed, because SiRcB remained in its fluorescent zwitterionic form after covalent binding to the BL-tag. By contrast, the free dye tended to aggregate, resulting in unspecific binding to hydrophobic substances and the formation of non-fluorescent spirolactone29. However, despite the non-fluorescent nature of SiRcB in hydrophobic environments, the observed SiRcB fluorescent signals were sufficiently strong to limit protein dynamics monitoring at the single-molecule level by SMI. The off-target signals might be caused by non-specific adsorption onto the glass surface or endogenous proteins due to the hydrophobicity of the probe. To increase hydrophilicity, sulfonate groups can be introduced into the fluorophore structure30, 31. However, their negative charges cause electrostatic repulsion between the fluorophores and the cell membrane.


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Figure 1. Design and fluorescence spectra of novel cell membrane permeable probes. (a) Structures of SiR-Me, SiR-carboxyl, SiRB, and SiRcB. (b) Structures of bacampicillin-based probes. SiRcB2, SiRcB4, and SiRcB6 contain di-, tetra-, and hexa-ethylene glycol, respectively. (c) The emission spectra of SiRcB, SiRcB2, SiRcB4, and SiRcB6 in PBS buffer (pH 7.4) containing 1.0% DMSO at 37 °C. The probe concentration was 500 nM, and the excitation wavelength 654 nm. (d) Specific labeling of BL-NLS expressed in HEK293T cells. The cells were incubated with 1.0 µM SiRcB, SiRcB2, SiRcB4, or SiRcB6 for 30 min at 37 °C. Scale bar: 10 µm.

To overcome this problem, we designed novel bacampicillin-based labeling probes by introducing a hydrophilic oligoethylenoxy linker, increasing hydrophilicity and the distance between the fluorescent dye and the BL-tag ligand in a stepwise manner: SiRcB(n) (n = 2, 4, 6) (Figure 1b, Table 1). Moreover, since oligoethylenoxy groups are both electroneutral and hydrophilic, the increased hydrophilicity of the probes was expected to preclude their accumulation onto hydrophobic cellular components, while maintaining their cell permeability. These probes were synthesized by conjugation of bacampicillin with oligoethylenoxy linkers, followed by condensation with Si-carboxyl (Schemes S3–6). Although the optical properties of the open zwitterionic forms of SiRcB(n) (n = 2, 4, 6) were nearly the same as those of SiRcB (Table 1), the emission spectra in aqueous buffer showed fluorescence enhancement with increasing oligoethylenoxy linker length (Figure 1c). This result indicates that the hydrophilic linker maintains the fluorescent structure of the SiR dye by preventing probe aggregation. As shown in Figure 1d, the SiRcB(n) probes can label nuclear BL-tag. To evaluate labeling selectivity, the S/B ratio was determined from the ratio of nuclear to cytosolic fluorescence intensities. However, the S/B ratio of the SiRcB(n) probes was similar to that of SiRcB probes, possibly because unreacted SiRcB can form non-fluorescent spirolactone, decreasing background signals (Figure S1). Next, we compared the la-

beling rates of intracellular (BL-NLS) or cell surface (BL-GPI) BL-tag fusion proteins by the SiRc-based probes to evaluate their cell permeability (Supplementary result, Figure S2). SiRcB2, SiRcB4, and SiRcB6 labeling rates were largely increased compared with the SiRcB labeling rate. Additionally, the relative intensity of SiRcB6 was lower than that of SiRcB4, especially for BL-NLS labeling (Figure S2c, d), suggesting that increasing the probe hydrophilicity decreased its cell permeability. To determine whether SiRc-based probes can be used for SMI of intracellular proteins, we performed SMI of Lyn11-BL using three of the novel probes. Labeling with SiRcB2, SiRcB4, and SiRcB6, unlike with SiRcB, resulted in numerous fluorescent spots on the images of cells expressing Lyn11-BL (Figure S5c, e, g), and few spots in non-transfected cells (Figure S5d, f, h). The bright spots in cells expressing Lyn11-BL were mobile (Movie S2), indicating diffusion of the labeled proteins on the inner side of the plasma membrane. Tracking analysis revealed that a significantly larger number of fluorescent spots with higher diffusion coefficients was detected upon labeling with SiRcB(n) (n = 2, 4, 6) than with SiRcB (Figure 2a). These results clearly indicate that introduction of the linker improved protein-labeling efficiency and remarkably reduced background signals due to non-specific probe adsorption.



