Journal: Nature Communications

Article Title: Syn DLP is a dynamin-like protein of Synechocystis sp. PCC 6803 with eukaryotic features

doi: 10.1038/s41467-023-37746-9

Figure Lengend Snippet: a Analytical size exclusion chromatography of Syn DLP (black) revealed the formation of oligomeric structures that are larger than common standard proteins (standard proteins’ peak positions and molecular masses in kDa are indicated, v = void volume). Absorption values at 280 nm were normalized (0–1). b Cryo-EM micrograph of Syn DLP oligomers. The data set of 8322 micrographs was measured one time (no independent replicates). c Class averages of Syn DLP oligomers with an enlarged oligomer side view including the curvature radius (left) and focused views of side, top, and slice view. d Local resolution map and FSC curve (with auto-masking) of the Syn DLP reconstruction. e Model of a Syn DLP octamer with GD (red), BSE1-3 (purple) and stalk (blue). f Secondary structure topology plot of Syn DLP with additional α-helices of the enlarged Syn DLP GD colored in orange. g Model of a Syn DLP octamer highlighting the sequential arrangement of monomers within the oligomer. Alternating monomers are colored in green and orange, respectively. h Model of the Syn DLP monomer, including structural features. GD, BSE and stalk are colored as in ( e ). Zoomed insets show the HPRN loop with the electrostatic surface of neighboring monomers, two putative lipid-binding sites at the tip of the stalk colored in magenta and green, respectively, and conserved motifs in the nucleotide-binding site of the GD: P-loop, switch I, switch II and G4 motif colored in green, orange, cyan and pink, respectively.

Article Snippet: Syn DLP samples were prepared by applying 3.5 μl Syn DLP (~3.0 mg/mL in 5 mM MgCl 2 , 7.5 mM KCl, 20 mM HEPES pH 7.4) to glow-discharged (PELCO easiGlow Glow Discharger, Ted Pella Inc., Redding, CA, USA) Quantifoil grids (R1.2/1.3 Cu 200 mesh; Electron Microscopy Sciences, Hatfield, PA, USA).

Techniques: Size-exclusion Chromatography, Cryo-EM Sample Prep, Binding Assay

Journal: Nature Communications

Article Title: Syn DLP is a dynamin-like protein of Synechocystis sp. PCC 6803 with eukaryotic features

doi: 10.1038/s41467-023-37746-9

Figure Lengend Snippet: a A surface conservation plot showed amino acids in the Syn DLP structure that are conserved in other DLP sequences. The surface conservation plot was produced using ConSurf – . The color key ranges from 1 (cyan, variable regions) to 9 (red-violet, conserved regions). The evaluation of single residues conservation in the primary sequence is shown in Supplementary Fig. . The position of GD, BSE, and stalk are highlighted in the first side view. The oligomerization interfaces 1–3 are labeled in the second side view. b Structural side-by-side comparison of dynamin-like GDs from Syn DLP (gray) with a bacterial ( Np BDLP, PDB: 2J69, green) and a eukaryotic representative (Dyn3, PDB: 5A3F, red). The table summarizes the size of different GDs ( Syn DLP, Np BDLP (PDB: 2J69), Ms IniA (PDB: 6J73), Dyn3 (PDB: 5A3F), Drp1 (PDB: 5WP9) and MxA (PDB: 3SZR)). c Structural arrangement of α-helices in the stalk domains of Syn DLP (gray) and four other DLPs was compared by structural alignment of the domains leading to Cα-RMSD values larger than 10. The areas used for the structural alignments are highlighted in green ( Np BDLP), orange ( Ms IniA), red (Dyn3), blue (MxA) or magenta (Drp1) and the remaining structural elements not used for the alignment are colored in light gray. For details about the number of aligned residues, see Supplementary Fig. .

Article Snippet: Syn DLP samples were prepared by applying 3.5 μl Syn DLP (~3.0 mg/mL in 5 mM MgCl 2 , 7.5 mM KCl, 20 mM HEPES pH 7.4) to glow-discharged (PELCO easiGlow Glow Discharger, Ted Pella Inc., Redding, CA, USA) Quantifoil grids (R1.2/1.3 Cu 200 mesh; Electron Microscopy Sciences, Hatfield, PA, USA).

