Cysteine-rich protein 2 accelerates actin filament cluster formation

Filamentous actin (F-actin) forms many types of structures and dynamically regulates cell morphology and movement, and plays a mechanosensory role for extracellular stimuli. In this study, we determined that the smooth muscle-related transcription factor, cysteine-rich protein 2 (CRP2), regulates the supramolecular networks of F-actin. The structures of CRP2 and F-actin in solution were analyzed by small-angle X-ray solution scattering (SAXS). The general shape of CRP2 was partially unfolded and relatively ellipsoidal in structure, and the apparent cross sectional radius of gyration (Rc) was about 15.8 Å. The predicted shape, derived by ab initio modeling, consisted of roughly four tandem clusters: LIM domains were likely at both ends with the middle clusters being an unfolded linker region. From the SAXS analysis, the Rc of F-actin was about 26.7 Å, and it was independent of CRP2 addition. On the other hand, in the low angle region of the CRP2-bound F-actin scattering, the intensities showed upward curvature with the addition of CRP2, which indicates increasing branching of F-actin following CRP2 binding. From biochemical analysis, the actin filaments were augmented and clustered by the addition of CRP2. This F-actin clustering activity of CRP2 was cooperative with α-actinin. Thus, binding of CRP2 to F-actin accelerates actin polymerization and F-actin cluster formation.


Introduction
Cysteine-rich proteins (CRPs; also referred to as cysteine and glycine-rich proteins; CSRPs) are muscle cell differentiation related proteins [1][2][3][4][5][6][7] and they consist of two LIM domains, which are double zinc-finger-like structures that mediate protein-protein interactions, and two glycine-rich regions. CRP family members (CRP1, CRP2/SmLIM, and CRP3/MLP) share a highly sequence homology, but their expression patterns differ depending on the type of tissue [8]. For instance, CRP1 is expressed in multiple smooth muscle organs and CRP2 is expressed in vascular smooth muscle cells (SMCs) [9][10][11], and these proteins act as cofactors for SMC differentiation [4]. In proepicardial cells, CRP2 forms complex with serum response factor (SRF) and GATA proteins in the nucleus, and this complex strongly activates SMC-a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 storage ring at the Photon Factory (KEK, Tsukuba, Japan), operated at 2.5 GeV with a ring current between 350 and 450 mA, was selected and collimated with the double focusing optics installed at beamline 6A. In order to allow small-angle measurements required for the present experiments, we used a beam of dimensions 0.20 mm (vertical) × 0.20 mm (horizontal) at the detector plane with a specimen-to-detector distance of 2280 mm. Scattered X-ray were recorded by a XRII-CCD detector (C7300) (Hamamatsu Photonics K.K., Shizuoka, Japan). The beam intensity incident at the specimen was monitored with an ion chamber placed in front of the specimen. The sample was inserted in a temperature-controlled cell with two mica windows, through which X-ray passed, and the temperature was maintained at 25˚C. Normally, 12-18 measurements for each prepared sample were taken at different concentrations. Each exposure time of X-ray was 10 s to minimize radiation damage of the sample. Buffer scattering measurements were performed periodically throughout the measurement sequence. Each intensity image was integrated in the circumferential direction. Then, after correction for variations in the beam intensity, as measured by the current values from the ion chamber, we obtained net intensity data, I(Q,c), by subtracting the buffer scattering from the sample solution scattering, where Q is the momentum transfer vector (= 4πsinθ/λ, in which 2θ and λ are the scattering angle and the wavelength of the monochromatized X-ray used, respectively) and c is the protein concentration.

Analysis of SAXS intensity data
First, we calculated the average intensity data, IðQ; cÞ, from the 12-18 measured net intensity data, I(Q,c), for each protein concentration. The Kratky plot, I(Q,c)ÁQ 2 vs. Q plot [20], was used for each IðQ; cÞ=c data. The apparent cross-sectional radius of gyration, R c (c), was determined from the slope of the modified Guinier plot for a rod-like particle (ln[I(Q,c)ÁQ] vs. Q 2 ) [20], in which we used IðQ; cÞ=c, in the narrow Q range (up to Q = 0.78/R c ). The slope of the Guinier plot (ln[I(Q,c)ÁQ] vs. Q 2 ) shows À R 2 cðcÞ 2 . The final R c value at c = 0 was determined by means of a least-squares fitting to the data points at various protein concentrations.
