Actin Filament Mechanics and Structure in Crowded Environments

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Actin Filament Mechanics and Structure in Crowded Environments Nicholas Castaneda, Myeongsang Lee, Héctor J. RiveraJacquez, Ryan R. Marracino, Theresa R. Merlino, and Hyeran Kang J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b12320 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on March 2, 2019

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The Journal of Physical Chemistry

Actin Filament Mechanics and Structure in Crowded Environments Nicholas Castanedaa,b, Myeongsang Leea, Hector J. Rivera-Jacqueza, Ryan R. Marracinoa,b, Theresa R. Merlinoa, Hyeran Kanga,c,d*

aNanoScience bBurnett

Technology Center, University of Central Florida, Orlando, FL 32826, USA

School of Biomedical Sciences, College of Medicine, University of Central

Florida, Orlando, FL 32827, USA cDepartment dDepartment

of Physics, University of Central Florida, Orlando, FL 32816, USA of Materials Science and Engineering, University of Central Florida,

Orlando, FL 32816, USA * To whom correspondence should be addressed. Email: [email protected]

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The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract The cellular environment is crowded with high concentrations of macromolecules that significantly reduce accessible volume for biomolecular interactions. Reductions in cellular volume can generate depletion forces that affect protein assembly and stability. The mechanical and structural properties of actin filaments play critical roles in various cellular functions including structural support, cell movement, division, and intracellular transport. Although the effects of molecular crowding on actin polymerization have been shown, how crowded environments affect filament mechanics and structure is unknown. In this study, we investigate the effects of solution crowding on the modulations of actin filament bending stiffness and conformations both in vitro and in silico. Direct visualization of thermally fluctuating filaments in the presence of crowding agents is achieved by fluorescence microscopy imaging. Biophysical analysis indicates that molecular crowding enhances filament’s effective bending stiffness and reduces average filament lengths. Utilizing the all-atom molecular dynamics simulations, we demonstrate that molecular crowding alters filament conformations and inter-subunit contacts that are directly coupled to the mechanical properties of filaments. Taken together, our study suggests that the interplay between excluded volume effects and non-specific interactions raised from molecular crowding may modulate actin filament mechanics and structure.


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Introduction The intracellular environment is crowded with numerous types of solutes such as small ions, organic compounds, and other macromolecules. Crowding effects raised from the excluded volume of macromolecules and/or non-specific chemical interactions modulate protein assembly kinetics, protein stability, protein structure, and protein-protein interactions.1-10 The assembly and mechanics of an essential cytoskeletal protein actin plays a key role in many biological processes including cell movement, division, cytokinesis, and force generation.11-15 Most in vitro biochemical and biophysical studies on actin have been performed in dilute buffer conditions, therefore not accurately reflecting its physical and mechanical properties in complex cellular environments. Although several reports have shown crowding effects on actin polymerization kinetics and filament stability16-24, molecular mechanisms that underlie how filament mechanics and structure are modulated in crowded cellular environment are not fully established. Molecular

crowding

generates

highly

constrained

environment

and

depletion

interactions25-27, which may affect the thermal fluctuations of filaments. Theoretical models predicted the effects of crowding on polymer dynamics.28-29 Such theories have been investigated in a recent study on individual single-walled carbon nanotube’s bending and diffusion in a crowded environment.30 In this study, we investigate how molecular crowding modulates the mechanical properties and conformations of actin filaments, combining fluorescence microscopy imaging and all-atom molecular dynamics (MD) simulations. Our hypothesis is that highly constrained environment and depletion forces induced by molecular crowders reduces actin filament bending dynamics as illustrated in Figure 1. We demonstrate that molecular 3 ACS Paragon Plus Environment


crowding alters filament thermal bending dynamics, enhances filament’s effective bending stiffness, and reduces average filament lengths. MD simulations allow for examining structural variations within a constructed filament model in the presence of molecular crowders. Our study suggests molecular mechanisms by which crowding modulates filament mechanics and structure in vivo, which can in turn alter interactions between actin filaments and actin regulatory proteins.

Methods Protein sample preparation Actin was purified from rabbit skeletal muscle acetone powder (Pel-Freeze Biologicals Inc., (Rogers, AR) and gel filtered over Sephacryl S-300 equilibrated in buffer A (0.2mM CaCl2, 1mM NaN3, 2mM Tris-HCl pH 8.0, 0.2mM ATP, 0.5mM DTT) as described.31 Gactin fluorescently labeled with rhodamine (>99% purity, purified from rabbit skeletal muscle) was purchased from Cytoskeleton, Inc. (Denver, CO). Ca2+-G-actin was subjected to cation exchange to Mg2+-G-actin by the addition of 0.2mM EGTA and MgCl2, equal to the initial concentration of G-actin plus 10μM.31 Rhodamine-labeled actin was polymerized by addition of 0.1 volume of 10X polymerization (KMI) buffer (500mM KCl, 20mM MgCl2, 100mM Imidazole pH 7.0, 10mM ATP and 10mM DTT) at room temperature (∼22 °C) for 1-2 hrs. Fluorescence microscopy imaging Actin filaments (50% rhodamine labeled) were equilibrated in buffers containing crowding agents (sucrose or polyethylene glycol (PEG) 8kDa in 1X KMI buffer) at desired 4 ACS Paragon Plus Environment

