PTM-associated condensation of homopolymeric (poly A) RNA by MDP1

Abstract

Mycobacterial DNA-binding protein 1 (MDP1) is a histone-like protein which carries endogenous post-translational modifications (PTMs) and contributes to nucleic acid organization in Mycobacterium tuberculosis (Mtb). Our previous work using total RNA (a heterogeneous mixture of RNA substrates) established that MDP1 induces RNA condensation, but the structural diversity of total RNA makes it difficult to fully resolve protein-intrinsic condensation behavior and the interactions stabilizing the resulting condensates. To minimize substrate-dependent effects, we used polyadenylic acid (poly A) RNA as a uniform model substrate and compared native MDP1 purified from Mtb (nMDP1-mtb), which retains its natural PTMs, with recombinant MDP1 expressed in Escherichia coli (eMDP1-mtb), which majorly lacks these modifications. Optical microscopy and high-speed AFM revealed that native and recombinant MDP1 induce distinct condensate morphologies: nMDP1-mtb induces compact globular condensates, whereas eMDP1-mtb induces elongated chain-like condensates. Increasing ionic strength rapidly dissolved condensates formed by both proteins, while 1,6-hexanediol had minimal effects, demonstrating that electrostatic interactions dominate condensate stabilization. These findings show that PTM-associated differences in MDP1 shape poly A RNA condensate morphology, while both protein forms rely on a shared electrostatic mechanism, contributing to a better understanding of MDP1-mediated RNA organization.

Significance

Our previous study demonstrated that MDP1 induces condensation of total RNA and revealed protein construct-dependent differences in condensate morphology. However, the heterogeneity of total RNA makes it difficult to distinguish intrinsic protein effects from contributions arising from RNA sequence and structure. Here, using a simplified homopolymeric poly A RNA substrate, we combined optical microscopy and high-speed AFM to visualize the condensate morphologies formed by PTM-enriched and PTM-deficient MDP1. Despite their distinct morphologies, condensates formed by both proteins were predominantly stabilized by electrostatic interactions. These findings provide a perspective into how a major mycobacterial nucleoid-associated protein influences the morphology and organization of RNA condensates.

Introduction

Mycobacterial DNA-binding protein 1 (MDP1), also known as Rv2986c, MtHU, HU-homolog, or HupB [1], is a major histone-like nucleoid-associated protein in Mycobacterium tuberculosis (Mtb) involved in genome organization [2,3], transcriptional regulation [4], and bacterial dormancy [5–8]. MDP1 contains a conserved N-terminal HU-like region (HUR) and a C-terminal intrinsically disordered region (IDR) enriched in post-translational modifications (PTMs). Native MDP1 carries multiple PTMs, including predominantly lysine monomethylation, together with lysine di- and trimethylation, acetylation, succinylation, and phosphorylation at additional residues, all of which have been identified and quantified in previous studies [1,9,10]. These PTMs can alter not only the electrostatic properties of MDP1 but also its intermolecular interactions and conformational behavior. Furthermore, PTMs such as acetylation, succinylation, and phosphorylation have been shown to modulate MDP1-mediated DNA binding and compaction [3,11,12]. Given these established effects, PTMs may also influence how MDP1 reorganizes RNA, motivating a mechanistic examination of PTM-associated RNA condensation.

MDP1 has been extensively studied for its role in DNA compaction, and our recent study using high-speed atomic force microscopy (HS-AFM), which enables nanoscale imaging of macromolecular dynamics [13–15], provided direct nanoscale visualization of this process [16]. Increasing evidence also links RNA organization to the dormant state of Mtb [17,18], raising the possibility that MDP1 may contribute to higher-order RNA organization. Our previous study [19] demonstrated that MDP1 forms PTM-associated RNA condensates using total RNA as a model substrate; however, the structural diversity of total RNA makes it difficult to fully resolve protein-intrinsic condensation behavior and the physical forces stabilizing these condensates.

To address this, we used polyadenylic acid (poly A) RNA as a sequence-uniform substrate to minimize sequence- and structure-dependent heterogeneity. Native MDP1 purified from Mtb (nMDP1-mtb), which retains endogenous PTMs, was compared with recombinant MDP1 expressed in Escherichia coli (eMDP1-mtb), which has almost no modifications. By combining optical microscopy (OM) and HS-AFM, we visualized RNA condensate morphologies across micrometer and nanometer scales and examined the physicochemical properties of the resulting condensates.

Materials and methods

1. RNA and 1,6-Hexanediol

Polyadenylic acid (poly A; MW 700–3500 kDa) RNA was purchased from Roche Sigma Aldrich (catalogue number, 10108626001), and 1,6-Hexanediol (1,6-HD) was purchased from Sigma Aldrich (catalogue number, 240117).

