Single-Molecule Rotation for EGFR Conformational Dynamics in Live

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Single-Molecule Rotation for EGFR Conformational Dynamics in Live Cells Youngchan Park, Sangwon Shin, Hyeonggyu Jin, Jiseong Park, Yeonki Hong, Jaemin Choi, Byunghyuck Jung, Hyunjoon Song, and Daeha Seo J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b09037 • Publication Date (Web): 31 Oct 2018 Downloaded from http://pubs.acs.org on November 1, 2018

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Journal of the American Chemical Society

Single-Molecule Rotation for EGFR Conformational Dynamics in Live Cells Youngchan Park,† Sangwon Shin,‡ Hyeonggyu Jin,‡ Jiseong Park,‡ Yeonki Hong,‡ Jaemin Choi,§ Byunghyuck Jung,*,§, Hyunjoon Song,*, † & Daeha Seo*,‡ †Department

of Chemistry, KAIST, Daejeon, 34141, Republic of Korea, ‡Department of Emerging Materials Science, DGIST, Daegu, 42988, Republic of Korea, §School of Undergraduate Studies, DGIST, Daegu, 42988, Republic of Korea

Supporting Information Placeholder ABSTRACT: Monitoring the dynamics of proteins in live cells on appropriate spatiotemporal scales may provide key information regarding long-standing questions in molecular and cellular regulatory mechanisms. However, tools capable of imaging the conformational changes over time have been elusive. Here, we present a single-molecule stroboscopic imaging probes by developing gyroscopic plasmonic nanoparticles, allowing for replication of protein-protein interactions and the conformational dynamics based on rotational and lateral velocities. This study fundamentally monitors the rotational motion of a membrane protein, epidermal growth factor receptor (EGFR), to decipher undiscovered structural dynamics in live cells without any molecular perturbations. This method offers a strategy to visualize assemblies and conformational changes, and provides unique insights into the mechanism underlying the molecular dynamics for receptors.

Rotational and lateral mobilities of proteins in living cells are propelled by thermal energy, and perturbed by biochemical and electrostatic interactions between inter- and intra-biomolecules during biological processes.1,2 Therefore, real-time observation of the orientation and the movement of each molecule reveals both of the homogeneous or heterogeneous molecular interactions and their conformational dynamics. Recent investigations based on single-molecule tracking and Förster resonance energy transfer illustrate protein-protein interactions and conformational changes, respectively, during biological processes.3-6 Such microscopic methods acquire structural and kinetic data concealed by ensemble averaging by deconvoluting the temporal trajectories of individual biomolecular dynamics.7,8 However, the regulation of conformational dynamics and their transition during signal transduction remains unclear. Rotational dynamics may provide novel insights into the underlying molecular mechanism. An ideal probe for imaging membrane receptor rotational and lateral dynamics at the single-molecule level would comprise a i) bright imaging domain that is sensitive to an angular displacement,9,10 ii) rigid linker preventing structural perturbations, and iii) specific targeting domain for the exact location and orientation at a principal rotational axis. Here, we synthesized monovalent (m)-, Gold dimer assembled with a Y-shaped oligonucleotide structure as a ROtational probe (mGYRO) (Figure

Figure 1. (a) Synthesis of mGYROs, conjugating two monofunctionalized Au nanoparticles (mAu NPs) (b) TEM image of mGYRO showing controlled locus of functionalization. (c) Darkfield (DF) images of two mGYROs at distinct angles (top, left), and the corresponding SEM images (top, right). The linearly polarized light is irradiated at an angle (arrows). With angular variations, the scattering intensities of the two particles are plotted and fitted to sine functions. Based on differences in the two periods, the particles are at 1.32 different angles in radian, well matched with SEM. (d) Angular error distribution (θOM-θSEM, rad) with 0.14 deviation (n = 21). (e) Angle sensitivity is calculated as: (Imax-Imin)/ Imax yields values of 0.14 ± 0.03 (mean ± SD) for Au spheres, 0.61 ± 0.08 for mGYROs, and 0.60 ± 0.09 for Au rods. (f-i) Monitoring of lipid molecules on a 0.2 mg/mL BSA cushioned SLB. (f) Lipid molecular trajectory (diffusion coefficient, 0.52 μm2/s) and (g) corresponding intensity and DF images. (h) Cumulative square displacement (CSD) and cumulative square angular displacement (CSAD) are plotted. (i) Mean diffusion coefficient and angular speed of lipid molecules were 0.35 ± 0.19 μm2/s, 52 rad/s, (n=47) respectively.