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concentrations prior to imaging for 1-2 hours and proceeded to be diluted using optical imaging buffer (1X KMI buffer with 1mM ATP, 1mM DTT, 0.015M glucose, 1mg/ml catalase, 0.2 mg/ml glucose oxidase, and varying molecular crowder concentrations). Molecular crowder concentrations included sucrose 2% w/w, 10% w/w, 30% w/w, 40% w/w, and 50% w/w along with PEG 8kDa 1% w/w, 2% w/w, and 3% w/w. Time-lapse images of thermally fluctuating rhodamine-labeled actin filaments in the absence or presence of crowding agents were obtained using a Nikon Eclipse Ti fluorescence microscope outfitted with a Hamamatsu Image EM X2 CCD camera and a 100X oil immersion objective (N.A. 1.49) at room temperature (∼22 °C). Pixel size was determined to be 0.16 μm/pixel. Nikon imaging software Elements (ver. 4.50) was used to image the filaments and perform analysis and video capture interval of filament fluctuations was 100msec. Microscope slides were cleaned by absolute ethanol, sonication bath, incubated in 0.1M KOH for one hour, and then rinsed with ddH2O as described.32 Filament adsorption onto the glass slides was prevented by coating the slides with various concentrations of fatty-acid free BSA (bioWORLD, Dublin, OH) in 1X buffer solution (50mM Tris-HCl pH 7.0, 1mM NaN3, and 150mM NaCl), or mPEG-silane 5k (Laysan Bio, Inc., Arab, AL) solubilized in a solution of ethanol for 18hrs at room temperature, with gentle shaking and extensively rinses with ddH2O.33 Experiments using mPEG-silane 5k passivation required slides and coverslips to be plasma treated using a PE-50 (Plasma Etch) cleaning system for 2 mins and immediately incubated with passivating agent. Viscosity measurement



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Viscosity measurements of the molecular crowding solutions were conducted by use of a miniature ball drop device.34 Briefly, approximately 100 microliter sample was filled into a glass capillary into which a precision steel ball of 0.5 mm diameter was dropped. The speed of the ball drop was measured by recording the time for the ball to travel 8.0 cm along the capillary and compared with that for the ball to drop in a capillary filled with pure water. The shear viscosity is obtained by calibrating the ball drop speed with that of water, using 0.97 cP as its viscosity at room temperature. The viscosity value of each sample was calculated by averaging three measurements. Filament bending mechanics and length distribution analysis The bending modes of the selected filaments were obtained by time-lapse recording of thermal fluctuations over a one-minute time-span. The change in angle with respect to the position along the filament is processed by Fourier decomposition analysis as described.35-37 Decomposition of filament modes is in accordance to eq 1: 𝜃(𝑠) =

2 𝐿

∞

(

∑𝑛 = 0 𝑎𝑛𝑐𝑜𝑠

)

𝑛𝜋𝑠 𝐿

(1)

Here 𝑎𝑛 denotes the amplitude, 𝑛 is the mode number, and 𝐿 is the contour length of the filament. The various bending modes are indicated by the tangent angles of different segments of the filament with respect to the arc length up to the mid-point of each segment. Filament amplitudes for each of the bending modes were plotted with respect to time (0.1sec intervals for 600 frames analyzed in 1 min) for each mode, and the variance of each amplitude for each mode provides an estimate of flexural rigidity of the filament.35-36


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The bending Lp of filaments was measured by analysis of thermal fluctuations in timelapse recording. Filaments were manually selected and digital images were processed using NIH ImageJ and analyzed by the Persistence38 and OriginLab 8.5 software. Amplitude variance var(𝑎𝑛) is calculated as the total population variance within each mode amplitude, L indicates filament length, n is the mode number. The persistence length for the filaments selected is calculated using eq 2: 𝐿𝑝 =

𝐿2

(2)

𝑛2𝜋2(𝑣𝑎𝑟(𝑎𝑛))

Polymer bending rigidity of filaments can be defined by using eq 3 (3)