2. Purification of MDP1 proteins

2.1. Native MDP1 purification

Native MDP1 (nMDP1-mtb) was purified from Mtb based on the method previously described [6]. Mtb H37Rv was cultured in Sauton medium at 37°C. After harvesting the bacteria by filtration, the bacterial cells were ground in a mortar with a pestle using quartz sand as an abrasive. The resulting lysates were then soaked in 1.5 l of 0.25 N HCl at 4°C for two nights. Acid-soluble proteins were obtained by sequential filtration through 0.45 μm and then 0.22 μm membrane filters. The filtrates were subsequently neutralized to approximately pH 7 using NaOH. Next, the extract was purified using a cation exchange column (HiPrep CM FF 16/10) (Cytiva). Gradient elution based on increasing salt concentration was performed, and the fractions containing the target protein were collected. After concentrating the eluted fractions, further purification was carried out by gel filtration chromatography using a HiLoad 26/60 Superdex 200 pg column (Cytiva), and the fractions containing MDP1 were again collected. The collected samples were then concentrated and dialyzed against a solution containing 8 M urea in 50 mM phosphate buffer (pH 6.8) with 300 mM NaCl. Refolding was performed by gradually reducing the urea concentration to 4 M, 2 M, 1 M, 0.5 M, and finally 0 M. The refolded protein was then used for further experiments. Approximately 7.8 mg of nMDP1-mtb was purified from 1 kg of wet bacterial cells.

2.2. Untagged recombinant eMDP1-mtb preparation

Recombinant MDP1 (eMDP1-mtb) was prepared by expressing MDP1 plasmid without any tag by inserting a TAA stop codon immediately after the MDP1 coding sequence in the pSO-ACE-HupB (Rv2986c)-His plasmid, which expresses His-tag (6 histidine tag) MDP1 in E. coli (eMDP1-mtb) [20]. This modified plasmid, designated pET-22b-MDP1(op, no-tag), was introduced into Rosetta2(DE3) pLysS. The recombinant bacteria were cultured in Luria-Bertani (LB) medium using a jar fermenter. A soluble protein fraction was prepared from the cultured bacterial cells and purified using a cation exchange column (HiPrep CM, Cytiva). The cation exchange binding buffer consisted of 25 mM phosphate buffer (pH 6.8) containing 150 mM NaCl, while the cation exchange elution buffer consisted of 50 mM phosphate buffer (pH 6.8) containing 2 M NaCl. Stepwise elution was performed, and the target protein was eluted in the fraction corresponding to 35% cation exchange elution buffer. The collected fraction was subsequently diluted three-fold with 50 mM phosphate buffer (pH 6.8) and further purified using a heparin column (HiPrep Heparin FF, Cytiva). The heparin binding buffer consisted of 50 mM phosphate buffer (pH 6.8) containing 300 mM NaCl, while the heparin elution buffer consisted of 50 mM phosphate buffer (pH 6.8) containing 2 M NaCl. Gradient elution of the target protein based on increasing salt concentration was then performed by increasing the proportion of the heparin elution buffer, resulting in the isolation of highly purified eMDP1-mtb protein. Protein refolding was carried out using urea, following the same procedure used for nMDP1-mtb, and the refolded protein was used for further observations. Approximately 1.4 mg of eMDP1-mtb was purified from 1 l of recombinant E. coli culture.

2.3. His-tagged recombinant eMDP1-mtb preparation

eMDP1-mtb with His-tag at C-terminus (eMDP1-mtb-His) was also prepared. pET-22b-MDP1(op)-His, described above, was introduced into Clear coli BL21(DE3) (Bioresearch Technologies, Hoddesdon, UK). Frozen stock of a bacterial strain harboring this plasmid was cultured in LB broth containing 50 μg/ml ampicillin at 37°C with vertical shaking (120 rpm) for 20–24 h. Then, Isopropyl β-D-1-thiogalactopyranoside (final 1 mM) was added to the culture, followed by an additional 4 h incubation at 37°C with vertical shaking (120 rpm). Finally, bacterial cells were harvested, washed, and stored at –80°C until use.

His-tag protein was purified from the frozen bacterial pellets as described previously [16]. Purity of the protein was determined by SDS-PAGE. Fractions containing His-tagged proteins were combined and dialyzed with phosphate-buffered saline (PBS [–]) for subsequent experiments. Purified proteins were then concentrated using Amicon Ultra-15 Centrifugal Filter with appropriate pore size (Merck Millipore, Burlington, MA). Protein concentrations were determined using PierceTM BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA).

2.4. Recombinant mMDP1-mtb preparation

Preparation of His-tag MDP1mtb (mMDP1-mtb) was described previously [1]. Briefly, sequences encoding MDP1 (hupB, Rv2986c) were amplified using Mtb H37Rv DNA as a template, incorporating Haemophilus influenzae restriction endonuclease D, number III (HindIII, TaKaRa Bio, Kusatsu, Shiga, Japan) and Neisseria denitrificans enzyme I (NdeI, TaKaRa Bio) restriction enzyme sites at the N-terminus and adding 6× histidine tag and Klebsiella pneumoniae restriction endonuclease N, number I (KpnI, TaKaRa Bio) restriction enzyme site at the C-terminus. The amplified DNA fragments (HindIII-NdeI-hupB-His6-KpnI) was excised with KpnI and HindIII and then inserted between KpnI and HindIII sites of E. coli-mycobacteria shuttle vector pSO246 (kanamycin (Km) resistance). The constructed plasmid was designated as pSO246-hupB-His6. DNA cassette (ACE unit) which involved the loci from acetamidase regulator gene (accession No. U63095) and acetamidase promoter region (accession No. X57175) of Mycolicibacterium smegmatis (Msm) mc²_155 genomic DNA (CP000480) in pCRII-TOPO [2,21,22] was excised with BamHI (Bacillus amyloliquefaciens H strain, enzyme I, TaKaRa Bio) and NdeI. ACE unit was inserted in pSO246-hupB-His6 which was excised with BamHI and NdeI (pSO264-ACE-hupB-His6). The self MDP1 deficient Msm [Δmdp1 (hygromycin B (Hyg) resistance)] [23] was then transformed with the constructed plasmid (pSO264-ACE-hupB-His6) and transformants selected on the 7H11-OADC agar plates containing 50 μg/ml Hyg and 10 μg/ml Km.