The pair-distance distribution function, p(r), was calculated by the program GNOM [21] using the merged data of low concentrated and high concentrated IðQ; cÞ=c data; in the narrow Q range (Q 0.04834), we used low concentrated IðQ; cÞ=c data, and in the wide Q range (Q > 0.04834), we used high concentrated IðQ; cÞ=c data. The maximum chord length, D max , of the molecule was determined according to the total estimation value of the GNOM program [21]. The program DAMMIF [22] was used to construct three-dimensional molecular envelopes that fit to the merged IðQ; cÞ=c data. The program was run 10 times independently and the obtained models were sorted and averaged by the DAMAVER program [23]. Finally, we refined the averaged model by the DAMMIN program [24].
For the F-actin analysis, we obtained net intensity data, I(Q,c), by subtracting the buffer and CRP2 scatterings from the sample solution scattering, where c is the actin concentration. We calculated the average intensity data, IðQ; cÞ, from the 12-18 measured net intensity data, I(Q, c), in each actin protein concentration. The apparent cross-sectional radius of gyration, R c , was determined from the slope of the modified Guinier plot for rod-like particles (ln[I(Q,c)ÁQ] vs. [20], in which we used IðQ; cÞ=c, in the narrow Q range (up to Q = 1.7/R c ). The slope of the modified Guinier plot (ln[I(Q,c)ÁQ] vs. Q 2 ) shows À R 2 c 2 . The SAXS analyzed raw data is available in supplementary S1 File.
Confocal laser scanning microscopy F-actin was formed by incubating 0.042 mg/mL of actin protein (an equivalent mixture of Alexa Fluor 488-labeled actin and non-labeled actin) with F-buffer containing 8% PEG and various concentrated CRP2 at 23˚C for 4 h. The solution was spread onto the slide glass and covered with cover glass. The Alexa Fluor-labeled F-actin was observed by confocal laser scanning microscopy (Nikon C2, Nikon, Tokyo, Japan). For analyze single bundled F-actin length, F-actin was formed by incubating 0.17 mg/mL of actin protein (an equivalent mixture of Alexa Fluor 488-labeled actin and non-labeled actin) with F-buffer containing 8% PEG and 0.022 mg/mL of CRP2 at 23˚C for 4 h. The Alexa Fluor-labeled F-actin was observed by confocal laser scanning microscopy. From the obtained fluorescence images, contour length of single bundled F-actin were measured using Image J software (NIH, Bethesda, MD). The filament lengths for each group were compared by analysis of variance followed by Mann-Whitney U test. For the experiments in presence of α-actinin, F-actin was formed by incubating 0.042 mg/ mL of actin protein (an equivalent mixture of Alexa Fluor 488-labeled actin and non-labeled actin) with F-buffer containing 8% PEG, with or without 0.022 mg/mL CRP2 and 0.01 mg/mL α-actinin at 23˚C for 2.5 h.

Actin polymerization assay
Actin monomers (0.4 mg/mL) containing 10% of pyrene labeled in G-buffer were induced to polymerize by addition of a 1/10th volume of 10 × F-buffer (5 mM Tris-HCl, pH 8.0, 1.0 M KCl, 20 mM MgCl 2 , 0.2 mM CaCl 2 , 10 mM ATP, and 0.5 mM DTT) in the absence or in the presence of CRP2 (0.026, 0.11, 0.22 mg/mL). The solution was mixed and transferred into quartz micro-cell, and then the fluorescence of the pyrene (excitation: 365nm; emission: 407 nm) was recorded using a F4500 spectrofluorometer (Hitachi, Tokyo, Japan) at 23˚C. The time from mixing to starting to measure was fixed at 90 sec.
Low-speed actin co-sedimentation assay 0.17 mg/mL of actin protein was copolymerized with 0.088 mg/mL of CRP2, 0.04 mg/mL of N-LIM, and 0.04 mg/mL of C-LIM for 2 h at 23˚C in F-buffer containing 8% PEG. After polymerization, the solution was centrifuged at 20,000 × g for 30 min at 23˚C. The resulting pellets were analyzed by SDS-PAGE and silver staining (2D Silver Stain-II, Cosmo Bio, Tokyo, Japan).