𝜅 = 𝐿𝑝 𝑘𝐵𝑇

, where Lp represents the persistence length, κ is the flexural rigidity of the filament, and 𝑘𝐵𝑇 is the thermal energy.38 Angular cosine correlation analysis is an alternative method to calculate filament Lp. The calculations were performed on digitized images of filaments using the Persistence38 and OriginLab 8.5 software. The two-dimensional cosine correlation (C) of tangent angles (𝜃) along the segment length (s) of the filament is given in eq 4 as described in detail in ref (38) (A is scaling factor) 31, 38-40: < 𝐶(𝑠) > = < 𝑐𝑜𝑠[𝜃(𝑠) ― 𝜃(0)] > = 𝐴 ∗ 𝑒 ―𝑥/2𝐿p (4) Length distributions of fluctuating filaments in various crowding conditions was fitted using log-normal modeling in eq 5.41



―(ln

𝑦 = 𝑦0 +

𝐴 2𝜋 𝜔𝑥

𝑒

() 𝑥

𝑥𝑐

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2 )

2𝜔2

(5)

, where probability (y) is the distribution function of filament lengths, 𝑦𝑜 is offset value, 𝑥𝑐 is the mean center, ω represents standard deviation, and A is the area under the curve. Statistical significance of filaments in the presence of crowding agents was determined using OriginLab 8.5 software by multiple analysis of variance (ANOVA) and post-hoc Tukey Test. Probability (p-value) between the samples indicated that there is a significant difference between filaments for various crowder concentrations with a p-value < 0.05 considered as statistically significant. Molecular dynamics (MD) simulations To evaluate the experimentally observed crowding effects on actin filament mechanics, we computationally constructed the atomistic models of actin filaments in presence of PEG and sucrose. Here, we used the recently resolved filament model that was revealed by high resolution (4.7 Å) cryo-electronmicroscopy (PDB ID: 3J8I).42 Using this model, we comprised three computational molecular models (control, F-actin with PEG, F-actin with sucrose) consisting of five subunits. To mimic the experimental crowding conditions of the 3% w/w PEG and 50% w/w sucrose, we randomly solvated 10 PEG or 20 sucrose molecules near the constructed filament with minimum 2.5 Å distance constraints using Packmol.43 We considered the analogous molecular weight of 9 repetitive ethylene glycol chains of PEG (Mw = 385.25) and sucrose molecules (Mw = 389.52). Prior to adding the crowding molecules with the actin subunits, 1000 times of conjugate gradient method of energy minimization were performed for both crowding


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molecules. The all-atom built actin filaments were solvated to TIP3P water box with 15 Å of water padding condition. Subsequently, 2 mM concentrations of MgCl2 were solvated to actin filaments to neutralize each system. Based on the constructed system, we performed 2000 times energy minimization to filaments to avoid bad contacts prior to 50 ns equilibrium MD simulations. The equilibrium simulations were performed over a timeframe of 50 ns using NAMD 2.12. version with CHARMM27 forcefield including CMAP correlations.44 To consider the crowding molecules such as PEG and sucrose, we used the CHARMM35 ether45 and CHARMM36 carboxyl forcefield.46 Equilibrium MD simulations were performed with condition of 2 fs per time step and applications of SHAKE algorithms. NPT ensemble condition was applied to the actin subunits with and without crowding agents at the 300K of temperature and 1 bar of pressure, respectively. Filament conformation and structure analyses from MD simulations After 50 ns MD simulations, we analyzed how crowding molecules modulate filament conformations and structure. To analyze the conformational changes of actin filaments, we measured the filament length and width as shown in Figure S1.47 Filament width was calculated by averaging the differences of distance between x-y plane projected 1st and 2nd actin subunit (G1 and G2), and 3rd and 4th actin subunit (G3 and G4). Filament length was measured as the distance between center mass of G1 and G5. The radius of gyration (Rg) of actin filaments was measured using visual molecular dynamics (VMD) program 1.9.1.48 Conformational changes of actin subunits affected by crowding agents were analyzed in regard to variations of helical twist angle. Twist angle within a filament was calculated based on eq 647:

 (t) = cos-1(nplus(t) • nminus(t)) / |nplus(t)||nminus(t)|) 9 ACS Paragon Plus Environment

(6)


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, where nplus(t) is 𝑪𝑮𝟏(𝒕) ― 𝑪𝑮𝟐(𝒕) and nminus(t) is 𝑪𝑮𝟒(𝒕) ― 𝑪𝑮𝟓(𝒕). 𝑪𝑮𝒊(t) is the position vector of the center of mass from i-th subunit, as illustrated in Figure S1. For the structural characteristics of filaments, root mean square fluctuations (RMSF), root mean squared displacement (RMSD), the number of hydrogen bonds, buried solvent accessible surface (SASA), and contact area based on SASA were analyzed from MD simulations. RMSD of each residue is defined as

< ∆𝑟2𝑖 (𝑡) > , which follows in

accordance to eq 7: < ∆𝑟2𝑖 (𝑡) > =

< |𝑟𝑖(𝑡) ― 𝑟𝑖(0)|2 >

(7)