Msm inducibly expressing Mtb MDP1 (pSO246-ACE-hupB-His/Msm) was grown in Sakaguchi flask with Mueller HintonII media (BD, NJ) containing 0.05% (v/v) Tween 80, 50 μg/ml Hyg, and 10 μg/ml Km with shaking at 120 r/min, 37°C in a TAITEC BioShaker G•BR-300 (Tokyo, Japan). When the OD600 reached around 1.0, acetamide was added to a final concentration of 2%. After 38–40 h incubation with acetamide, cells were harvested, washed, and stored at –80°C until use.

mMDP1-mtb was purified from the frozen bacterial pellets as described previously [16]. Purity of the protein was determined by SDS-PAGE. Fractions containing mMDP1-mtb were combined and dialyzed with PBS [-] for subsequent experiments. Purified mMDP1-mtb was then concentrated using Amicon Ultra-15 Centrifugal Filter with appropriate pore size (Merck Millipore, Burlington, MA). Protein concentration was determined using PierceTM BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA).

2.5. SDS-PAGE analysis of MDP1 samples

Electrophoresis was performed using 15% polyacrylamide gels in a Mini-PROTEAN Tetra Cell system (Bio-Rad Laboratories, Hercules, CA, USA). Each lane was loaded with 100 ng of purified MDP1 protein. Following electrophoresis, the gel was stained by silver staining using 2D-Silver Stain II (Cat# 423413, Cosmo Bio Co., Ltd., Tokyo, Japan) according to the manufacturer’s instructions. Gel images were acquired using iBright1500 imaging system (Thermo Fisher Scientific, Waltham, MA, USA).

Apparent molecular weights were estimated from SDS-PAGE migration distances using a molecular weight marker. Migration distances of marker bands were measured relative to the 10 kDa reference band and fitted with a saturating exponential calibration curve. The molecular weights of the target proteins were then estimated by interpolating their migration distances using this calibration curve.

3. Observation of RNA-protein complexes by OM

OM experiments were performed to visualize MDP1-mediated RNA condensation. Poly A RNA was used at a final concentration of 54 ng/μl. Prior to imaging, RNA and protein samples were diluted in PBS [-]. For each observation, 4 μl of RNA solution and 4 μl of protein solution (final protein concentration: 5 μM) were gently mixed directly in individual wells of a CORNING 384-well imaging plate (Cat No. 3540, Corning, Corning, NY), which features an untreated polystyrene surface. The samples were imaged using a phase-contrast optical microscope (BZ-X10, Keyence, Japan), at room temperature (23–25°C). Phase-contrast images were captured using a 20× objective lens combined with 3× digital zoom. Each well was monitored continuously for a total duration of ~60 min and around 15 snapshots were taken at ~30 min and ~60 min for both conditions. Each experiment was performed in triplicate.

3.1. Data analysis of OM images

For image quantification, experiments were performed in three independent replicates for each condition. From each experiment, two representative images were selected for analysis (total n=6 images per condition). These images of TIFF-format with a resolution of 1,920×1,440 pixels corresponding to 240 μm×180 μm were analysed using ImageJ [24] to evaluate condensate properties following background and noise removal. First, Gaussian Blur (σ=3) was applied to reduce noise and smooth particle contours. Background subtraction was then performed using the rolling ball algorithm (radius=20 pixels) to enhance contrast between particles and the background. Automatic thresholding was used to binarize the images, minimizing misidentification or subjective interpretation. The images were masked, eroded 2 times, dilated 6 times, followed by 4 times erosion to refine particle boundaries.

Then, we used the command of “analyze particles” to obtain number, area, and circularity of individual condensates. The percentage area covered by condensates compared to total observation area was calculated by summing up areas of individual condensates and then dividing by whole imaging area (240 μm×180 μm), for one image. These calculations were repeated for 6 different images per condition, and then the corresponding box-and-whisker plot was generated.

To assess circularity, average circularities of individual condensates were calculated for one image. The box-and-whisker plot was generated similarly using 6 different images per condition. The area distribution of individual condensates was analysed using all obtained areas of individual condensates from 6 images, per condition.

For contrast analysis, we quantified pixel intensity of images. The same preprocessing steps were applied as above (except binarization), followed by extraction of pixel intensity values using the histogram function in ImageJ to generate intensity distributions. Background color of each image was set to 0 and the value increases with intensity.