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1a). This dimerized gold nanoparticle (Au NP) is optimized to 25nm size and 4-nm in inter-particle distance, considering the plasmonic coupling and nucleotide stability11,12 (Note S1). The probe is distinguishable from cytosolic organelles upon dark-field microscopy (DF, Figure S1) and the double stranded DNA (dsDNA) and the linkers display adequate stability and rigidity without any potential perturbation (Figure S2), consistent with previous in-situ transmission electron microscope (TEM) and Xray observations.13,14 The size and distance between two NPs are assessed experimentally and theoretically via the Finite-Difference Time-Domain method (FDTD, Figure S3). TEM analysis provided direct evidence that probes are indeed generated in dimer form (Figure 1b). Advantages in using the DNA include ease of targeting molecular assemblies at the precise center of a Au dimer, modularity to conjugate further functionalities (Figure 1a, bottom), and commercial availability. We observed the assemblage of NPs comprising small Au NPs at the center of Au dimers, designating the probe targets in a site-specific and stoichiometric manner, whereas a similar linear plasmonic structure, Au nanorod, revealed non-site-specific (Figure S4). To examine the orientation and angle sensitivity of a single mGYRO, probes were immobilized on a coverslip, using a biotinstreptavidin linker. Thereafter, we observed the brightness of each spot while changing the incident linear polarized angle, Δθ (Figure 1c). Scattering intensity of each single probe was measured as a function of θ and fitted to the sine-function with a period of π. The orientation was determined with the fitted function and was well matched with the orientation from scanning electron microscope (SEM) imaging (Figure 1c, right), thereby showing the angledependent aspect with an ~8° error (Figure 1d). Angle sensitivity was 4.4 folds that of spheres and comparable to that of rods, which are commonly used for linear particles (Figure 1e). Consequently, mGYROs can easily measure the 2-dimensional orientation under polarized light. We applied these rotational probes to visualize the diffusive dynamics of lipid molecules on a 2-dimensional model membrane, supported lipid bilayer (SLB), and confirmed that mGYRO served as a single molecule imaging probe without diffusion perturbation by comparing the diffusion coefficients measured using various imaging probes (Movie S1, and Figure S5). The lipid molecules, however, were too fast to observe their rotational mobility, thus 0.2 mg/mL BSA was applied to slow the diffusion dynamics on SLB.15 Consequently, lipid molecules slowed down to 0.35 ± 0.19 µm2/s with observable rotational dynamics, showing continuous intensity changes (Figure 1f-i, and Movie S2). As EGFR proteins are much slower than lipid molecules (0.16 ± 0.14 µm2/s), there was no difficulty observing the proteins. The mGYROs served as bona fide non-perturbing single molecule imaging agents, displaying Brownian rotational and lateral mobility of lipid molecules with any significant slope changes.16 To apply the monovalent nanoprobes for single EGFR imaging in live cells, we generated a stable cell line expressing SNAP-EGFRGFP and specifically labeled the receptors with benzylguanine (BG), linker DNAs, and mGYROs (Figure S6). The traces of the scattering intensities and x-y position were obtained via DF microscopy.17 Based on the recent studies, EGFR shows dynamic changes in lateral diffusion, which may result from protein-protein interaction, mainly from reversible dimerization.18 The existence of dimers, however, remains controversial.19-21 To validate the oligomeric states of EGFR and their corresponding diffusivity, long-time observation is essential (>1 min, temporal resolution: 20 ms).22 We first followed colocalization events (n = 541) of single receptors using monovalent Au NPs (mAu) in live cells (Figure 2ae). To validate the oligomerization states, we simultaneously