, where ri (t) and ri (0) are atomistic coordinates of i-th Cα atoms from actin residues at simulation time and initial points, respectively. The RMSF, the number of hydrogen bonds, and SASA were measured every 250 ps through the VMD 1.9.1 package.48 The number of hydrogen bonds was measured at the longitudinal and lateral contact region as shown in the recent study by Galkin et al.42 The buried SASA was calculated by measuring the average SASA differences of D-loop between G1 and G3 (or between G2 and G4). The inter-subunit contact areas (As) were calculated as follows in accordance to eq 8: As=(Si+Sj-Sij)/2

(8)

, where Si, Sj, and Sij are the surface areas of i-th subunit, j-th subunit, and the combined subunits (i-th and j-th) respectively, using the VMD program. The area of specific residue pairs identified by Galkin et al.42 was calculated in a similar way as the inter-subunit


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contact areas. Each conformation of actin subunit was obtained every 250 ps from the entire 50 ns MD simulations. Excluded volume fraction calculation The excluded volume fraction (𝜙l) was assessed to show how the two different molecular crowders (PEG or sucrose) affect actin filaments based on eq 9. 𝜙𝑙(𝑛𝑐) =

𝑉𝑒𝑥𝑐𝑙 𝑉𝑡𝑜𝑡

1 4

3

= 𝑉𝑡𝑜𝑡3𝜋(𝑅𝑁 𝑔 ) 𝑛𝑐

(9)

, where nc is the number of molecular crowder, Vtot is total simulation volume and Vexcl is 10 the excluded volume of system, and 𝑅𝑁 𝑔 is the radius of molecular crowders . Here, we

considered equal number of PEG and sucrose molecules for excluded volume fraction to visualize the distinct crowding effect on the volume fractions. 𝑅𝑁 𝑔 was obtained from the area of each crowder based on SASA.

Results Molecular crowding modulates filament thermal bending To determine how molecular crowding affects filament bending dynamics, we directly visualized thermally fluctuating filaments using epi-fluorescence microscopy. Analysis of filament bending modes allows for measurements of bending persistence length (Lp), a measure of filament stiffness. PEG of molecular weight 8kDa and sucrose were chosen as an inert polymeric crowder and a small molecular crowder, respectively (PEG 8k 1-3% w/w and sucrose 2-50% w/w).16,

19, 22-23, 49-51

Filaments were constrained in a shallow

chamber (zavg ~ 1 µm) that restricts rotational motion and keeps filaments within twodimensional focal plane. We monitored the thermal fluctuations of filaments in the 11 ACS Paragon Plus Environment


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presence of crowding agents at varying concentrations. Our data show that filament thermal bending decreases with increasing concentrations of molecular crowders (Figure 2 and Videos S1-3). The filament fluctuations obtained from videos were analyzed by the Fourier decomposition of filament shapes as a sum of ten cosine modes (eq 1).35-36,

38, 52-53

Bending mode analysis indicates that mode amplitude variance (var(an)) (eq 2) reduces with increasing crowder concentrations (Figure 3).35-37 In the case of sucrose, we observe significant reductions in both mode amplitudes and variance from the lowest (2% w/w) up to the highest (50% w/w) concentrations (Figure 3e-i and Figure S2). Molecular crowding enhances filament bending stiffness and shortens average filament length We measured the bending Lp of filaments in the presence of molecular crowding from mode analysis (eq 1 and 2).35-36 Average bending Lp of filaments increases ~2.0-fold with PEG and ~1.5-fold with sucrose compared to control (Figure 4a). In the case of PEG, average filament flexural rigidity κ (eq 3) ranges from ~7.68 x 10-26 Nm2 – 8.80 x 10-26 Nm2, while sucrose κ on average is ~6.77 x 10-26 Nm2 – 8.42 x 10-26 Nm2. Our results show representative filaments in the highest crowding concentrations obtain a flexural rigidity (κ) ≈ 7.74 x 10-26 Nm2 in 3% w/w PEG and 7.45 x 10-26 Nm2 in 50% w/w sucrose with filament lengths of 9.8 µm (3% w/w PEG) and 7.1 µm (50% w/w sucrose), respectively. We averaged the Lp of filament modes 1 and 2 for each of the crowding conditions and plotted filament bending Lp as a function of length (Figure 4b-c). The bending Lp is independent of filament length in the presence of crowding (Figure 4b-c), consistent with previous studies that showed the analysis of microtubules and actin 12 ACS Paragon Plus Environment

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filaments in dilute buffer conditions.35-36, 54 These lower modes can accurately provide an estimation of filament κ, dependent on sampling interval time.35-37, 54 Thermal fluctuations are shown to dominate at lower order modes (n = 1 or 2), however, higher order modes cannot accurately reflect the bending stiffness of the filaments due to the rapid reduction of amplitude variance.35,