4. Observation of RNA-protein complexes by HS-AFM

HS-AFM imaging in tapping mode was performed in solution at room temperature (24–26°C) using a custom-built HS-AFM, as described previously [16]. We used small cantilever (BL-AC10DS-A2, Olympus; spring constant, ~0.1 N/m; resonant frequency and quality factor in liquid, ~500 kHz and ~1.5, respectively). The free oscillation amplitude (A₀) was set to 1.5–2.5 nm, and the set-point amplitude was maintained at ~90% of A₀ for optimal tip-sample interaction. A small cylindrical glass stage (2 mm diameter×2 mm height, Japan Cell) was affixed to the top of a Z-scanner using nail polish, and a thin muscovite mica disc (1.5 mm diameter, 0.05 mm thickness, Furuuchi Chemical Corporation) was glued onto the stage with epoxy.

To visualize individual RNA molecules, poly A RNA was deposited onto 3-aminopropyltriethoxysilane (APTES)-modified mica prepared as described previously [25]. APTES was diluted 1:10,000 in MilliQ water, and 2 μl of the fresh solution was applied to freshly cleaved mica for 5 min. The surface was rinsed five times with 20 μl MilliQ water, followed by a final rinse with imaging buffer (10 mM Tris-HCl (pH 7.5), 20 mM NaCl, 60 mM KCl). Poly A RNA (2 μl; ~1 ng/μl in imaging buffer) was then deposited for 3 min, rinsed with 20 μl imaging buffer, and the surface was immersed in the imaging chamber containing 65 μl imaging buffer for HS-AFM imaging. APTES-modified mica was used exclusively for RNA-only imaging to reduce molecular fluctuation and enable quantification.

For time-lapse RNA-protein interaction experiments, poly A RNA was instead deposited onto freshly cleaved bare mica to allow greater molecular mobility during condensate formation. Poly A RNA (2 μl; final concentration ~2 ng/μl) was diluted in imaging buffer and incubated on the mica surface for 3 min, followed by gentle rinsing with 20 μl imaging buffer to remove unbound molecules. The surface was then immersed in the imaging chamber containing 65 μl imaging buffer, and HS-AFM imaging was initiated. Proteins (nMDP1-mtb or eMDP1-mtb with and without His-tag) were added directly to the imaging chamber to a final concentration of ~100 nM, and time-lapse imaging was performed under identical buffer conditions.

For perturbation experiments, RNA-protein condensates were first allowed to form on the mica surface. Subsequently, either 1,6-HD (final concentration ~5% w/v) or KCl (final salt concentration ~230 mM, consisting of 150 mM KCl supplemented to the buffer containing 20 mM NaCl and 60 mM KCl) was injected into the liquid pool from the side of the imaging chamber. Imaging was temporarily paused during reagent addition and resumed ~1 min later to monitor condensate morphology after perturbation. Morphological changes in condensates were recorded in real time. All experiments were conducted more than three times. The representative images and movies for each experimental condition in this study originate from a single experiment, showing sequential imaging of the observed events.

4.1. Data analysis of HS-AFM images

We analyzed HS-AFM images using UMEX, a custom-built software. As a pre-treatment of analysis, we applied a low-pass filter to eliminate spike noise and a flattening filter to level the xy-plane. The maximum height of each RNA molecule was measured by calculating the difference between the maximum height of RNA, and the average height of the substrate. For time-lapse observations under standard conditions, well-resolved individual condensates for both nMDP1-mtb and eMDP1-mtb were manually traced throughout the image sequence, and their volume and circularity were calculated.

For perturbation analysis, nMDP1-mtb condensates retained sufficiently distinct boundaries to allow individual condensates to be traced. In contrast, eMDP1-mtb formed interconnected chain-like assemblies that became increasingly dispersed after reagent addition, making reliable tracking of individual condensates difficult. Therefore, for eMDP1-mtb, condensate volume was quantified by analyzing all detectable condensate regions present within the field of view at each time point rather than tracing individual condensates. For height measurements, five condensate-containing regions were randomly selected within each field of view. The maximum height within each measurement region was determined relative to the surrounding mica surface. This approach enabled consistent comparison of condensate height changes over time while avoiding selective measurement of only the tallest structures or specific regions within the field of view. The average of the five measurements was used as the representative height value for each time point. For perturbation analysis, imaging was paused during the addition of 1,6-HD or KCl and resumed ~1 min later. Consequently, pre-treatment and post-treatment image sequences were analyzed independently. Within each sequence, the condensate volume in the first frame was assigned a value of 1, and subsequent volumes were normalized relative to this value.

All statistical analyses were performed using OriginPro 2024 (OriginLab Corporation, USA). A significance cutoff of p=0.05 was used for all tests. The normality of distributions was assessed using the Shapiro-Wilk and Kolmogorov-Smirnov tests. Comparisons were conducted using two-sample t-test to determine group-wise differences between the control (no protein) and experimental conditions (after protein addition).

Results

Mesoscale RNA condensation by MDP1 observed with OM

The modification states of MDP1 have been characterized previously [1,9], with PTMs including monomethylation of lysine residues observed in nMDP1-mtb, but they are rare in eMDP1-mtb (Figure 1). Consistent with these differences, SDS-PAGE confirmed the purity of protein preparations and revealed a clear mobility difference between nMDP1-mtb and eMDP1-mtb (Figure S1). The untagged eMDP1-mtb migrated lower than untagged nMDP1-mtb, consistent with the lower mass expected from the absence of endogenous PTMs. Introduction of a His-tag produced the expected upward shift in eMDP1-mtb, reflecting the added molecular weight of the tag. These PTM differences define the two MDP1 forms examined in this study. We note that, for OM experiments, we used the His-tagged eMDP1-mtb because it could be prepared more practically in the quantities required for imaging; the untagged protein was tested under HS-AFM only, which requires considerably smaller amounts of protein.