Techniques: Produced, Sequencing, Labeling, Comparison

Journal: Nature Communications

Article Title: Syn DLP is a dynamin-like protein of Synechocystis sp. PCC 6803 with eukaryotic features

doi: 10.1038/s41467-023-37746-9

Figure Lengend Snippet: a Ribbon representation of a Syn DLP monomer. C8 and C777, forming an intramolecular disulfide bridge that connects BSE1 and BSE3, are highlighted in green. BSE1, which consists of two α-helices, BSE2 and BSE3 are colored purple, gray and magenta, respectively. GD colored in red, stalk in blue. b Syn DLP was incubated with different DTT concentrations (the final concentrations after adding SDS sample buffer are given). The amount of Syn DLP with an intact disulfide bridge depended on the DTT concentration (band with intact bridge at ~180 kDa, band with reduced cysteines at ~100 kDa). M = marker. Representative gel from two independent experiments with the same result. c Analytical size exclusion chromatography of Syn DLP C777A (red) compared to Syn DLP wt (black) revealed the formation of oligomeric structures for both proteins. A 280 values were normalized (0–1) for better comparison. d CD spectra of 1 µM Syn DLP wt (black) and Syn DLP C777A (red). Mean of three measurements is shown. For comparison, the spectra were normalized (θ value at 250 nm set to 0, minimum θ set to −1). e The thermal stability of Syn DLP (black) and Syn DLP C777A (red) was monitored using CD spectroscopy. The ellipticities at 222 nm were plotted against the temperature, normalized (0–1) and fitted with an adapted Boltzmann fit (Eq. ). The fit curves are shown as lines. Mean of independent measurements ( n = 3) and error bars (S.D.) are shown. f Measured ANS-FTSA of Syn DLP wt (black) and Syn DLP C777A (red). ANS fluorescence intensities at 470 nm were plotted against the temperature and normalized (0–1) for better comparison. A temperature range capturing the transition phase was fitted with an adapted Boltzmann fit (Eq. ). Fit curves are shown as lines. The mean of three independent experiments and error bars (S.D.) are displayed. g The GTPase activity of Syn DLP wt (black) and Syn DLP C777A (red) were measured in a continuous, regenerative, coupled assay. The mean and error bars (S.D.) of three independent experiments are shown. The data points were fitted using Eq. .

Article Snippet: Syn DLP samples were prepared by applying 3.5 μl Syn DLP (~3.0 mg/mL in 5 mM MgCl 2 , 7.5 mM KCl, 20 mM HEPES pH 7.4) to glow-discharged (PELCO easiGlow Glow Discharger, Ted Pella Inc., Redding, CA, USA) Quantifoil grids (R1.2/1.3 Cu 200 mesh; Electron Microscopy Sciences, Hatfield, PA, USA).

Techniques: Incubation, Concentration Assay, Marker, Size-exclusion Chromatography, Comparison, Circular Dichroism, Fluorescence, Activity Assay

Journal: Nature Communications

Article Title: Syn DLP is a dynamin-like protein of Synechocystis sp. PCC 6803 with eukaryotic features

doi: 10.1038/s41467-023-37746-9

Figure Lengend Snippet: Kinetic parameters describing the GTPase activity of Syn DLP variants

Article Snippet: Syn DLP samples were prepared by applying 3.5 μl Syn DLP (~3.0 mg/mL in 5 mM MgCl 2 , 7.5 mM KCl, 20 mM HEPES pH 7.4) to glow-discharged (PELCO easiGlow Glow Discharger, Ted Pella Inc., Redding, CA, USA) Quantifoil grids (R1.2/1.3 Cu 200 mesh; Electron Microscopy Sciences, Hatfield, PA, USA).

Techniques: Activity Assay

Journal: Nature Communications

Article Title: Syn DLP is a dynamin-like protein of Synechocystis sp. PCC 6803 with eukaryotic features