54

Two-dimensional average cosine correlation analysis is an

alternative method used to measure filament bending Lp (eq 4) (Figure S3). The values obtained display a similar trend to those shown with bending mode analysis, although slightly lower, we found bending stiffness increases stepwise with PEG and displays maximal Lp with sucrose 50% w/w. It is interesting that Lp shows only slight increase although solution viscosity varies several-fold (Figure S4). The range of PEG concentrations were chosen up to 3% w/w because we noted above this range (e.g. 5% w/w PEG) mainly induces actin bundles, consistent with a previous study.27 Our results show a decrease in the steady-state actin filament lengths with increasing concentrations of PEG and sucrose (Figure 5). The steady-state filament length distributions in the presence of crowders are best fit with a log-normal function (eq 5)41 (Figure S5) with fitting parameters provided in Table S1. Average filament length in the presence of PEG (1-3% w/w) ranges from Lavg ≈ 4.5 - 5.2 µm. The highest concentration of PEG (3% w/w) results in filament length reduction of 20% compared to control filaments (Lavg ≈ 6.0 µm). Average filament length in the presence of sucrose (2-50% w/w) ranges from Lavg ≈ 4.0 - 5.1 µm. The highest concentration of sucrose (50% w/w) exhibits a reduction of 28% compared to control. A previous study21 reported that specific viscosity of nucleotide-free actin decreases at high sucrose concentrations, suggesting this result



may be due to filament length reduction. We confirmed their postulation demonstrated by our characterization of filament length in the presence of sucrose. Molecular crowding induces conformational changes in actin filaments To evaluate how molecular crowding affects filament conformations, we performed equilibrium all-atom molecular dynamics (MD) simulations of actin filaments in the absence or presence of molecular crowders. A molecular model of actin filaments composed of 5 subunits was constructed based on recently resolved high-resolution (4.7 Å) filament structure from cryo-electron microscopy (PDB ID: 3J8I) (Figure 6a).42 Either PEG or sucrose molecules were randomly solvated near the constructed filament (Figure 6b-c). Upon completion of 50 ns simulation, we analyzed the distributions of filament length (L), width (w), radius of gyration (Rg), and filament twist (θ) (N = 200, Figure 6d-g) in the presence of PEG or sucrose molecules (Figure S6, see details in Methods).55 Filament lengths in the presence of crowders are shorter than control filaments (Lavg,Control = 112.10 Å, Lavg,PEG = 110.33 Å, Lavg,sucrose = 109.35 Å) (Figure 6d). PEG reduces filament width, while sucrose slightly increases filament width compared to control filament (Wavg,Control = 35.64 Å, Wavg,PEG = 32.82 Å, Wavg,sucrose = 36.07 Å) (Figure 6e). In addition, both PEG and sucrose molecules slightly reduce the average Rg of actin filaments (Rg,Control = 48.05 ± 0.18 Å, Rg,PEG = 47.69 ± 0.17 Å, Rg,sucrose = 47.65 ± 0.21 Å; Figure 6f and Figure S6a). Interestingly, both molecular crowders increase average filament twist angle (θ) compared to control actin filament (θavg,Control = 84.32 ± 1.44º, θavg,PEG = 90.27 ± 1.83º, θavg,sucrose = 88.71 ± 2.15º; Figure 6g and Figure S6b). Based on the excluded volume fractions calculation (Table S2), filaments in the presence of PEG exhibit larger


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excluded volume than those with sucrose, which may be related to the significant increase of filament twist in the presence of PEG (Figure 6g).

Molecular crowding affects structural properties of actin filaments To investigate the role of molecular crowders in filament structural stability, we analyzed inter-subunit (longitudinal and lateral) contact areas in the presence of crowding (Figure S7). Both longitudinal and lateral contact areas increase in the presence of PEG or sucrose (Table 1). Sucrose increases lateral contact areas by 10.1% and longitudinal contact areas by 5.5% compared to control filament, whereas PEG enhances lateral contact areas by 5.7% and longitudinal contact areas by 7.1%. Molecular crowders affect the areas of the specific residue pairs shown in the recent study42 formed at longitudinal interfaces (M44:Y143, M44:G168, M47:F352, K61:E167, R62:D288, I64:Y166, E241:T324, D244:M325, G245:P322) and lateral interfaces (N111:T194, K113:E195, H173:I267, V201/T202:S271) (Figure S7 and Table S3). Overall, the interactions between the residue pairs are strengthened in the presence of PEG and sucrose, evidenced by increased longitudinal and lateral contact areas (Table S3). In particular, we observed significant increase of contact areas, encompassing hydrogen bonds between M44:G168 (at longitudinal contact) and V201/T202:S271 (at lateral contact) pairs, and salt bridges between K61:E167 and R62:D288 (at longitudinal contact) pairs. These stabilized hydrogen bonds and salt bridges at inter-subunit contacts are consistent with previous reports42, 56, and may explain the enhanced filament bending 15 ACS Paragon Plus Environment