Figure 1.

Schematic and lysine monomethylation profile of native and recombinant MDP1. (A) Schematic representation of native (nMDP1-mtb) and recombinant (eMDP1-mtb) MDP1 illustrating the N-terminal HUR domain (red) and C-terminal intrinsically disordered region (IDR; yellow). Blue dots indicate methylated lysine residues. (B) Relative abundance of lysine monomethylation across MDP1 residues based on previously reported mass spectrometry data [1,9]. Blue and yellow bars represent nMDP1-mtb and eMDP1-mtb, respectively. Yellow bar at position 38 is shown with reduced opacity to reveal the blue bar behind it.

When mixed with RNA, nMDP1-mtb induced discrete, globular condensates (Figure 2Ai–ii), whereas eMDP1-mtb formed elongated condensates that became increasingly interconnected over time (Figure 2Aiii–iv). nMDP1-mtb condensates remained spherical over time, while eMDP1-mtb condensates progressively merged into irregular networks. Poly A RNA alone did not form visible condensates, and neither buffer, nMDP1-mtb nor eMDP1-mtb produced condensates in the absence of RNA (Figure S2), indicating that both components are required for condensate formation.

Figure 2.

OM imaging of RNA condensation induced by native and recombinant MDP1. (A) Left (both sets of images), phase-contrast images taken at ~30 or ~60 min after mixing poly A RNA and indicated MDP1 protein. Right (both sets of images), corresponding binary images for quantification (see Methods 3.1). (i–ii) nMDP1-mtb and (iii–iv) eMDP1-mtb. Time points of incubation are denoted by _30 or _60 on protein labels, scale bars, 10 μm. (B–E) Quantification of RNA-protein interactions based on three independent experiments. (B) Percentage area covered by condensates compared to total observation area. (C) Circularity of condensates. Statistical values are indicated as mean±SD (SD in brackets) from three replicates. The orange horizontal line within each box marks the median, and the cross (×) indicates the mean. p≤0.05 was considered significant; ns=not significant, (*p≤0.05, **p≤0.01, ***p≤0.001), p values are based on two sample t-test. (D) Distribution of the number and area of individual condensates at ~30 and ~60 min. Arrows mark the median area values corresponding to those described in the text. (E) Contrast intensity distributions for each experimental condition.

Quantitative analysis of OM images showed that nMDP1-mtb condensates increased in abundance and area over time, occupying ~0.6% of the observed field at ~30 min and ~1.3% at ~60 min (Figure 2B). Circularity of these condensates remained high (~0.9) throughout the observation period (Figure 2C), consistent with the maintenance of globular morphology. The area of larger condensates increased from ~10 μm² to ~20 μm² between ~30 min and ~60 min, and the median area increased from ~1.92 μm² to ~2.82 μm² over the same period (Figure 2D), consistent with gradual condensate growth.

By contrast, the area coverage of eMDP1-mtb-formed condensates increased from ~6.2% at ~30 min to ~10.4% at ~60 min (Figure 2B), while circularity decreased over time (~0.76 to ~0.72; Figure 2C), reflecting the transition to more elongated, interconnected condensates. The size distribution shifted toward larger and more irregular assemblies, with condensate areas increasing from ~40 μm² to ~90 μm² between ~30 min and ~60 min, and the median area of individual condensates increased from ~1.93 μm² to ~2.54 μm² over the same time interval, with shapes becoming larger and more irregular (Figure 2D). Brightness distributions further supported differences in condensate morphology. nMDP1-mtb condensates exhibited a broader distribution extending toward higher brightness values than eMDP1-mtb condensates (Figure 2E), indicating distinct optical properties between the two types of condensates. Together, these observations show that both forms of MDP1 reorganize poly A RNA but produce distinct mesoscale morphologies.

Nanoscale RNA condensation by MDP1 observed with HS-AFM

Next, to directly visualize RNA-protein interactions at the nanoscale, we employed HS-AFM. Unlike OM, HS-AFM enables real-time imaging of biomolecules in aqueous solution with nanometer-scale spatial resolution and has been widely used to investigate the dynamic behavior of proteins, nucleic acids, and their complexes as reviewed in [13,15,26]. This capability allows the formation, structural evolution, and dynamic interactions of individual molecules and assemblies to be followed over time. Because HS-AFM requires well-separated molecules for accurate nanoscale analysis, experiments were performed at substantially lower RNA and protein concentrations than those used for OM (see Methods 4). At higher molecular densities, neighboring structures overlap, making it difficult to distinguish individual molecules and accurately track their behavior.

All RNA-protein interaction experiments were performed on bare mica surface using 10 mM Tris-HCl (pH 7.5), 20 mM NaCl, 60 mM KCl buffer, which allowed us to observe dynamic assembly behavior. However, under these conditions, poly A RNA remained dispersed and highly fluctuating, preventing reliable structural quantification, although individual molecules could still be faintly observed (Figure 3; No protein).

Figure 3.