doi: 10.1038/s41467-023-37746-9

Figure Lengend Snippet: a Purified Syn DLP HPRN-AAAA was analyzed via SDS-PAGE in the presence of 0.1 mM DTT (lane −) or 100 mM DTT (lane +). The calculated molecular mass of the protein is 93 kDa. The SDS-PAGE analysis revealed a single band at ~100 kDa (lane +) without showing further protein bands, thus the protein was ≥95% pure. M = marker. Representative gel of two independent experiments showing the same results. b CD spectra of Syn DLP wt (black) and Syn DLP HPRN-AAAA (red). The mean of three measurements is shown. The spectra were normalized (θ value at 250 nm set to 0, minimum θ set to −1) for better comparison. c Analytical size exclusion chromatography of Syn DLP wt (black) and Syn DLP HPRN-AAAA (red). The elution peak positions of the standard proteins and the corresponding molecular masses are indicated (v = void volume). A 280 values were normalized (0–1) to better compare the chromatograms. d CD spectra of Syn DLP wt (black) and Syn DLP HPRN-AAAA (red) were recorded at increasing temperatures. The ellipticities at 222 nm were plotted against the temperature, normalized (0–1) and fitted with an adapted Boltzmann fit (Eq. ). The fit curves are displayed as lines. Mean of three independent experiments and error bars (S.D.) are shown. e ANS-FTSA measurements of Syn DLP wt (black) and Syn DLP HPRN-AAAA (red). ANS fluorescence intensities at 470 nm were plotted against the temperature and normalized (minimum set to 0, main peak set to 1). The temperature range that captured the transition phase was fitted with an adapted Boltzmann fit (Eq. ). Fit curves are shown as lines. Error bars represent S.D., n = 3 (mean of independent measurements). f GTPase activity of 0.5 µM Syn DLP (black) or Syn DLP HPRN-AAAA (red). Mean of independent experiments ( n = 3) and error bars (S.D.) are shown. Data points were fitted using the Michaelis-Menten equation (Eq. ). The fit curves are displayed as lines.

Article Snippet: Syn DLP samples were prepared by applying 3.5 μl Syn DLP (~3.0 mg/mL in 5 mM MgCl 2 , 7.5 mM KCl, 20 mM HEPES pH 7.4) to glow-discharged (PELCO easiGlow Glow Discharger, Ted Pella Inc., Redding, CA, USA) Quantifoil grids (R1.2/1.3 Cu 200 mesh; Electron Microscopy Sciences, Hatfield, PA, USA).

Techniques: Purification, SDS Page, Marker, Circular Dichroism, Comparison, Size-exclusion Chromatography, Fluorescence, Activity Assay

Journal: Nature Communications

Article Title: Syn DLP is a dynamin-like protein of Synechocystis sp. PCC 6803 with eukaryotic features

doi: 10.1038/s41467-023-37746-9

Figure Lengend Snippet: a Fluorescence spectroscopy using Laurdan as a fluorescent probe. LUVs were prepared with 50% DOPC and 50% of the indicated TM lipid ( w / w ) mixed with Laurdan at a 1:500 molar ratio. Laurdan fluorescence emission was measured after 30 min incubation of Syn DLP and LUVs and the ΔGP value was calculated from the spectra. Mean of three independent experiments (single measurements shown as circles) and error bars (S.D.) are shown. ns = not significant ( P > 0.05), * P < 0.05, ** P < 0.01, *** P < 0.001 based on a two-sided unpaired Student’s t-test. The ΔGP values of MGDG ( P = 0.52), SQDG ( P = 0.055), DOPG ( P = 0.00028) or DGDG ( P = 0.14) are compared to DOPC. Arrow indicates increasing membrane interaction. b LUVs were prepared with different DOPG/DOPC molar ratios (Laurdan added at a 1:500 molar ratio). Laurdan fluorescence spectra were recorded after 30 min and ΔGP values (black) were determined from the spectra. Mean of independent measurements ( n = 4) and error bars (S.D.) are shown. Arrow indicates increasing membrane interaction. c Progression of the surface pressure (black) measured over time in parallel to the SFG intensity shown in ( d ). SFG spectroscopy of 0.5 µM Syn DLP was carried out on a DMPG monolayer under reaction buffer conditions (without DTT). t = 0 s corresponds to the moment of Syn DLP addition. Mean of three independent measurements and error bars (S.D.) are shown. d The SFG spectrum in the amide I region of the DMPG monolayer (black) and after Syn DLP addition and equilibration (red) is shown. A fit of the amide I band is displayed as a line. The mean of three independent measurements is shown. SFG intensity was normalized (0–1) for better comparison.

Article Snippet: Syn DLP samples were prepared by applying 3.5 μl Syn DLP (~3.0 mg/mL in 5 mM MgCl 2 , 7.5 mM KCl, 20 mM HEPES pH 7.4) to glow-discharged (PELCO easiGlow Glow Discharger, Ted Pella Inc., Redding, CA, USA) Quantifoil grids (R1.2/1.3 Cu 200 mesh; Electron Microscopy Sciences, Hatfield, PA, USA).