Figure 2. (a) Scheme of EGFR colocalization. (b) Relative scattering intensity of Au dimers depending on interparticle distance. Three main types of observable colocalizations of EGFR; (c) association, (d) transient encounter, and (e) passing by without physical touching (3, 31, 66%, respectively, from 541 colocalization events out of 12 cells pooled from 3 independent experiments). Scattering intensity changes of each Au NP and their HMM analysis (f-h). Cumulative square displacement (CSD)HMM analysis from a lateral diffusion data (i-k). We observed constant CSD slope before and after the colocalization (f, i) whereas two EGFRs show a simple collision (g, j) or passing by without physical contact (h, k). measured coupled plasmonic intensities (Figure 2f-h), a function of the distance (d) between two Au NPs (Figure 2b), and lateral diffusivities (Figure 2i-k) that is function of associated protein number, (Figure S7). To detect discrete states from the stochastic transition of oligomers from the trajectories, we employed statistical machine learning analysis, a Bayesian model incorporating hidden Markov model (HMM-Bayes) and HaMMy.23,24 We evaluated the temporal accuracy of the HMM methods by simulating Brownian lateral and rotational trajectories (Figure S8, Movie S3), and confirmed the consistency of results. The HMM analysis of the EGFR trajectories revealed reversible EGFR dimerization (Figure 2f-k, green line) with rare observation of higher ordered oligomerization in ligand(EGF)-free conditions. Furthermore, we investigated EGFR dynamics after ligand binding (Figure S9). Clustered Au NPs were observed as well as fluorescent protein (GFP) and organic dye (Texas red), indicating EGFR oligomerization. Next, we also confirmed that the mGYROs target SNAP-EGFRGFP specifically (Figure 3a, b) and do not alter the EGFR mobility in live cells, in contrast to multi-conjugated NPs (Figure S10). At low labeling density to minimize optical cross-talks between neighboring probes, polarized DF revealed sparse labeling of diffuse EGFR, with fluctuations in the scattering intensity (IEGFR) of the mGYROs (Figure 3c). Based on these results, we


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Journal of the American Chemical Society around the TM domain should be constants, and changes in morphological factors (r, h) are negligible owing to the intrinsic structural stability of the helical TM domain.27 We therefore assumed that there exist additional factors to change the morphological factors. In EGFR dimerization, the asymmetric association of kinase domains (KDs) is a critical step, and recent nuclear magnetic resonance and molecular dynamics studies confirmed that the KD dimerization is inhibited when KDs are buried in the plasma membrane on their own.28-30 Ligand-driven EGFR dimerization stabilizes the active conformation of intracellular domains (ICD) by allosterically disentangling membrane-interacting elements (specifically, the LRRLL motif within the juxtamembrane segment, and the KD) from the plasma membrane. Therefore, we interpret the origin of stochastic change in angular displacement of EGFR as the interaction between ICD and lipid membrane. Additionally, this is relatively more dominant in rotational mobility than in lateral mobility owing to their dependency in r ( r-2 vs ln(r-1)), suggesting that the hydrophobic interaction between ICD and the lipid bilayer is sufficient to shift rotational but not oligomeric state. Figure 3. Oligomeric and conformational states of EGFR in live cells and their transition dynamics. (a) Schematic representation of targeting strategies of mGYROs and (b) their corresponding darkfield (DF) image showing specific labeling of mGYROs. (c) Scattering intensity of mGYRO fluctuates in linearly polarized DF imaging. (d) CSD (orange) and the two separated states analyzed via HMM analysis (red) indicate that EGFRs reversibly form monomers and dimers (top). CSAD (cyan) and the three separated states analyzed via HMM (blue) indicate that EGFRs change their conformations with regard to intracellular domains (ICD) (bottom). The corresponding trajectory is displayed with changes in intensity and contrast (inset). (e) Transitions in oligomeric states and conformational states are expressed with state symbols and proposed models. Dwell time (τ) for each state is listed in the table. A total of 400 distinct states were observed from 20 different cells (M1=100, M2=70, M3=0 (forbidden), D1=190, D2=20, and D3=11). investigated rapid changes in both lateral and rotational diffusion, revealing that several states exist (Movie S4). To investigate the origins of the fluctuation, we measured the intensity among immobilized mGYROs with different polarization angles (parallel, Ill and perpendicular, I+). Herein, i) all immobilized probes under non-polarized (Figure S2e) and polarized irradiation (Figure 3c, gray line) show stable brightness, ii) diffusing probes under non-polarized light also maintained stability (Figure S11), iii) IEGFR was always between Ill and I+, and iv) IEGFR fluctuated in a stochastic manner (Figure 3c, red line). Hence, changes in IEGFR from diffusing probes were not due to artifacts or vibrations between the Au NPs or translocations along the z-axis, but rather from angular displacement of mGYRO under polarized light. In principle, angular and lateral diffusion coefficients (DR, DL) of the conventional cylinder model of transmembrane (TM) protein in cellular membrane follows the Saffman-Delbruck relationship,25,26 𝐷𝑅 =