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a

λem

b

(nm)

(nm)

-1

(M cm-1)

Φfl

0

654

671

100,000

0.39

7.20

9.6

651

670

100,000

0.38

−0.08

6.85

16.7

651

670

100,000

0.39

SiRcB6

−0.43

6.45

23.8

651

670

100,000

0.37

SiR-carboxyl

−0.88

5.86

−

645

661

100,000

0.39

TMR-HaloTag ligand

−

−

−

554

579

70,000

0.22

c

a

probes

CLogP (open form)

CLogP (closed form)

Linker length (Å)

SiRcB

1.05

7.98

SiRcB2

0.27

SiRcB4

b

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λabs

b

ε

b

Table 1. Physicochemical parameters and spectral properties of SiRc-based probes and TMR-HaloTag ligand a

calculated by ChemDraw 15 (PerkinElmer), b measured in PBS containing 0.1% sodium dodecyl sulfate (SDS) (for SiRcB and SiRcB(n) (n = 2, 4, 6) as open zwitterionic forms), c optical properties are described in reference 29

Figure 2. Evaluation of non-specific adsorption of SiRcB and SiRcB(n) (n = 2, 4, 6), and comparison of SiRcB4 and SiRcB6 labeling efficiency by TIRFM. (a) Distribution of the diffusion coefficients of Lyn11-BL labeled with 1.0 nM SiRcB and SiRcB(n). The data shown are summed over three cells. (b) Comparison of the number of bright spots per unit area between the cells labeled with SiRcB4 and SiRcB6. Lyn11-BL expressing cells were incubated with 50 pM, 100 pM, 500 pM, and 1.0 nM SiRcB4 or SiRcB6 and observed by TIRFM. The data shown are means ± standard error of the mean (S.E.M.) (N = 5).

The number of fluorescent spots with lower diffusion coefficients was slightly increased in SiRcB2-labeled samples than in SiRcB4- and SiRcB6-labeled samples (Figure 2a), indicating that the bright spots reflected non-specific adsorption (Figure S5d). Moreover, when the cells were incubated with subnanomolar concentrations of SiRcB6, the number of bright spots was lower than that in SiRcB4-labeled cells (Figure 2b, Figure S5i, j), indicating decreased membrane permeability. Based on these results, SiRcB4 was the most suitable of the four SiRc derivatives for intracellular protein SMI. Analysis of receptor-adaptor interactions in response to ligand stimulation by confocal microscopy. We performed multicolor imaging using the BL-tag and SiRcB4 labeling system to study interactions between

proteins of the innate immune system in living cells. Toll-like receptor 4 (TLR4)38 is a crucial sensor of lipopolysaccharide (LPS), which is the major constituent of the outer membrane of gram-negative bacteria, and is one of the pathogen-associated molecular patterns (PAMPs)38. The recognition of LPS by TLR4 requires several other proteins, including LPS binding protein (LBP), CD14, and MD2 (Figure 3a). TLR4 utilizes Tollinterleukin-1 receptor (TIR) domain-containing adaptor protein (TIRAP)39 and myeloid differentiation factor-88 (MyD88) to activate the NF-κB pathway, and TIR domain-containing adaptor protein inducing IFN−β (TRIF) and TRIF-related adaptor molecule (TRAM) to activate the interferon regulatory transcription factor-3 (IRF3) pathway and induce the production of the appropriate pro-inflammatory cytokines38. Here, we focused on the receptor-adaptor interaction between TLR4 and TIRAP upon LPS stimulation to analyze their molecular dynamics in living cells. TIRAP was selected as the target adaptor protein because it localizes in the plasma membrane, where it interacts with TLR440. Thus, the dynamic interactions between TLR4 and TIRAP occur at the appropriate depth for SMI using TIRFM. To analyze the TLR4-TIRAP dynamics, the BLtag protein-labeling system was used in combination with a commercial HaloTag system. BL-tag and HaloTag were attached to the N-terminus of TIRAP (BLTIRAP) and the C-terminus of TLR4 (TLR4-Halo), respectively. To test the function of these fusion proteins, we transfected HEK293T cells with BL-TIRAP or Hemagglutinin (HA)-tagged TIRAP (HA-TIRAP), along with a luciferase reporter for NF-κB. Overexpression of BL-TIRAP or HA-TIRAP increased luciferase activity at a comparable level, suggesting that fusion with BL-tag did not perturb the capacity of TIRAP to activate NF-κB (Figure S7a).