SAXS analysis of CRP2
The structure of quail CRP2 in solution was determined by NMR [25]. The two LIM domains of CRP2 are structurally and dynamically independent from each other. To understand the structural interaction between CRP2 and actin filaments in solution, we first determined the structural characteristics of CRP2 in solution by SAXS analysis. In this study, we used recombinant mouse CRP2 with (His) 6 tag at the C-terminus, which was expressed in E. coli and purified for determining the interaction of CRP2 with F-actin [6]. Fig 1A shows the SAXS data, IðQ; cÞ, of each concentrated CRP2 solution. The IðQ; cÞ in the small-angle region (Q 1.7 × 10 −2 Å -1 ) showed an upturn, which indicates some aggregation of the molecules in solution. The concentration of the CRP2 in this study was 0.20-0.50 mM, and these high concentrations of CRP2 slightly aggregated in solution.
First, we checked the structural fold of CRP2 using Kratky plot (I(Q,c)ÁQ 2 vs. Q plot), where I(Q,c) is the IðQ; cÞ=c and Q is the momentum transfer vector (4πsinθ/λ). In the case of a wellfolded globular protein, the Kratky plot will exhibit a "bell-shaped" peak at low Q and converge to the Q axis at high Q, while for an unfolded protein, the plot will lack the peak and slightly increase at high Q [20]. The Kratky plot of CRP2 showed gradual peak that was slightly decreased at high Q ( Fig 1B). Thus, CRP2 appears to be partially unfolded in solution.
The decreased ratio of SAXS intensity for Q provides the general shape of the materials. The I(Q) of CRP2 roughly correlated with Q -1 , indicating that CRP2 was a rod or ellipsoidal object. Furthermore, the ellipsoidal particle, whose ratio of diameter to length was more than 2, shows the Guinier linearity of the cross section curves [20]. We then checked the Guinier linearity of CRP2 in the cross sectional modified Guinier plot (ln[I(Q,c)ÁQ] vs. Q 2 plot) for each CRP2 concentration ( Fig 1C). In the region of 7.5 × 10 −4 Q 2 Å -2 , the intensities decreased linearly, and in the smaller angle region, 4.0 × 10 −4 Q 2 7.5 × 10 −4 Å -2 , the intensities showed lower values than the values of the extended lines ( Fig 1C). This trend of the cross sectional plot is observed in ellipsoidal particles [20]. Thus, the structural character of CRP2 was hypothesized to be ellipsoidal. The apparent radii of cross sectional gyration, R c (c), was then calculated from the slopes of the lines in Fig 1C. The fitting range of a straight line for the modified Guinier plot was 0.9 × 10 −3 Å -2 Q 2 2.5 × 10 −3 Å -2 , which is 0.4 QÁR c 0.8. The apparent R c value, extrapolated to c = 0, was 15.9 (0.74) Å.
We then merged low concentrated and high concentrated IðQ; cÞ=c data to predict the shape of CRP2. Using the merged data, the pair-distance distribution function, p(r), was calculated by the program GNOM (Fig 1D) [21]. The maximum chord length, D max , of the molecule was determined to be 118 Å according to the total estimation value of GNOM program. The CRP2 shape was then modeled with the use of the ab initio shape prediction program DANM-MIF [22] from the merged data, and the D max , 118 Å. The obtained 10 dummy models were averaged by the program DAMAVER [23] and then the final ab initio model was refined by the DAMMIN program [24]. Fig 1E shows the culminating dummy model of CRP2 in solution. The CRP2 model contains roughly four tandem cluster structures. The NMR-determined structure of the full-length quail CRP2 shows that the folded LIM domains are connected via a structurally disordered flexible linker [25]. Furthermore, our Kratky plot of CRP2 showed partial unfolded structure ( Fig 1B). Thus, both end clusters in our model indicate folded LIM domains and the middle clusters show a flexible linker of LIM domains (Fig 1E).