𝑘𝑇 2

4𝜋𝜂ℎ𝑟

(1),

𝐷𝐿 =

𝑘𝑇

(

4𝜋𝜂ℎ

ln

𝜂ℎ 𝜂𝑤𝑟

)

― 𝛾 (2)

where, T, η, h, and r, respectively denote the temperature, viscosity of the lipid membrane, height, and radius of the TM protein. In live cell experiments of single molecular EGFR diffusion, T and η

To determine the number of conformational states resulting from ICD docking and its correlation with oligomeric states, we analyzed both of the CSAD and CSD from the trajectories with HMM (Figure 3d). The results were deduced from measured trajectories; two oligomeric (M, D) states from CSD, and three conformational states (1, 2, 3) from CSAD. From tens of trajectories, three notable observations were obtained: i) two monomeric (M1, M2), and three dimeric (D1, D2, D3) states were observed, with nearly identical angular velocities in the same rotational states, albeit significant differences among rotational states (M1-D1 vs M2-D2 vs D3, ~10, 50, 90 rad2/s, respectively, Figure S12). ii) Considering the dwell time (τ) of each state, the slowest rotational states (M1 and D1), expecting the KD buried form of EGFR, are energetically stable. iii) Considering the state symbols, further detailed investigations of temporal transitions are required (Figure 3e). Each observation requires independent consideration. First, the rotational state is maintained while the oligomeric state changes (i.e. M1 to D1 and vice versa), which possibly represent dimerization occurring when the rotational direction of two EGFRs is matched (Note S2). Second, the M3 state was not detected among hundreds of dynamic transitions, although disentangled ICD from membrane is an important conformation of EGFR activation. The undetected M3 state could be interpreted in a manner wherein subsequent dimerization further stabilizes the disentangled ICD form, which is also consistent with the proposed conformation of the EGFR KD.30 Finally, including conventional observations showing that the transition occurs only between monomers and dimers, the present results revealed that with an approximate approach, most transitions occur between M1 and D1 states. In summary, this study revealed the conformational dynamics of ICD of EGFR, which was previously undetectable in live cells, at a single-molecule level, using a site specific plasmonic nanoparticles, stroboscopic imaging methods, and mathematical algorithms. This method allowed not only to visualization of molecular diffusion and rotation as well as analysis of structural transitions of the protein during signaling. The present success in imaging the rotation and transition lies in robust optical stability and precisely controlled nanomaterials surface chemistry.31-32

ASSOCIATED CONTENT



Supporting Information Materials and methods, additional simulations and calculations, optical and electron microscopy images, and their statistics and analysis (PDF), single-molecule tracking and their analysis video (MOV)

AUTHOR INFORMATION Corresponding Author Email: [email protected]; [email protected]

[email protected];

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the national research foundation of South Korea (NRF) grant (to Y.P., S.S, H.J., J.P., Y.H., and D.S.) funded by the Korea government (MSIP; Ministry of Science, ICT & Future Planning) (No. 2017R1C1B2010945). Y.P and H.S. was supported by the National Research Foundation (NRF) funded by the Korea government (MSIP) (NRF-2018R1A2B3004096). B.J. was supported by the DGIST Undergraduate Group Research Program (UGRP) grant. We also thank the CCRF of DGIST for technical support of TEM, SEM, and NMR.