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Figure 3. Mechanism of multicolor imaging and computational analysis of SMI results. (a) Overview of LPS/TLR4 signaling. (b) Imaging of HEK293T cells expressing TLR4-Halo and BL-TIRAP by confocal microscopy. The cells were incubated with 100 nM HaloTag-TMR Ligand and 500 nM SiRcB4 at 37 ºC for 30 min. Images were acquired before and after 10 min stimulation with 10 µg/mL of LPS. Scale bar: 20 µm. (c) Multicolor SMI of HEK293T cells expressing TLR4-Halo and BL-TIRAP. The cells were incubated with 50 pM HaloTag-TMR Ligand and 100 pM SiRcB4 for 30 min at 37 °C. Scale bar: 5 µm. (d, e) Fluorescent labeling of TLR4 and TIRAP (d), and TLR4(∆TIR)-Halo and BL-TIRAP (e) with the HaloTag-TMR ligand and SiRcB4, respectively. (f–i) Transient response of the diffusion of TLR4-Halo (f), and BL-TIRAP (g) in TLR4-Halo and BL-TIRAP expressing cells, and TLR4(∆TIR)-Halo (h), and BL-TIRAP (i) in TLR4(∆TIR)-Halo and BL-TIRAP expressing cells, upon LPS stimulation (10 µg/mL). N > 140,000 (5 cells).

Similarly, TLR4-Halo and FLAG-tagged TLR4 (FLAGTLR4) increased luciferase activity to a similar degree in response to LPS stimulation, suggesting no effect of the HaloTag on the TLR4 function as an LPS receptor (Figure S7b). Then, the receptor-adaptor molecular dynamics induced by LPS stimulation were analyzed in living cells. HEK293T cells expressing both target proteins were labeled with SiRcB4 and HaloTag-TMR ligand, and stimulated with 10 µg/mL LPS for 10 min. Fluorescence

images were acquired before and after LPS stimulation. Confocal microscopic images showed the localization of TLR4 in plasma membranes and endoplasmic reticulum and TIRAP in plasma membranes (Figure 3b), which are consistent with those of previous studies40, 41. This result confirms that the tags did not influence protein localization. However, localization of the fluorescence protein fusions was not altered, even after ligand stimulation. Thus, it is not possible to monitor TLR4-TIRAP dynamics by conventional microscopy.



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Live cell multicolor SMI of TLR4 and TIRAP, or TLR4(∆TIR) and TIRAP, and statistical analysis of protein dynamics. We used SiRcB4 and the HaloTagTMR ligand to perform multicolor SMI in living cells expressing BL-TIRAP and TLR4-Halo, after stimulation with 10 µg/mL LPS, and quantitatively analyzed the protein dynamics by statistical analysis. The dynamics of fluorescently-labeled TLR4-Halo and BL-TIRAP were successfully captured with an EM-CCD camera (Figure 3c, Movies S3–6), demonstrating the capacity of our protein-labeling system for multicolor SMI of intracellular proteins in living cells, using cell-permeable fluorescent probes (BL-tag and HaloTag probes). From the statistical analysis of the intensities of each fluorescent spot, we verified that the detected mobile fluorescent spots originated from a single spot labeled with SiRcB4 or HaloTag-TMR ligand (Figure S9). Lateral molecular mobility is of particular importance for investigating intracellular signal transduction mechanisms because, in certain cases, it can be correlated with molecular states and environmental conditions. For example, intermolecular interactions are thought to decrease diffusive mobility within the membrane42. Indeed, altered mobility was noted for some membrane receptors, such as EGFR, after ligand stimulation and subsequent binding to downstream signaling molecules, such as Grb21, 43. Furthermore, intracellular signaling molecules exist in multiple states with different diffusion coefficients44, 45. Here, we divided the molecular states into two fractions (slow or fast state), and quantified the influence of LPS stimulation for different time periods on their ratio. The lateral mobility of diffusive molecules was analyzed with a probability density function (PDF) described in Eq. (1)46. Based on the PDF for a two-state model, we estimated the ratio between states and calculated the diffusion coefficients of each state, where state 1 and state 2 represent groups of fast-moving and slowmoving molecules, respectively. Then, the rate of increase of the population of molecules in state 2 was calculated using Eq. (2), and summarized as the time course Rn min, which is defined as the ratio between slowmoving molecules after and before stimulation (Figure 3f, g, Table S1). As shown in Figure 3f, the TLR4 population with small diffusion coefficients increased upon LPS stimulation (N > 140,000; 5 cells). Similarly, the TIRAP population with slower diffusion rates increased only after LPS stimulation. These results suggest that LPS stimulation alters TLR4 and TIRAP mobility. Furthermore, the diffusion rate of the slow TIRAP molecules was comparable to that of the slow TLR4 molecules (Table S1), implying interactions between TLR4 and TIRAP, which lead to decreased diffusion constants upon LPS stimulation. To confirm that the population increase in slowmoving TLR4 and TIRAP molecules after LPS stimulation was due to TLR4-TIRAP interactions, multicolor SMI was performed using a TIR-domain deletion mutant