Techniques: Fluorescence, Spectroscopy, Incubation, Membrane, Comparison

Journal: Nature Communications

Article Title: Syn DLP is a dynamin-like protein of Synechocystis sp. PCC 6803 with eukaryotic features

doi: 10.1038/s41467-023-37746-9

Figure Lengend Snippet: Syn DLP GTPase activities in the presence and absence of LUVs were determined in a continuous, regenerative, coupled assay. Comparison of Syn DLP under standard measurement conditions (black) and with 50 µM DOPG LUVs (dissolved in reaction buffer, extrusion to 100 nm) added with the assay components (red). Mean of independent experiments ( n = 3) and error bars (S.D.) are shown. Data points were fitted using the Michaelis-Menten equation (Eq. ). Fit curves are shown as lines.

Article Snippet: Syn DLP samples were prepared by applying 3.5 μl Syn DLP (~3.0 mg/mL in 5 mM MgCl 2 , 7.5 mM KCl, 20 mM HEPES pH 7.4) to glow-discharged (PELCO easiGlow Glow Discharger, Ted Pella Inc., Redding, CA, USA) Quantifoil grids (R1.2/1.3 Cu 200 mesh; Electron Microscopy Sciences, Hatfield, PA, USA).

Techniques: Comparison

Journal: Nature Communications

Article Title: Syn DLP is a dynamin-like protein of Synechocystis sp. PCC 6803 with eukaryotic features

doi: 10.1038/s41467-023-37746-9

Figure Lengend Snippet: a Size distribution of protein and LUVs was analyzed using DLS and relative intensities of the respective sizes are shown. MGDG/DOPG (60%/40%, w / w ) LUVs (black), Syn DLP (red) or a mixture of LUVs and Syn DLP (blue) were incubated in reaction buffer and measured. From each condition the size distribution of three independent measurements (+ three technical replicates of each measurement) are shown. b Fusion of MGDG/DOPG (60%/40%, w / w ) LUVs was measured in the presence of increasing Syn DLP concentrations using a FRET-based fusion assay. Curves showing the donor fluorescence over the first 100 s after mixing LUVs with protein. The relative fusion activities were calculated as described in the methods section. The curves represent the mean of three independent measurements. The whole measurement as well as the positive control containing 2 µM of the fusogenic protein IM30 are shown in Supplementary Fig. . c Initial fusion rates (black) were calculated from the curves in ( b ) defined as the slope of a linear regression of the first 20 s. Mean of independent measurements ( n = 3) and error bars (S.D.) are shown.

Article Snippet: Syn DLP samples were prepared by applying 3.5 μl Syn DLP (~3.0 mg/mL in 5 mM MgCl 2 , 7.5 mM KCl, 20 mM HEPES pH 7.4) to glow-discharged (PELCO easiGlow Glow Discharger, Ted Pella Inc., Redding, CA, USA) Quantifoil grids (R1.2/1.3 Cu 200 mesh; Electron Microscopy Sciences, Hatfield, PA, USA).

Techniques: Incubation, Single Vesicle Fusion Assay, Fluorescence, Positive Control

Journal: Nature Communications

Article Title: Syn DLP is a dynamin-like protein of Synechocystis sp. PCC 6803 with eukaryotic features

doi: 10.1038/s41467-023-37746-9

Figure Lengend Snippet: Transition temperatures of Syn DLP variants determined via CD spectroscopy and ANS-FTSA

Article Snippet: Syn DLP samples were prepared by applying 3.5 μl Syn DLP (~3.0 mg/mL in 5 mM MgCl 2 , 7.5 mM KCl, 20 mM HEPES pH 7.4) to glow-discharged (PELCO easiGlow Glow Discharger, Ted Pella Inc., Redding, CA, USA) Quantifoil grids (R1.2/1.3 Cu 200 mesh; Electron Microscopy Sciences, Hatfield, PA, USA).