SAXS analysis of CRP2 bound F-actin
To examine the structural interaction between CRP2 and actin filaments in solution, we analyzed the structural characters of rod like F-actin and CRP2 bound F-actin in solution by SAXS analysis. Fig 2A shows the SAXS data, IðQ; cÞ=c, of F-actin with each CRP2 ratio. At the region around 0.1 Å -1 of the F-actin scattering data, the scattering valley and peak were slightly observed at 0.105 Å -1 (Λ = 60 Å, where Λ is the Bragg distance) and 0.11 Å -1 (Λ = 57 Å), respectively (Fig 2A). These valley and peak were characteristic of the SAXS profile for F-actin, indicating the actin periodical structure [26][27][28]. These characteristic profiles were also observed for CRP2 bound F-actin (Fig 2A). The decreased ratio of SAXS intensity for Q in each F-actin solution provides information on the shape of materials. The decreased ratio, d, between 0.01 < Q < 0.016 of each F-actin are shown in Table 1. From the scattering theory, d = 1  indicates a rigid rod material, and d = 2 indicates a flat disc material. The d of bare F-actin was approximately 1.4 indicating an intermediate object between rigid rod and flat disc shape. Factin is a filamentous material having high flexibility [29,30]. Furthermore, low regional upward scattering of cylindrical objects can be ascribed to the flexibility of the cylindrical objects, to branching, or to large amounts of aggregation [31]. Thus, the d value of F-actin would be construed as ascribing to the flexibility of F-actin. With respect to CRP2, it was observed that as the CRP2 concentration increased, the d values increased. Thus, the macroscopic morphology of F-actin would change with the binding of CRP2. Since CRP2 is an actin binding protein, one possibility of macroscopic change is the formation of filament branching. Previous reports calculate cross sectional radius of gyration, R c , of F-actin using a modified Guinier plot for rod-like materials [26,32]. These reports fit the slope of the scattering intensity in the Q 0.063 A -1 regions. We then calculated the R c of CRP2-bound F-actin. Fig 2B shows the modified Guinier plots for rod-like materials using the SAXS data (ln[I(Q)ÁQ] vs. Q 2 plot) [20]. In the inner part of the Guinier plot Q 2 < 0.002, there was upward scattering, and then we calculated the slope of the scattering in the 0.002 Å -2 Q 2 0.004 Å -2 region, which is the 0.045 Å -1 Q 0.063 Å -1 or 1.2 QÁR c 1.7 region. The R c of each F-actin solution is shown in Table 1. The R c value of bare F-actin was determined to be 26.7 Å. This value is similar to previous reports of 25.7 Å [26]. The R c values of F-actin were nearly constant with increasing CRP2 ratios (Table 1). Thus, it is assumed that CRP2 locates interface of the two adjacent actin subunits of cross sectional single F-actin; this location little affects the increase of R c of F-actin (see Discussion).

Cluster formation of CRP2-bound F-actin
Confocal laser scanning microscopy was used to observe F-actin structures and the formation of branches. We used Alexa Fluor 488-labeled actin monomers for polymerization. Fig 3A  shows the fluorescence images of F-actin with or without CRP2. The filament clustering and complexity were clearly accelerated by the addition of CRP2 (Fig 3A). This held true even with the addition of small amounts of CRP2. We compared the length of each single bundled actin filament with or without CRP2 (Fig 3B). The length of each single bundled F-actin was clearly elongated by addition with CRP2. We furthermore examined actin polymerization kinetics using pyrene-actin assay; this method allows detection of filament formation by increase in pyrene fluorescence (Fig 3C). Actin polymerization was clearly accelerated by addition of CRP2 (Fig 3C). Recent study reports that CRP2 promotes F-actin bundling [19]. Thus, CRP2 accelerates actin polymerization, elongates filament length and promotes F-actin cluster formation.