HS-AFM imaging of nanoscale RNA condensation induced by native and recombinant MDP1. (A–B) Time-lapse HS-AFM images of RNA condensates induced by (A) nMDP1-mtb (Movie S3) and (B) eMDP1-mtb (Movie S5) at ~20, ~40, and ~60 min. Individual condensates are encircled by white dashed lines. Top panels show low-contrast images normalized from –3 to 9 nm for direct comparison, whereas bottom panels show high-contrast renderings of the same frames normalized from –1 to 3 nm to enhance visualization. The white horizontal line in the color scale bar indicates 0 nm. All experiments were performed within a 350 nm×350 nm scanning area with 100×100 pixels. Frame rates are given in the corresponding movie captions. (C) Fusion process of nMDP1-mtb-induced RNA condensates observed ~55 min after incubation (Movie S4). White arrows indicate gaps and fusion sites, and the dashed circle highlights the fused condensate. Time points marked with “+” indicate elapsed time after 0 s; the same notation is used in subsequent figures. Images in (C) are normalized from –3 to 9 nm. All scale bars are 100 nm.

To enable clear visualization and quantitative characterization of individual RNA molecules for control comparisons, poly A RNA was additionally deposited on 3-aminopropyltriethoxysilane (APTES)-modified mica as described previously [25], which reduces molecular fluctuation through surface adsorption. Under these conditions, RNA appeared as extended but locally folded features exhibiting an apparent height of ~1.9 nm (Figure S3 and Movie S1). Volume and circularity were measured to be ~1600 nm³ and ~0.1, respectively. Because adsorption on APTES-modified mica can affect the apparent shape and extension of RNA molecules, these values were used primarily as reference measurements rather than for strict quantitative comparison with condensates observed on mica.

Upon addition of nMDP1-mtb, the protein rapidly interacted with poly A RNA (Figure S4A, Movie S2; left), leading to the immediate appearance of globular condensates that persisted throughout ~60 min of imaging (Figure 3A and Movie S3). These condensates gradually increased in size over time. Time-resolved imaging showed that larger condensates expanded at the expense of smaller ones, indicating redistribution of contents toward larger condensates (Figure 3C and Movie S4). Consistent with this growth, condensate height increased from ~7.5 nm at ~20 min to ~10 nm at ~60 min. Quantitative analysis further supported this trend, with individual condensate volume increasing from ~10,000 nm³ at ~20 min to ~33,000 nm³ by ~60 min (Figure 4A, left), while circularity remained high (~0.80; Figure 4B, left), consistent with a compact globular morphology.

Figure 4.

Quantitative analysis of HS-AFM observations of RNA condensation by native and recombinant MDP1. (A) Box plots showing the distribution of individual volumes measured from HS-AFM images of RNA alone (NP; no protein) and RNA-protein complexes at the indicated time points. (B) Box plots showing the corresponding circularity values derived from the same HS-AFM datasets. Boxes represent the interquartile range (IQR); the orange horizontal line marks the median, and the cross (×) indicates the mean. Whiskers extend to the most extreme data points within 1.5× IQR from the quartiles, and points outside this range are considered outliers. Statistical comparisons were performed using two-sample t-tests. p≤0.05 was considered significant; ns=not significant; (*p≤0.05, **p≤0.01, ***p≤0.001). Color coding follows the panel labels: blue denotes nMDP1-mtb, yellow denotes eMDP1-mtb and pink denotes poly A RNA observed on APTES-modified mica substrate.

In contrast, addition of eMDP1-mtb did not generate globular condensates with RNA. Instead, the protein bound to RNA and produced elongated, chain-like condensates decorated with protein (Figures 3B and S4B and Movie S2; right and S5). These condensates remained extended throughout the imaging period and did not undergo progressive compaction. Condensates exhibited minimal height changes, reaching only ~3 nm by ~20 min with no further increase. The volume of individual condensates increased modestly from ~5,000 nm³ at ~20 min to ~9,000 nm³ by ~60 min (Figure 4A right), while circularity remained low (~0.1; Figure 4B right). Importantly, eMDP1-mtb lacking the His-tag produced condensates with similar morphologies (Figure S5 and Movie S6), indicating that the His-tag does not substantially influence RNA condensation behavior.

This nanoscale behavior was qualitatively consistent with the OM observations: nMDP1-mtb formed compact globular condensates that grew through redistribution of contents, whereas eMDP1-mtb produced elongated condensates with limited compaction. Although similar morphologies were observed by both techniques, the dimensions of the resulting structures cannot be directly compared. OM visualized freely suspended condensates with dimensions of several micrometers, whereas HS-AFM captured surface-adsorbed assemblies on the nanometer scale, with nMDP1-mtb forming condensates typically below 100 nm and eMDP1-mtb producing more extended assemblies reaching several hundred nanometres. Because HS-AFM experiments were performed at considerably lower molecular concentrations and under surface-confined conditions, diffusion and structural rearrangement may be restricted relative to the freely suspended condensates observed by OM. Nevertheless, both OM and HS-AFM consistently revealed that PTM-enriched and PTM-deprived MDP1 promote distinct modes of higher-order RNA organization.