Techniques: Circular Dichroism

Journal: Stem Cell Research & Therapy

Article Title: Modulation of senescent Lepr + skeletal stem cells via suppression of leptin-induced STAT3‒FGF7 axis activation alleviates abnormal subchondral bone remodeling and osteoarthritis progression

doi: 10.1186/s13287-025-04342-1

Figure Lengend Snippet: Bone stromal cells, particularly SSCs, are significant markers of senescence in osteoarthritis. A , C , E Representative images of P21 ( A ), IL-1β ( C ), and IL-6 ( E ) IHC in articular cartilage and subchondral bone at different points in the progression of OA. Scale bar, 100 μm. B , D , F Quantitative analysis of the P21 ( B ), IL-1β ( D ), and IL-6 ( F ) areas in articular cartilage and subchondral bone (n = 3). G IF staining of Lepr and P21 in mouse subchondral bones in various treatment groups. The white arrows indicate P21-positive cells. Scale bar, 100 μm. H Proportion of Lepr + P21 + cells among Lepr + cells (n = 3). I IF staining of Grem1 and P21 in the subchondral bones of mice in various treatment groups. The white arrows indicate the double-positive cells. Scale bar, 100 μm. J Proportion of Grem1 + P21 + cells among Grem1 + cells (n = 3). K , L Representative images of P21 ( K ) and IL-6 ( L ) IHC in the articular cartilage and subchondral bone of OA human samples. The N side represents the less severely diseased side of the source joint, whereas the O side represented the more severely diseased side of the joint. Scale bar, 100 μm. M , N Quantitative analysis of P21 ( M ) and IL-6 ( N ) levels in articular cartilage and subchondral bone (n = 3). Quantitative analyses were conducted using the IHC Profiler plug-in for Image J. All data are presented as the mean ± S.D. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant

Article Snippet: The following primary and secondary antibodies were used: Lepr (Proteintech, Wuhan, China; 20,966-1-AP, 1:100), Grem1 (Affinity, Cincinnati, OH, USA; DF15419, 1:100), P21 (Proteintech, Wuhan, China; 10,355-1-AP, 1:100), HRP-labeled goat anti-rabbit IgG (Servicebio, Wuhan, China; GB23303), and Alexa Fluor® 488-labeled goat anti-rabbit IgG (Servicebio, Wuhan, China; GB25303).

Techniques: Staining

Journal: Stem Cell Research & Therapy

Article Title: Modulation of senescent Lepr + skeletal stem cells via suppression of leptin-induced STAT3‒FGF7 axis activation alleviates abnormal subchondral bone remodeling and osteoarthritis progression

doi: 10.1186/s13287-025-04342-1

Figure Lengend Snippet: Inhibition of the leptin-Lepr signaling pathway mitigated SSC senescence and OA progression. A GO enrichment of pathways associated with significantly upregulated genes (p.adjust < 0.05) in the normal versus OA 8W groups. B GSEA enrichment of pathways associated with significantly upregulated genes (p.adjust < 0.05) in the normal versus OA 8W groups. C , D mRNA levels of leptin in osteochondral tissues from human ( C ) and OA mice ( D ) samples. The N side represents the less severely diseased side of the source joint, whereas the O side represents the more severely diseased side of the joint. E Schematic of the experimental design. The mice were subjected to ACLT surgery and received intra-articular treatment (n = 3). F , G Representative images of safranin O/Fast Green staining and OARSI grades of the mice that received different treatments at 8 weeks after ALCT surgery. Scale bars, 100 μm. H Representative images of COL II IHC in the articular cartilage and subchondral bone of mice that received different treatments at 8 weeks after ALCT surgery. Scale bar, 100 μm. I Quantitative analysis of the COL II area in articular cartilage and subchondral bone. J IF staining of Grem1 and P21 in the subchondral bones of mice that received different treatments at 8 weeks after ALCT surgery. The white arrows indicate P21-positive cells. Scale bar, 100 μm. K Proportion of Grem1 + P21 + cells among Grem1 + cells. L Micro-CT scans of knee joints from various treatment groups after ALCT surgery. M , N Microarchitectures of tibial subchondral bones showing BV/TV ( M ) and SBP.th ( N ). Quantitative analyses were conducted using the IHC Profiler plug-in for ImageJ. All data are presented as the mean ± S.D. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant

Article Snippet: The following primary and secondary antibodies were used: Lepr (Proteintech, Wuhan, China; 20,966-1-AP, 1:100), Grem1 (Affinity, Cincinnati, OH, USA; DF15419, 1:100), P21 (Proteintech, Wuhan, China; 10,355-1-AP, 1:100), HRP-labeled goat anti-rabbit IgG (Servicebio, Wuhan, China; GB23303), and Alexa Fluor® 488-labeled goat anti-rabbit IgG (Servicebio, Wuhan, China; GB25303).

Techniques: Inhibition, Staining, Micro-CT