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of TLR4 (TLR4(∆TIR)-Halo). Since the TIR domain of TLR4 is necessary for TLR4-TIRAP interactions39, we expected that the TIRAP diffusion coefficients would not change after LPS stimulation. Confocal imaging of HEK293T cells transiently expressing BL-TIRAP and TLR4(∆TIR)-Halo showed that the localizations of the proteins did not change regardless of LPS-stimulation (Figure S8b). TIRFM SMI was then performed following incubation with SiRcB4 and the HaloTag TMR ligand (Figure S10). Single-molecule fluorescence spots from TLR4(∆TIR) and TIRAP were captured, and the data were statistically analyzed as described above (Table S1). The results reveal that the relative ratio of slowmoving TIRAP molecules did not change even after ligand stimulation (Figure 3i), indicating that since TIRAP did not interact with TLR4(∆TIR) (Figure 3e), binding of TIRAP to TLR4 through the TIR domain is essential for changing its mobility. On the other hand, the population of slow-diffusing TLR4(∆TIR) molecules still increased after stimulation (Figure 3h), indicating that the change of TLR4 mobility upon LPS stimulation does not require the TIR domain or interactions with TIRAP or other intracellular molecules, and binding to LPS or other extracellular components, such as MD-2 and CD14, is sufficient. Discussion SMI often suffers from off-target signals caused by cellular autofluorescence and non-specific adsorption of the dyes, even when imaging membrane proteins by TIRFM25. Our designed probe with NIR fluorescence overcame these limitations by utilizing an NIR-emitting fluorescent dye and chemical modification to decrease probe hydrophobicity. Introduction of an oligoethylenoxy group linker improved the hydrophilicity and suppressed non-specific adsorption of the probes while retaining cell permeability. The result of SMI showed that labeling with SiRcB(n) (n = 2, 4, 6) remarkably reduced the background signals and blinking observed for SiRcB (Figure 2a; Figure S5, Movies S1, S2). In addition, fine-tuning of the linker length is essential because the high hydrophilicity of the probe interferes with cellular uptake, resulting in decreased labeling efficiency of intracellular proteins (Figure 2b, Figure S2c, d). Thus, stepwise control of probe hydrophilicity allows for efficient labeling of intracellular proteins with a remarkably high S/B ratio. Our approach is critical for protein labeling with probes using protein tags in SMI. Moreover, this method can be applied to fluorescent probes for use in superresolution imaging based on single-molecule localization microscopy. Multicolor SMI is a useful method for visualizing intermolecular interactions and analyzing molecular dynamics in living cells. However, FP-based labeling is not optimal for SMI because of the necessity to control labeling density by controlling FP expression levels; thus,