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stiffness by molecular crowding demonstrated in our study (Figure 4). To evaluate the stability at longitudinal interfaces, we measured buried solvent accessible surface area (SASA) for the DNase I binding loop (D-loop) residues 40-51 of actin subdomain 242, which plays a critical role in modulating the mechanical properties of filaments.11,

57-58

Consistent with increased contact areas, the D-loop buried SASA increases with both molecular crowders compared to control filaments (Table 1). Hydrogen bonding is a key factor to determine the mechanical properties of filaments that are formed through non-covalent interactions.59 We analyzed the number of hydrogen bonds for residue pairs located at inter-subunit contacts based on 50 ns MD simulations (Figure 7 and Table 2). From our calculations we found four specific residue pairs that sustain hydrogen bond interactions including M44:G168 identified by Galkin et al42 (Figure 7 and Table 2). Overall, molecular crowders increase the number of hydrogen bonds for longitudinal contacts, while the number of hydrogen bonds for lateral contacts decrease in the presence of crowding. Importantly, our simulation demonstrates molecular crowders significantly enhance hydrogen bond interactions of the K61:E167 salt bridge42 by ~2.0-fold among the residue pairs at longitudinal contacts (Figure 7b and Table 2). To determine the effects of crowding on dynamics and conformational changes of actin filaments, we further analyzed the root mean square fluctuation (RMSF) and root mean squared displacements (RMSD) per each residue (Figure S8). RMSF near the Dloop, a highly mobile region of subdomain 260-62, is significantly lower with PEG than in the absence of crowder or with sucrose (Figure S8a). This reduced fluctuation of the Dloop in the presence of PEG allows for stable longitudinal contacts between subunits, 16 ACS Paragon Plus Environment

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consistent with enhanced bending mechanics (Figure 4). Significant changes in RMSD were observed in the D-loop region, C-terminal (residues 368 to 375) and partial domain of subdomain 4 (residue 218 to 250) in the presence of PEG (Figure S8b).63-65 The increase of RMSD in the D-loop and C-terminal may affect longitudinal inter-subunit interactions within F-actin (Figure S8b).

Discussion The motivation for the current study was to investigate how the mechanical and structural properties of actin filaments are modulated under conditions mimicking crowded cellular environments. Biophysical characterization indicates that molecular crowding reduces filament thermal bending, which leads to the increase of effective filament bending stiffness. All-atom MD simulations demonstrate that crowding agents significantly affect filament conformations including twist angle and enhances intersubunit interactions that are linked to the mechanical properties of filaments. Molecular crowding results in altered excluded volume fractions10 (Table S2), thus modulating filament compactness (Figure 6) and mechanics (Figure 4). The conformational and mechanical modulations of filaments in the presence of crowding may be attributed to the excluded volume effect2, 6, 50, 66 and/or thermodynamic factors such as enthalpy and “soft” non-specific interactions.3,

26, 67

Filament twist increase associated

with crowding agents (Figure 6) indicates the excluded volume effect plays an important role in controlling filament conformations. The interplay between excluded volume effects and enthalpic interactions can contribute to filament assembly3,

22

influence average steady-state filament lengths (Figures 5 and 6). 17 ACS Paragon Plus Environment

such that they may


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The enhanced filament stiffness generated by molecular crowding (Figure 4) can be explained by stabilization of filament structure. Actin filament inter-subunit interactions have been shown to play a critical role in controlling overall filament mechanical properties.11, 15, 47, 56, 58 Filament compactness evidenced by radius of gyration and twist angle (Figure 6) could result in altered longitudinal and lateral contacts (Figure S7, Table S3). The increased twist of actin filaments in the presence of molecular crowders implicate that crowders reinforce the local inter-subunit interactions within the filaments. Hydrogen bond interactions observed at both longitudinal and lateral contact sites (Figure 7) may contribute to the D-loop stabilization and enhanced filament mechanics.42, 59 In addition, our all-atom MD simulation results demonstrate that molecular crowding enhances salt-bridge interactions, in particular K61:E167 residue pair (Figure 7 and Table 2). Galkin et al. has shown E167 forming an ionic bridge with K61 of an adjacent subunit by high-resolution cryo-electron microscopy.42 In the presence of molecular crowding, the K61:E167 salt-bridge exhibits sustained hydrogen bonds that affect inter-subunit contacts, potentially linked to filament bending stiffness observed in this study.31-32, 56 Filament stiffening and conformational modulations driven by molecular crowding potentially affect the biological functions of actin filaments inside the cell. Recent studies demonstrate that filaments in solution exist in multiple structural states with variable twist and D-loop conformations, known as structural polymorphism42,

62.