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dynamics and fluctuations of DNA-nanogold conjugates by individualparticle electron tomography Nat. Commun. 2016, 7, 11083. (13) Chen, Q.; Smith, J. M.; Park, J.; Kim, K.; Ho, D.; Rasool, H. I.; Zettl, A.; Alivisatos, A. P. 3D Motion of DNA-Au Nanoconjugates in Graphene Liquid Cell Electron Microscopy Nano Lett. 2013, 13, 45564561. (14) Mastroianni, A. J.; Sivak, D. A.; Geissler, P. A.; Alivisatos, A. P. Probing the Conformational Distributions of Subpersistence Length DNA Biophys. J., 2009, 97, 1408-1417. (15) Diaz, A. J.; Albertorio, F.; Daniel, S.; Cremer, P. S. Double cushions preserve transmembrane protein mobility in supported bilayer systems Langmuir, 2008, 24, 6820-6826. (16) Farlow, J.; Seo, D.; Broaders, K. E.; Taylor, M. J.; Gartner, Z. J.; Jun, Y.-W. Formation of targeted monovalent quantum dots by steric exclusion Nat. Methods 2013, 10, 1203-1205. (17) Ueno, H.; Nishikawa, S.; Iino, R.; Tabata, K. V.; Sakakihara, S.; Yanagida, T.; Noji, H. Simple Dark-Field Microscopy with Nanometer Spatial Precision and Microsecond Temporal Resolution Biophys J. 2010, 98, 2014-2023. (18) Chung, I.; Akita, R.; Vandlen, R.; Toomre, D.; Schlessinger, J.; Mellman, I. Spatial control of EGF receptor activation by reversible dimerization on living cells Nature 2010, 464, 783-787. (19) Huang, Y.; Bharill, S.; Karandur, D.; Peterson, S. M.; Marita, M.; Shi, X.; Kaliszewski, M. J.; Smith, A. W.; Isacoff, E. Y.; Kuriyan, J. Molecular basis for multimerization in the activation of the epidermal growth factor receptor eLife 2016, 5, e14107. (20) Belyy, V.; Shih, S.-M.; Bandaria, J.; Huang, Y.; Lawrence, R. E.; Zoncu, R.; Yildiz, A. PhotoGate microscopy to track single molecules in crowded environments Nat. Commun. 2017, 8, 13978. (21) Low-Nam, S. T.; Lidke, K. A.; Cutler, P. J.; Roovers, R. C.; van Bergen en Henegouwen, P. M. P.; Wilson, B. S.; Lidke, D. S. ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding Nat. Struct. Mol. Biol. 2011, 18, 1244-1249. (22) Tsunoyama, T. A.; Watanabe, Y.; Goto, J.; Naito, K.; Kasai, R. S.; Suzuki, K. G. N.; Fujiwara, T. K.; Kusumi, A. Super-long singlemolecule tracking reveals dynamic-anchorage-induced integrin function Nat. Chem. Biol. 2018, 14, 497-506. (23) McKinney, S. A.; Joo, C.; Ha, T. Analysis of Single-Molecule FRET Trajectories Using Hidden Markov Modeling Biophys. J. 2006, 91, 1941-1951. (24) Monnier, N.; Barry, Z.; Park, H. Y.; Su, K.-C.; Katz, Z.; English, B. P.; Dey, A.; Pan, K.; Cheeseman, I. M.; Singer, R. H.; Bathe, M. Inferring transient particle transport dynamics in live cells Nat. Methods 2015, 12, 838-840. (25) Saffman, P. G.; Delbruck, M. Brownian motion in biological membrane Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 3111-3113. (26) Gambin, Y.; Lopez-Esparza, R.; Reffay, M.; Sierecki, E.; Gov, N. S. Lateral mobility of proteins in liquid membranes revisited Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 2098-2102. (27) Psachoulia, E.; Marshall, D. P.; Sansom, M. S. P. Molecular Dynamics Simulations of the Dimerization of Transmembrane α-Helices Acc. Chem. Res. 2010, 43, 388-396. (28) Arkhipov, A.; Shan, Y.; Das, R.; Endres, N. F.; Eastwood, M. P.; Wemmer, D. E.; Kuriyan, J.; Shaw, D. E. Architecture and Membrane Interactions of the EGF Receptor Cell 2013, 152, 557-569. (29) Endres, N. F.; Das, R.; Smith, A. W.; Arkhipov, A.; Kovacs, E.; Huang, Y.; Pelton, J. G.; Shan, Y.; Shaw, D. E.; Wemmer, D. E.; Groves, J. T.; Kuriyan, J. Conformational Coupling across the Plasma Membrane in Activation of the EGF Receptor Cell 2013, 152, 543-556. (30) Zhang, X.; Gureasko, J.; Shen, K.; Cole, P. A.; Kuriyan, J. Allosteric Mechanism for Activation of the Kinase Domain of Epidermal Growth Factor Receptor Cell 2006, 125, 1137-1149. (31) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Quantum dot bioconjugates for imaging, labelling and sensing Nat. Mater. 2005, 4, 435-446. (32) Jin, D.; Xi, P.; Wang, B.; Le Zhang; Enderlein, J.; van Oijen, A. M. Nanoparticles for super-resolution microscopy and single-molecule tracking Nat. Methods 2018, 15, 415-423.