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dual-color SMI using two FPs with similar fluorescence intensity is challenging, often making NIR-FPs impractical for use in SMI of intracellular proteins. Thus, we used two orthogonal protein tag systems, the BL-tag and HaloTag systems, with NIR-fluorescent probes for multicolor SMI. In contrast to FP-based systems, labeling density can be easily optimized with two different probe concentrations in the picomolar range (see Figure 2b, Figure S5). Because these protein-labeling probes have the advantage of efficient plasma membrane permeability, the experimental doses can be reduced to low concentrations for labeling of intracellular proteins. The combination of BL-tag and HaloTag probes with low spectral overlap of highly photostable fluorophores enabled dual-color SMI of two intracellular proteins and time-lapse analysis of protein diffusion within the same live-cell context. Analysis of protein dynamics based on multicolor SMI showed an increase in the population of slow-moving TLR4 and TIRAP upon ligand stimulation by LPS (Figure 3f). The mobility of both proteins was decreased by their interaction. This result is consistent with the current model of TLR4 dynamics in response to ligand stimulation40, which demonstrates the reliability of our imaging system for detecting protein-protein interactions. In this study, we adopted a method for estimating molecular diffusion from the position change of the molecules by calculating the momentary travel distances. Co-localization analysis by dual-color simultaneous measurement might be a more direct way to estimate the binding rate between the molecule pair of interest at the molecular level47. However, there are difficulties in colocalization analysis, such as aligning color channels or a low possibility of events in which a molecule stained by a dye interacts with another molecule also stained by a dye because of partial staining of molecules. Thus, it is difficult to distinguish intermolecular interactions from only co-localization in a small space with a diameter of a few hundred nanometers. Our method can reveal the changes in molecular mobility upon the receptor-adaptor interaction from the sequential image acquisition of different two colors even without two color simultaneous measurement to detect molecular interaction directly using molecular tracking. Statistical analysis showed that regardless of whether the TIR domain was present or not, TLR4 diffusion slowed down following stimulation, although the diffusion rate of TIRAP was dependent on the presence of the TIR domain (Figure 3f–i, Table S1). These results indicate that TLR4 immobilization in response to LPS stimulation depends not only on interactions with TIRAP, but also on clustering with MD2 and CD14. However, the increased population of TLR4 with small diffusion coefficients was more prominent than that of TLR4(∆TIR), suggesting that the decrease in TLR4 diffusion was enhanced by interactions with TIRAP, and TIRAP immobilization may activate the interaction with

other adaptor or downstream signalling molecules such as MyD88. Conclusion In conclusion, we developed novel NIR-fluorescent probes for intracellular protein labeling using the BL-tag system and applied these probes in multicolor SMI. SiRcB4 showed superior properties for labeling of intracellular proteins and an excellent S/B ratio, even at the single-molecule level. Our multicolor SMI system enabled quantitative analysis of LPS-induced TLR4-TIRAP interactions in living cells at the single-molecule level for the first time. Our system may also be useful for quantitative analysis of other receptors in response to various PAMPs and investigation of the immune response of downstream adaptors localized at the cytoplasmic membrane, such as TRAM48. Furthermore, this probe can be used to study other intracellular proteinprotein interactions involved in signal transduction by wide-field fluorescence microscopy. Our intracellular protein-labeling platform may be valuable for use in screening studies based on quantitative single-molecule analysis of molecular dynamics. Materials and Methods Materials and analytical instruments. Unless otherwise specified, all reagents were purchased from the chemical suppliers Tokyo Chemical Industries (Tokyo, Japan), Wako Pure Chemical (Osaka, Japan), or SigmaAldrich Chemical Co. (St. Louis, MO, USA), and used without further purification. Bacampicillin hydrochloride was a gift from Nichi-Iko Pharmaceutical Co. Ltd. HaloTag TMR Ligand was purchased from Promega. All labeling probes were dissolved in dimethyl sulfoxide (DMSO) (biochemical grade; Wako Pure Chemical). LPS from Salmonella minnesota Re595 was purchased from Wako Pure Chemical (121-05291). Analytical thinlayer chromatography was performed on 60F254 silica plates (Merck & Co., Inc., Kenilworth, NJ, USA) and visualized under UV light. Silica gel column chromatography was performed using BW-300 (Fuji Silysia Chemical Ltd., Kasugai, Japan). Nuclear magnetic resonance (NMR) spectra were recorded on a JNM-AL400 instrument (JEOL, Tokyo, Japan) at 400 MHz for 1H NMR and at 100 MHz for 13C NMR, or an AVANCE500HD instrument (Bruker, Billerica, MA, USA) at 500 MHz for 1H NMR and at 125 MHz for 13C NMR, using tetramethylsilane as an internal standard. Mass spectra were measured on a JMS-700 mass spectrometer (JEOL) for FAB and a LCT-Premier XE mass spectrometer (Waters, Milford, MA, USA), for electrospray ionization (ESI). High-performance liquid chromatography (HPLC) analyses were performed on an Inertsil ODS-3 column (4.6 × 250 mm; GL Sciences) using an HPLC system composed of a pump (PU-2080; JASCO) and a detector (MD-2010 and FP-2020; JASCO). Preparative