Physicochemical properties of MDP1-induced RNA condensates

To examine the physicochemical interactions underlying condensate stabilization, we first investigated the effects of 1,6-HD, which is commonly used to disrupt weak hydrophobic interactions [27–29]. Pre-formed RNA-protein condensates were generated prior to reagent addition, and ~5% 1,6-HD was subsequently injected into the liquid pool during HS-AFM observation. Because ~1 min elapsed between reagent addition and restart of image acquisition, the post-treatment time course described below represents the period after imaging resumed rather than the immediate response to injection.

nMDP1-mtb-induced globular condensates remained mainly intact following 1,6-HD treatment, showing only limited structural changes throughout the observation period (Figure 5A and Movie S7). Similarly, eMDP1-mtb-induced chain-like condensates remained extended and showed minimal disruption after 1,6-HD addition (Figure 5B and Movie S8). Quantitative analysis showed that absolute and normalized volumes as well as height of condensates, remained close to initial values for both forms throughout the observation period (Figures S6 and 5Ei–iv). These observations indicate that weak hydrophobic interactions were not the dominant factor governing condensate stability under these conditions.

Figure 5.

HS-AFM imaging of the response of MDP1-RNA condensates to 1,6-HD and KCl. (A–B) Time-lapse HS-AFM images before and after addition of 1,6-HD (~5%, purple arrow) to condensates formed by (A) nMDP1-mtb (Movie S7) and (B) eMDP1-mtb (Movie S8). (C–D) Time-lapse HS-AFM images before and after addition of KCl (~150 mM, green arrow) to condensates formed by (C) nMDP1-mtb (Movie S9) and (D) eMDP1-mtb (Movie S10). Images are shown at ~20, ~40, and ~60 s after restart of image acquisition. Corresponding normalized volume quantification is shown below each image series. Image contrasts in (A, C) were normalized from –3 to 9 nm and in (B, D) from –1 to 3 nm for better visualization. The white line in the color scale bar indicates 0 nm. Scale bars, 100 nm. Because ~1 min elapsed between reagent injection and restart of HS-AFM scanning, displayed time points do not represent the immediate response after addition. “Before” panels show representative condensates prior to reagent addition and are not necessarily the final frame before acquisition was stopped. For quantification, “Before” and “After” datasets were analyzed independently, each with separate 0 s reference points. (E) Time-dependent changes in normalized condensate volume following treatment with 1,6-HD (i–iv) or KCl (v–viii). Panels i, ii, v, and vi correspond to nMDP1-mtb condensates, whereas panels iii, iv, vii, and viii correspond to eMDP1-mtb condensates.

We next examined the effects of increased ionic strength by adding an additional ~150 mM KCl (final salt concentration ~230 mM, consisting of 150 mM KCl supplemented to the buffer containing 20 mM NaCl and 60 mM KCl) to pre-formed condensates. In contrast to 1,6-HD treatment, KCl progressively disrupted condensates induced by both forms. nMDP1-mtb condensates gradually lost their compact morphology after KCl addition (Figure 5C and Movie S9). Quantitative analysis showed that absolute volumes as well as height of condensates decreased progressively following KCl addition (Figure S6). The normalized volume decreased from 1.0 at the start of post-treatment imaging to ~0.77, ~0.52, and ~0.35 after ~10, ~20, and ~30 s, respectively (Figure 5Ev–vi). Similarly, eMDP1-mtb condensates became progressively dispersed following KCl treatment (Figure 5D and Movie S10), with normalized volume decreasing from 1.0 to ~0.78, ~0.46, and ~0.35 over the same period (Figures S6 and 5Evii–viii). Because HS-AFM images only surface-associated condensates, we cannot completely exclude contributions from changes in condensate attachment to the mica surface or surface rearrangement that may also influence the measured dimensions. Together, the resistance to 1,6-HD and destabilization upon increasing ionic strength indicate that electrostatic interactions are the primary contributors to condensate stabilization.

Discussion

The present study builds on our earlier demonstration that MDP1 induces RNA condensation using heterogeneous RNA substrate [19]. Although total RNA better reflects the compositional diversity of cellular RNA, its variable sequences, and secondary structures complicate the analysis of intrinsic protein-driven condensation behavior. By using poly A RNA as a uniform-sequence substrate, the present study demonstrates that the distinct condensation behaviors of nMDP1-mtb and eMDP1-mtb are intrinsic to the protein and are not dependent on RNA sequence heterogeneity.

Using this simplified RNA system, we found that nMDP1-mtb and eMDP1-mtb generate distinct higher-order morphologies: nMDP1-mtb forms compact globular condensates, whereas eMDP1-mtb produces elongated chain-like condensates (Figures 2 and 3). These behaviors were consistently observed by both OM and HS-AFM, indicating that they arise from intrinsic differences between the two protein forms rather than imaging-specific effects.

HS-AFM observations further revealed distinct nanoscale assembly dynamics (Figure 6). nMDP1-mtb condensates increased progressively in size while maintaining high circularity (Figure 3A), and larger condensates frequently grew at the expense of smaller condensates. Fusion events followed by relaxation into larger globular condensates were also observed (Figure 3C). In contrast, eMDP1-mtb formed elongated condensates with low circularity that remained dispersed throughout the observation period under the same conditions (Figure 3B). These differences in morphology and assembly dynamics were accompanied by distinct optical properties. OM analysis showed broader brightness distributions and brighter interiors for nMDP1-mtb condensates than for eMDP1-mtb condensates (Figure 2E). Together with the fusion and contents redistribution observed by HS-AFM, the higher brightness of nMDP1-mtb condensates suggests a greater degree of condensate compaction than that of eMDP1-mtb condensates.