Our buried SASA,

RMSF, and RMSD results (Table 1 and Figure S8) indicate molecular crowding modulates D-loop conformations. These conformational changes induced by molecular crowding suggest that intracellular filaments may adopt different polymorphic structural states compared to filaments under in vitro conditions. We predict that filament stiffening 18 ACS Paragon Plus Environment

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associated with the enhanced D-loop stability in crowded cellular environments most likely influences the interactions between filaments and actin binding proteins (ABPs) such as cofilin or myosin. For example, D-loop reorganization is closely linked to the binding and severing activities of the essential ABP cofilin.32, 58, 68 Discrete polymorphic states of D-loop under crowded conditions may have an impact on cofilin binding and/or filament severing. It is interesting to note, molecular crowding results in over-twisted filament conformations involving mechanical strain within the filament. Such changes in filament twist generated by crowding may affect crosslinking proteins binding, thereby controlling the geometry and organization of actin bundles in cells.69

Conclusions In summary, we have demonstrated through biophysical characterization and all-atom MD simulations, that molecular crowding significantly alters the mechanical and structural properties of actin filaments. Our experimental data indicate that molecular crowding enhances the effective bending stiffness of filaments and reduces steady-state filament lengths. MD simulations have shown that molecular crowding modulates filament conformations and inter-subunit interactions, which supports the experimentally observed increase of filament stiffness raised by crowding. This study suggests, the molecular mechanisms of how filament mechanics and structure are modulated in crowded intracellular environments and will aid in the understanding of in vivo regulatory processes of actin cytoskeleton.



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Supporting Information Description Document containing eight additional figures and three tables (PDF). Acknowledgements This study was supported by the UCF start-up fund and In-House grant (to H.K.). The authors would like to thank the National Science Foundation for support of this work through REU site EEC 1560007 and acknowledge the computational resources from UCF STOKES cluster. We thank Dr. Jay X. Tang and Eric Girardi at Brown University for assistance with viscosity measurements and Dr. Jay X. Tang for his critical reading and suggestions for the manuscript. We thank anonymous reviewers for their critical reviews and valuable suggestions. We thank Aaron B. Marbin for his help with data analysis for an earlier stage of this work. Competing Interests Statement The authors declare no competing financial interests.



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Main Tables Lateral

Longitudinal 2

Buried SASA 2

2

Contact area (Å )

Contact area (Å )

(Å )

F-actin (Control)

1238.55

2336.40

873.92

F-actin with PEG

1308.05

2501.72

895.18

F-actin with Sucrose

1363.66

2464.65

877.86

Table 1. Comparison of calculated averaged longitudinal contact area, lateral contact area, and solvent accessible surface buried area (SASA) of DNase binding loop (D-loop) from last 5ns of MD simulations. Data indicates modulations in D-loop conformation in the absence and presence of molecular crowding agent polyethylene glycol (PEG) or sucrose. Buried SASA values are determined by the outside and inside regions of the Dloop (see details in Methods).


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M44:G168 K61:E167 R62:D288 K113:E195 F-actin (Control) F-actin with PEG F-actin with Sucrose

0.4

0.7

7.9

4.0

0.4

1.9

8.2

2.7

0.6

1.6

8.7

2.9

Table 2. The average number of hydrogen bonds formed by residue pairs at longitudinal and lateral contacts after 50 ns equilibrium MD simulations.



Main Figures

Figure 1. Schematic of actin filament bending in dilute and crowded environments. Left image demonstrates a thermally fluctuating filament in dilute buffer condition. Right image represents a filament in a highly crowded environment consisting of various proteins and other molecules.


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Figure 2. Representative time-lapse images of thermally fluctuating filaments in the presence of molecular crowding agents. Filaments were constrained to a shallow chamber (~1 µm) which prevented rotation and kept them in plane of focus. Images are separated by 6 sec time intervals for a total of 1 min time-lapse. Polymerization of filaments by buffer: 1X KMI (50mM KCl, 2mM MgCl2, 10mM Imidazole at pH 7.0, 1mM ATP and 1mM DTT). Concentration of molecular crowding solutions are in mass % (w/w%). Scale bars indicate 5 μm.



Figure 3. Filament bending mode amplitudes are modulated by molecular crowding agents. The amplitudes of 10 bending modes from filaments represented in Figure 2 as a function of time within each subpanel (t = 60sec). The variance within each of the mode amplitudes demonstrates an independent estimate of the filament flexural rigidity. (a) control, (b) PEG 1% w/w, (c) PEG 2% w/w, (d) PEG 3% w/w, (e) sucrose 2% w/w, (f) sucrose 10% w/w, (g) sucrose 30% w/w, (h) sucrose 40% w/w, and (i) sucrose 50% w/w.