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HPLC was performed with an Inertsil ODS-3 column (10.0 × 250 mm; GL Sciences, Inc.) using an HPLC system with a pump (PU-2087; JASCO) and a detector (UV-2075; JASCO). Buffer A was composed of 0.1% formic acid in H2O; Buffer B consisted of 0.1% formic acid in acetonitrile. Fluorescence spectra were measured using a Hitachi F4500 spectrometer. Ultraviolet-visible absorbance spectra were measured using a Shimadzu UV1650PC spectrometer. The pKmcl-BL-GPI plasmid was a kind gift from Dr. Atsushi Miyawaki. Synthesis. The synthetic procedures and characterization of compounds including SiRcB and SiRcB(n) (n = 2, 4, 6) are described in the Supporting Information. Cell culture. HEK293T cells were cultured in highglucose Dulbecco’s modified Eagle medium (DMEM) + Gluta Max-I (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Gibco), penicillin (100 U/mL), and streptomycin (100 µg/mL) (Invitrogen). Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. A subculture was performed every 2–3 days from subconfluent (< 80%) cultures using a trypsinethylenediaminetetraacetic acid solution (Invitrogen). Transfection of plasmids was carried out in a glassbottomed dish (Matsunami) or 6-well (TPP) plate using Lipofectamine 3000 (Invitrogen) according to a standard protocol. Confocal fluorescence microscopy. Fluorescence microscopic imaging was recorded using a confocal fluorescence microscopic imaging system including a fluorescence microscope (IX71, Olympus), a cooled CCD camera (Cool Snap HQ, Roper Scientific) or an EMCCD (iXon3, ANDOR TECHNOLOGY), a confocal scanner unit (CSU-X1, Yokogawa Electric Corporation). Multispectral LED light sauce (Spectra X light engine, Lumencor) was used to excite fluorophores at 377 ± 25 nm (for Hoechst33342), 488 ± 3 nm (for MitoTracker® Green FM in Figure S3), 542 ± 13.5 nm (for LysoTracker® Red DND-99), and 643 ± 10 nm (for SiRA, SiRB, SiRB2, SiRcB, SiRcB2, SiRcB4, and SiRcB6). The filter sets were BP377 ± 25/DM405/BA447 ± 30 (for Hoechst33342), BP488 ± 3/DM488/BA520 ± 17.5 (for MitoTracker® Green FM), BP560 ± 12.5/DM561/BA624 ± 20 (for LysoTracker® Red DND99 in Figure S3), or BP628 ± 10/DM635/BA692 ± 20 (for SiRA, SiRB, SiRB2, SiRcB, SiRcB2, SiRcB4, and SiRcB6). The lens was UPlanSApo (60×, N.A. 1.35, Oil, Olympus). The whole system was controlled using MetaMorph 7.6 software (Molecular Devices). Confocal microscopic imaging of BL-NLS. HEK293T cells maintained in glass-bottomed dishes in DMEM containing 10% FBS at 37 °C under 5% CO2 were transfected with the pcDNA3.1 (+)-BL-NLS plasmid using Lipofectamine 3000. After 5 h, the culture medium was replaced with DMEM and the cells were incubated at 37 °C for 24 h. Next, the cells were washed three times with HBSS and incubated with 500 nM