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5


Figure 1. (a) Synthesis of mGYROs, conjugating two mono-functionalized Au nanoparticles (mAu NPs) (b) TEM image of mGYRO showing controlled locus of functionalization. (c) Dark-field (DF) images of two mGYROs at distinct angles (top, left), and the corresponding SEM images (top, right). The linear-ly polarized light is irradiated at an angle (arrows). With angular variations, the scattering intensities of the two particles are plot-ted and fitted to sine functions. Based on differences in the two periods, the particles are at 1.32 different angles in radian, well matched with SEM. (d) Angular error distribution (θOM-θSEM, rad) with 0.14 deviation (n = 21). (e) Angle sensitivity is cal-culated as: (Imax-Imin)/ Imax yields values of 0.14 ± 0.03 (mean ± SD) for Au spheres, 0.61 ± 0.08 for mGYROs, and 0.60 ± 0.09 for Au rods. (f-i) Monitoring of lipid molecules on a 0.2 mg/mL BSA cushioned SLB. (f) Lipid molecular trajectory (diffusion coefficient, 0.52 μm2/s) and (g) corresponding in-tensity and DF images. (h) Cumulative square displacement (CSD) and cumulative square angular displacement (CSAD) are plotted. (i) Mean diffusion coefficient and angular speed of lipid molecules were 0.35 ± 0.19 μm2/s, 52 rad/s, (n=47) respectively.


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Figure 2. (a) Scheme of EGFR colocalization. (b) Relative scat-tering intensity of Au dimers depending on interparticle dis-tance. Three main types of observable colocalizations of EGFR; (c) association, (d) transient encounter, and (e) passing by without physical touching (3, 31, 66%, respectively, from 541 colocalization events out of 12 cells pooled from 3 independent experiments). Scattering intensity changes of each Au NP and their HMM analysis (f-h). Cumulative square displacement (CSD)-HMM analysis from a lateral diffusion data (i-k). We ob-served constant CSD slope before and after the colocalization (f, i) whereas two EGFRs show a simple collision (g, j) or passing by without physical contact (h, k).



Figure 3. Oligomeric and conformational states of EGFR in live cells and their transition dynamics. (a) Schematic representation of targeting strategies of mGYROs and (b) their corresponding dark-field (DF) image showing specific labeling of mGYROs. (c) Scattering intensity of mGYRO fluctuates in linearly polarized DF imaging. (d) CSD (orange) and the two separated states analyzed via HMM analysis (red) indicate that EGFRs reversibly form monomers and dimers (top). CSAD (cyan) and the three separated states analyzed via HMM (blue) indicate that EGFRs change their conformations with regard to intracellular domains (ICD) (bottom). The corresponding trajectory is displayed with changes in intensity and contrast (inset). (e) Transitions in oligomeric states and conformational states are expressed with state symbols and proposed models. Dwell time (τ) for each state is listed in the table. A total of 400 distinct states were observed from 20 different cells (M1=100, M2=70, M3=0 (for-bidden), D1=190, D2=20, and D3=11).


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Single-Molecule Rotation for EGFR Conformational Dynamics in Live

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