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Figure 4. Molecular crowding agents modulate bending stiffness of actin filaments. (a) Bending Lp of filaments (mode number, 2) in various polyethylene glycol (PEG) 8k and sucrose concentrations. Bending Lp plotted as a function of filament length with crowding agents (b) PEG 8k and (c) sucrose. Dashed lines represent the average filament crowding Lp and dotted line is the average filament control Lp. (n.s. not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). N ≈ 7 filaments per condition and uncertainty bars represent the standard deviation (SD). 33 ACS Paragon Plus Environment


Figure 5. Average steady-state filament length is modulated in the presence of molecular crowding. The box represents the 25th–75th percentile, whiskers indicate standard deviation (SD), and the middle square is the mean. Tukey test indicated significant difference in comparison (p < 0.05). Total number of filaments analyzed per each condition N ≈ 100 – 400 (n.s. not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).


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Figure 6. Conformational and geometrical analyses of actin filaments (F-actin) in the presence of molecular crowders polyethylene glycol (PEG) and sucrose after 50 ns molecular dynamics (MD) simulations. Conformations of (a) F-actin without molecular crowders, (b) F-actin with PEG, and (c) F-actin with sucrose. Geometrical analyses analyzing the probability of filament (d) length, (e) width, (f) radius of gyration (Rg), and (g) twist angle (θ). Second order polynomial function of exponential model used for distribution curves (d)-(g).



Figure 7. Molecular crowders affect the number of hydrogen bonds formed by residue pairs at longitudinal and lateral contacts during 50 ns MD simulations. (a) M44:G168, (b) K61:E167, and (c) R62:D288 belong to longitudinal contacts, and (d) K113:E195 is located lateral contact.


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TOC Graphic



Figure 1. Schematic of actin filament bending in dilute and crowded environments. Left image demonstrates a thermally fluctuating filament in dilute buffer condition. Right image represents a filament in a highly crowded environment consisting of various proteins and other molecules. 144x81mm (300 x 300 DPI)

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Figure 2. Representative time-lapse images of thermally fluctuating filaments in the presence of molecular crowding agents. Filaments were constrained to a shallow chamber (~1 µm) which prevented rotation and kept them in plane of focus. Images are separated by 6 sec time intervals for a total of 1 min time-lapse. Polymerization of filaments by buffer: 1X KMI (50mM KCl, 2mM MgCl2, 10mM Imidazole at pH 7.0, 1mM ATP and 1mM DTT). Concentration of molecular crowding solutions are in mass % (w/w%). Scale bars indicate 5μm. 157x129mm (300 x 300 DPI)



Figure 3. Filament bending mode amplitudes are modulated by molecular crowding agents. The amplitudes of 10 bending modes from filaments represented in Figure 2 as a function of time within each subpanel (t = 60sec). The variance within each of the mode amplitudes demonstrates an independent estimate of the filament flexural rigidity. (a) control, (b) PEG 1% w/w, (c) PEG 2% w/w, (d) PEG 3% w/w, (e) sucrose 2% w/w, (f) sucrose 10% w/w, (g) sucrose 30% w/w, (h) sucrose 40% w/w, and (i) sucrose 50% w/w. 165x136mm (300 x 300 DPI)


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Figure 4. Molecular crowding agents modulate bending stiffness of actin filaments. (a) Bending Lp of filaments (mode number, 2) in various polyethylene glycol (PEG) 8k and sucrose concentrations. Bending Lp plotted as a function of filament length with crowding agents (b) PEG 8k and (c) sucrose. Dashed lines represent the average filament crowding Lp and dotted line is the average filament control Lp. (n.s. not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). N ≈ 7 filaments per condition and uncertainty bars represent the standard deviation (SD). 63x154mm (300 x 300 DPI)



Figure 5. Average steady-state filament length is modulated in the presence of molecular crowding. The box represents the 25th–75th percentile, whiskers indicate standard deviation (SD), and the middle square is the mean. Tukey test indicated significant difference in comparison (p < 0.05). Total number of filaments analyzed per each condition N ≈ 100 – 400 (n.s. not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). 109x83mm (300 x 300 DPI)


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Figure 6. Conformational and geometrical analyses of actin filaments (F-actin) in the presence of molecular crowders polyethylene glycol (PEG) and sucrose after 50 ns molecular dynamics (MD) simulations. Conformations of (a) F-actin without molecular crowders, (b) F-actin with PEG, and (c) F-actin with sucrose. Geometrical analyses analyzing the probability of filament (d) length, (e) width, (f) radius of gyration (Rg), and (g) twist angle (θ). Second order polynomial function of exponential model used for distribution curves (d)-(g). 100x133mm (300 x 300 DPI)



Figure 7. Molecular crowders affect the number of hydrogen bonds formed by residue pairs at longitudinal and lateral contacts during 50 ns MD simulations. (a) M44:G168, (b) K61:E167, and (c) R62:D288 belong to longitudinal contacts, and (d) K113:E195 is located lateral contact. 132x128mm (300 x 300 DPI)


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Actin Filament Mechanics and Structure in Crowded Environments

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