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SiRB, SiRcB, SiRcB2, SiRcB4, or SiRcB6 in DMEM for 30 min in a CO2 incubator. The cells were then washed three times with HBSS and fluorescence microscopic images were captured using appropriate filter sets. Labeled cells were observed soon after the washing step. Confocal microscopic imaging of TLR4-Halo, TLR4(∆ ∆TIR)-Halo, and BL-TIRAP. HEK293T cells maintained in a glass-bottomed dish with DMEM containing 10% FBS at 37 °C under 5% CO2 were transfected with the plasmids encoding TLR4-Halo (or TLR4(∆TIR)-Halo), BL-TIRAP and MD2 as described above. After 5 h, the culture medium was replaced with DMEM and the cells were incubated at 37 °C for 24 h. Next, the cells were washed three times with HBSS and incubated with 500 nM SiRcB4 or 100 nM HaloTag TMR Ligand for 30 min in a CO2 incubator. The cells were then washed three times with HBSS and microscopic images were acquired. Fluorescence images of the cells in HBSS were captured using appropriate filter sets. Labeled cells were observed soon after the washing step. The fluorescence images were captured in prestimulated cells and the cells after 10 min of LPS stimulation. Micro cover glass cleaning. The 25-mm glass coverslips (Matsunami) were extensively cleaned to remove background fluorescence. First, they were sonicated in a solution of 0.1 M KOH for 30 min. Next, they were sonicated in ethanol for 30 min three times. Coverslips were stored in ethanol until use. Set up of TIRFM. A TIRF microscope (Ti-E, Nikon) equipped with an EM-CCD camera (ImagEM C9100-13, Hamamatsu Photonics) and a 60× oil-immersion objective (Apo TIRF 60× oil, NA = 1.49; Nikon) was used. Laser diodes (Sapphire, COHERENT) were used to excite HaloTag TMR Ligand and SiR-carboxyl-based probes (SiRcB, SiRcB2, SiRcB4, and SiRcB6) at 561 and 640 nm, respectively. Specimens were excited with an evanescent light. The fluorescence signals from HaloTag TMR Ligand and SiR-based probes were imaged using dichroic mirrors (DM; Di01-R561 and Di02-R635, Semrock) and barrier filters (BF; FF01-609/54 and 676/29, Semrock), respectively. The laser power density is ~3.91 × 103 W/cm2, which was calculated from the field of illumination (256 µm2) and laser power (~10 mW). Image sequences (300 frames) were acquired with an exposure time of 32.82 ms. Frame rate was 30.48 fps. Experiments were performed at 37 °C. Single-molecule imaging of Lyn11-BL with a TIRF microscope. HEK293T cells maintained on glass coverslips in a 6-well plate were transfected with Lyn11-BL using Lipofectamine 3000. After 5 h, the culture medium was replaced with DMEM and the cells were incubated at 37 °C for 24 h. The culture medium was exchanged with Opti-MEM (Gibco), and the cells were incubated at 37 °C for 2 h. Next, the cells were washed three times


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with HBSS and incubated with 1.0 nM SiRcB or SiRcB(n) in Opti-MEM for 30 min in a CO2 incubator. The cells were then washed three times with HBSS and single-molecule imaging was performed using a TIRF microscope. Multicolor SMI of TLR4-Halo, TLR4(∆TIR)-Halo, and BL-TIRAP. HEK293T cells maintained on glass coverslips in a 6-well plate were co-transfected with TLR4-Halo, BL-TIRAP, and MD2 using Lipofectamine 3000. After 5 h, the culture medium was replaced with DMEM, and the cells were incubated at 37 °C for 24 h. Next, the culture medium was exchanged with OptiMEM and the cells were incubated at 37 °C for 2 h. The cells were then washed three times with HBSS and incubated with SiRcB4 (100 pM) and HaloTag TMR Ligand (50 pM) in Opti-MEM for 30 min in a CO2 incubator. The cells were stimulated with LPS (10 µg/mL) containing Opti-MEM, or LPS non-containing Opti-MEM, after washed three times with HBSS. SMI was performed before stimulation and 1, 5, 10, 15, 20, 25, 30 min after the stimulation using a TIRFM. Statistical PDF analysis. To describe lateral mobility with state transitions, we focused on molecules that move by simple diffusion, irrespective of whether they adopt multiple states or not. If a mobile molecule has two states but no state transitions, the population of the molecule can be analyzed as a mixture of two simple diffusion processes. Assuming that the proportion of molecules in state 1 (the group of the fast-moving molecules) is p, the PDF is written by a summation of PDFs for two simple diffusion processes as published previously46 and calculated as =

4 + 4 + 1− + − 4 + 4 +

−

(1) where the population, p, is constant with time, meaning that two independent populations of molecules exist irrespective of time. Here, σ is noise term, and D1 and D2 are the calculated diffusion coefficients of molecules, which belong to state 1 and the state 2, respectively. The ratio of molecules in state 2 (the group of slowmoving molecules) at each time point relative to the value of pre-stimulation molecules, Rn min, were calculated as =