We then examined the binding of CRP2 to bundled and clustered actin filaments by actin co-sedimentation assay. We used PEG for leading formation of higher order F-actin structure and then we precipitated the F-actin by low-speed (20,000 × g) centrifugation. G-actin protein was polymerized with or without full length, N-terminal LIM domain and glycine-rich region part (N-LIM), and C-terminal LIM domain and glycine-rich region part (C-LIM) of CRP2 proteins. It was considered that N-LIM or C-LIM of CRP2 influences the interaction between CRP2 and F-actin, because we previously revealed that N-LIM of CRP2 directly bound to Factin [6]. Fig 4 shows the SDS-PAGE image of the resulting pellets of co-sedimentation assay. CRP2 accumulated in the pellet together with bundled F-actin (Fig 4). Furthermore, the amount of CRP2 decreased or unchanged under coexistence of N-LIM or C-LIM, respectively, in the pellet together with the F-actin (Fig 4). However, it was obscure whether only N-LIM or C-LIM accumulated in the pellet together with the F-actin; probably it was because molecular weights of these proteins were too small to compare the density of bands (Fig 4). Therefore, CRP2 directly bound to bundled F-actin, and N-LIM part inhibited the binding of CRP2 with bundled F-actin. We finally examined the F-actin clustering activity of CRP2 in the presence of another actin crosslinking protein, α-actinin. Previous research has shown that CRP2 binds with α-actinin as well as actin filaments [6,10,14]. In our study, we first found that the formation and clustering activity of CRP2 was strongly augmented by the addition of α-actinin (Fig 5). Discussion CRP2 directly associates with F-actin through its N-terminal LIM domain and glycine-rich region [6,14]. CRP2 consists of two LIM domains and glycine-rich regions at both ends of a flexible linker region; the two LIM domains move freely and show independent space orientation [25]. The cross sectional radius of gyration R c of bare and CRP2-bound F-actin were nearly identical with the value being approximately 26.7 Å. On the other hand, the R c and D max of CRP2 in solution were 15.9 Å and 118 Å, respectively. Thus, it is assumed that the Nterminal LIM domain of CRP2 is the contact point with interface of two adjacent actin subunits of cross sectional single F-actin, and the C-terminal LIM domain moves freely from the F-actin surface (Fig 6). The result of our co-sedimentation assay supports this idea; N-terminal LIM and glycine rich region inhibited the binding of full length of CRP2 with F-actin. On the other hand, CRP3, which is a CRP family protein, binds to actin filaments via its C-terminal LIM domain and self-associates via its N-terminal LIM domain, resulting in bundling of F-actin [33]. Previous reports [14,19] and our result clearly show that CRP2 also associates with F-actin directly and facilitates actin polymerization, crosslinking and clustering. Thus, although CRP3 self-associates via its N-terminal LIM domain [33], CRP2 could self-associate via the freely movable C-terminal LIM domain. However, our SAXS analysis did not show clear dimerization but aggregation of CRP2 in solution. Thus, we speculate that the self-association of CPP2 would be loose not rigorous. The loose self-associated CRP2 would work to crosslink the actin filaments and cause cluster formation.
The superstructure of F-actin is highly regulated by numerous actin-binding proteins, such as α-actinin, filamin, fascin, fimbrin, and scruin. These proteins form a variety of actin structures, ranging from meshworks to networks of thick bundles [35][36][37][38]. Of these actin-binding network formation of actin filaments caused by CRP2 is augmented by the coexistence of αactinin. Interestingly, this augmentation effect could arise from even small amounts of α-actinin. α-actinin interacts with actin molecules with outside direction of F-actin (Fig 6) [34,39]. From our SAXS analysis, CRP2 will bind with the interface of the cross sectional two adjacent actin subunits of single F-actin. Thus, the interacting positions of CRP2 and α-actinin with Factin will be different but closely, and then these molecules can act cooperatively to bundle and crosslink actin filaments (Fig 6).
During vascular smooth muscle development CRP2 expression increases and it translocates from the nucleus into the cytoplasm [4,40]. Particularly, CRP2 localizes to the actin cytoskeleton in smooth muscle cells [4,6]. Thus, it is of significant interest to understand the role that CRP2 might play while bound to the cytoskeleton in smooth muscle cells. In this study, we found that CRP2 accelerates actin polymerization and cluster formation of F-actin through its direct interaction with F-actin. Moreover, CRP2's activity is cooperatively augmented by αactinin. Future studies will address the influence of CRP2-bound F-actin in smooth muscle cells.
In conclusion, we examined the structure of CRP2 and CRP2 bound F-actin in solution by SAXS analysis. The CRP2 structure was relatively extended and consists of four tandem clusters. The cross sectional radius of gyration of F-actin was nearly constant even with the addition of CRP2. However, we found that CRP2 accelerated actin polymerization and complexity of F-actin structure by binding directly with F-actin. This effect of CRP2 was strongly augmented by addition of α-actinin. Our findings provide additional information of the function of CRP2 in actin filaments formation.