Figure 6.

Schematic model of MDP1-RNA condensate formation and morphological response to physicochemical perturbations observed at nanoscale. Poly A RNA alone remains dispersed (grey; left). Upon addition of native MDP1 (blue) or recombinant MDP1 (yellow), distinct condensate morphologies form (right): globular condensates for nMDP1-mtb and chain-like condensates for eMDP1-mtb. Purple arrows indicate the effect of 1,6-HD, and green arrows indicate the effect of KCl, both applied to pre-formed condensates. Arrow directions represent the experimental sequence leading to each morphological outcome.

Physicochemical perturbation experiments further showed that condensates induced by both protein forms were rapidly disrupted by increased ionic strength but were comparatively resistant to 1,6-HD treatment (Figures 5 and 6). These observations suggest that electrostatic interactions make a major contribution to condensate stabilization under the present experimental conditions. This interpretation is consistent with studies showing that RNA-protein condensates are frequently stabilized through electrostatic interactions between positively charged protein regions and the negatively charged RNA backbone. Increased ionic strength weakens such interactions through electrostatic screening [30–32], whereas aliphatic alcohols such as 1,6-HD often have limited effects on condensates in which hydrophobic interactions are not the dominant stabilizing force [33,34]. The similar responses of nMDP1-mtb and eMDP1-mtb indicate that, despite their distinct morphologies, both condensate types share a common electrostatic basis of stabilization.

The major difference between the two protein forms is the presence of endogenous PTMs in nMDP1-mtb (Figure 1). These modifications are concentrated within the IDR, the region that supports dynamic multivalent intermolecular interactions governing biomolecular assembly [35,36]. Consequently, PTMs within the IDR have the potential to modulate these interactions. Previous studies have shown that PTMs such as acetylation, phosphorylation, and succinylation regulate MDP1-mediated DNA binding and compaction [3,10,37,38]. Importantly, although lysine monomethylation is the predominant modification identified in native MDP1, the protein also contains multiple additional PTMs, including di- and trimethylation, acetylation, succinylation, and phosphorylation [1,9,10]. More broadly, PTMs have been shown to remodel the conformational ensembles of intrinsically disordered proteins by shifting the balance between extended and more compact states while tuning their multivalent interaction potential and condensation behavior [39,40].

Taken together, the formation of more compact condensates by nMDP1-mtb compared with eMDP1-mtb under our experimental conditions cannot be explained solely by differences in net positive charge. Rather, the combined PTM profile may reorganize the network of weak multivalent interactions within the IDR, thereby influencing condensate architecture while maintaining electrostatic interactions as a major contributor to condensate stabilization. The distinct condensate morphologies observed here, together with the similar perturbation responses of both condensate types, suggest that PTMs primarily influence condensate morphology and assembly behavior rather than the dominant mechanism of stabilization.

MDP1 is well established as a DNA-organizing protein [3,12,16], and our previous work extended its functional scope to RNA condensation using total RNA [19]. MDP1 has also been strongly associated with bacterial dormancy and stress adaptation in Mtb [5,8,10,16]. During dormancy, nucleic acid organization undergoes substantial reorganization, and transcriptional activity is extensively remodeled [4,10,17,18]. The ability of MDP1 to induce higher-order RNA assemblies in a PTM-associated manner therefore raises the possibility that RNA condensation contributes to nucleic acid organization under metabolically reduced conditions. Although the present study was performed in vitro using a simplified RNA substrate, the observed PTM-associated differences in condensate morphology suggest that the chemical state of MDP1 could influence RNA organization during physiological adaptation in Mtb. Together, these findings suggest that the PTM state of MDP1 influences the organization of RNA condensates while preserving a common electrostatic basis of stabilization (Figure 6).

Several limitations remain. The experiments were performed in vitro using a simplified RNA substrate, and the present study does not identify which individual PTMs, or combinations of PTMs, are responsible for the observed differences in condensate morphology. Although the observed differences are consistent with an influence of endogenous PTMs, the two protein preparations were obtained from different expression systems and purified using different strategies; therefore, contributions from these experimental differences cannot be completely excluded. In addition, protein and RNA concentrations differ from those in cells, and the influence of other cellular components was not examined. Future studies will be required to determine how individual PTMs contribute to RNA condensation behavior under physiological conditions and how MDP1-mediated RNA organization operates in the cellular environment.

Conflict of interest

The authors declare no competing financial interest.

Author contributions

N.S., A.N., S.M., and N.K. designed the research; N.S., A.N., S.M., and N.K. performed the research; S.M., A.N. prepared the protein samples; N.S., K.N., H.I., K.U., and N.K. analyzed the data; N.S., and N.K. wrote the first draft, and the final draft was prepared after receiving input from all authors.

Data availability

Supporting Information is available free of charge.

Supporting images and analysis and supporting movie captions (PDF).

HS-AFM movies for RNA only, and RNA interaction with tested proteins (MP4).

The evidence data generated and/or analyzed during the current study are available from the corresponding author on reasonable request.