1 − 1 −

(2) where pn min and pbefore are the value of p in each time point. When we perform PDF analysis, we simultaneously analyzed all images that were captured at each time

point and determined the diffusion coefficients D1 and D2 at the first time. Then, we calculated the population of the state 1 at each time point, pbefore or pn min, by using the obtained D1 and D2. Finally, we calculated the increasing rate of the population of molecules in the state 2 by using Eq. (2), and summarized as the time course of Rn min, which is the ratio of the slow-moving molecules relative to the value of pre-stimulation molecules. ASSOCIATED CONTENT Supporting Information. Materials and Methods contains Chemical Synthesis; Detection of Protein Labeling; Evaluation of Membrane Permeability; Plasmid Construction; Luciferase Assay; and Statistical Analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * K.K. ([email protected]), M.U. ([email protected]), and Y.K. ([email protected]).

Author Contributions ‡

R.S., and J.K. contributed equally.

Funding Sources This research was supported by MEXT, Japan (Grants 25220207, 26102529, 16H00768, to K.K., and 24115513, 15H03120, 15H00818 to S.M.).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors would like to thank to S. Teraguchi for discussion and to M. Hata (RIKEN Quantitative Biology), A. Yoshimura, E. Kurumatani, and Y. Kimura (Osaka University, IFReC) for assistance in cell culturing and support experiments.

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Figure 1. Design and fluorescence spectra of novel cell membrane permeable probes. (a) Structures of SiRMe, SiR-carboxyl, SiRB, and SiRcB. (b) Structures of bacampicillin-based probes. SiRcB2, SiRcB4, and SiRcB6 contain di-, tetra-, and hexa-ethylene glycol, respectively. (c) The emission spectra of SiRcB, SiRcB2, SiRcB4, and SiRcB6 in PBS buffer (pH 7.4) containing 1.0% DMSO at 37 °C. The probe concentration was 500 nM, and the excitation wavelength 654 nm. (d) Specific labeling of BL-NLS expressed in HEK293T cells. The cells were incubated with 1.0 µM SiRcB, SiRcB2, SiRcB4, or SiRcB6 for 30 min at 37 °C. Scale bar: 10 µm. 194x110mm (300 x 300 DPI)


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Figure 2. Evaluation of non-specific adsorption of SiRcB and SiRcB(n) (n = 2, 4, 6), and comparison of SiRcB4 and SiRcB6 labeling efficiency by TIRFM. (a) Distribution of the diffusion coefficients of Lyn11-BL labeled with 1.0 nM SiRcB and SiRcB(n). The data shown are summed over three cells. (b) Comparison of the number of bright spots per unit area between the cells labeled with SiRcB4 and SiRcB6. Lyn11-BL expressing cells were incubated with 50 pM, 100 pM, 500 pM, and 1.0 nM SiRcB4 or SiRcB6 and observed by TIRFM. The data shown are means ± standard error of the mean (S.E.M.) (N = 5). 185x102mm (300 x 300 DPI)



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Figure 3. Mechanism of multicolor imaging and computational analysis of SMI results. (a) Overview of LPS/TLR4 signaling. (b) Imaging of HEK293T cells expressing TLR4-Halo and BL-TIRAP by confocal microscopy. The cells were incubated with 100 nM HaloTag-TMR Ligand and 500 nM SiRcB4 at 37 ºC for 30 min. Images were acquired before and after 10 min stimulation with 10 µg/mL of LPS. Scale bar: 20 µm. (c) Multicolor SMI of HEK293T cells expressing TLR4-Halo and BL-TIRAP. The cells were incubated with 50 pM Halo-Tag-TMR Ligand and 100 pM SiRcB4 for 30 min at 37 °C. Scale bar: 5 µm. (d, e) Fluorescent labeling of TLR4 and TIRAP (d), and TLR4(∆TIR)-Halo and BL-TIRAP (e) with the HaloTag-TMR ligand and SiRcB4, respectively. (f–i) Transient response of the diffusion of TLR4-Halo (f), and BL-TIRAP (g) in TLR4-Halo and BL-TIRAP expressing cells, and TLR4(∆TIR)-Halo (h), and BL-TIRAP (i) in TLR4(∆TIR)-Halo and BL-TIRAP expressing cells, upon LPS stimulation (10 µg/mL). N > 140,000 (5 cells). 226x185mm (300 x 300 DPI)


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Intracellular Protein-Labeling Probes for Multicolor Single-Molecule

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