Journal: Communications Biology

Article Title: Polarized ATP synthase in synaptic mitochondria induced by learning and plasticity signals

doi: 10.1038/s42003-025-08963-3

Figure Lengend Snippet: a – c Labeling of memory engram and non-engram cells in the hippocampal DG region by AAV viral vectors. a Expression of EGFP protein driven by the CaMKII promoter for labeling excitatory neurons in the hippocampal DG region. mCherry protein expression driven by the Syn promoter, with loxP sequences, labels activity-dependent memory engram cells. b Overview of the TRAP technique. By combining TRAP with tamoxifen (TM) injection, active neurons within a specific time window can be fluorescently labeled. c Schematic representation of the behavioral protocol for labeling in cFos-CreER mice. d Representative confocal images showing memory engram and non-engram cells. Blue: DAPI, Red: mCherry, Green: EGFP. Scale bar: 200 μm. e , f Observation and statistical analysis of dendritic spine density in memory engram and non-engram cells. e Cells with co-localized red and green signals, along with dendrites, are classified as memory engram cells, while cells with only green fluorescence are categorized as non-engram cells. Scale bar (cell images):10 μm, Scale bar (dendrite images): 2 μm. f Quantification of spine density and spine width (n-engram cells: 21 dendritic segments, n-non-engram cells: 36 dendritic segments, from three mice). Significance: ** P < 0.01, *** P < 0.001 (spine density: Unpaired t test, spine width: Mann–Whitney U test), P = 0.007 for spine density quantification and P = 0.0026 for spine width quantification. g 3D volume view of dendrites and mitochondria in each group. Mitochondria labeled with anti-TOMM20-AF647 (validated in Supplementary Fig. ). h Quantification of mitochondrial localization within dendritic spines of memory engram and non-engram cells (n-engram: 27 dendritic segments; n-non-engram: 35 dendritic segments; from 11 brain sections/4 mice). Significance: ** P < 0.01 (Mann–Whitney U test), P = 0.0015. i Representative 3D volume renderings of dendritic spines (with/without mitochondria) in engram cells. j 3D MINFLUX imaging of ATP5a protein in mitochondria located in mCherry-positive dendrite (left) and EGFP-positive dendrite (right). The z-axis is color-coded. ATP5a was first labeled using a primary antibody, followed by secondary antibody conjugation with AF647. Scale bar (confocal images): 2 μm, Scale bar (3D MINFLUX images): 500 nm. In the box-and-whisker plot, the box represents the interquartile range (IQR, 25th–75th percentiles), divided by the median. The whiskers span the full data range, terminating at the minimum and maximum observed values, individual data points are displayed in dot plots while violin plots depict probability density distributions.

Article Snippet: Recent advancements in single-molecule localization microscopy techniques, such as MINFLUX, offer high-resolution insights into cellular structures and molecular dynamics , .

Techniques: Labeling, Expressing, Activity Assay, Injection, Fluorescence, MANN-WHITNEY, Imaging, Conjugation Assay, Whisker Assay

Journal: Communications Biology

Article Title: Polarized ATP synthase in synaptic mitochondria induced by learning and plasticity signals

doi: 10.1038/s42003-025-08963-3

Figure Lengend Snippet: a , b Representative MINFLUX imaging of ATP5a in dendritic spines of memory engram cells. a Left: mCherry-labeled dendritic spines (Gray). Middle: ATP5a (red) and synaptophysin (green). Right: line intensity profile. b Z-projection of 3D MINFLUX data for mitochondrial ATP5a in the region indicated, green arrows highlight regions of high syn intensity. Confocal scale bar, 500 nm, MINFLUX scale bar, 200 nm. c , d Representative MINFLUX imaging of ATP5a in dendritic spines of non-engram cells. c Left: EGFP-labeled dendritic spines (Gray). Middle: ATP5a (red) and synaptophysin (green). Right: line intensity profile. d Z-projection of 3D MINFLUX data for mitochondrial ATP5a in the region indicated, green arrows highlight regions of high syn intensity. Confocal scale bar, 500 nm, MINFLUX scale bar, 200 nm. e , f Analysis pipeline for correlating ATP5a MINFLUX local density with syn intensity in dendritic spines. e Regions of interest (ROIs) containing mitochondria within dendritic spines of an engram cell were selected for MINFLUX imaging. Clusters of ATP5a molecules were identified using DBSCAN-based clustering, and local densities were calculated to assess ATP5a distribution. Scale bar, 200 nm. f ATP5a local density and syn intensity data were measured using an 8-segment division centered on the mitochondrial staining centroid. Regions with the highest synaptic intensity (syn+) and the lowest synaptic intensity (syn−) were compared to analyze differences in ATP5a molecular density. g Spatial redistribution of mitochondrial ATP5a relative to synaptic contact regions in dendritic spines of memory engram cells versus non-engram cells. Statistical analysis revealed significant differences in the spatial distribution of ATP5a between these two groups. (n-memory engram = 26 spine mitochondria from 12 brain sections from nine mice; n-non-engram = 13 spine mitochondria from 6 brain sections from 4 mice). Significance: ** P , ## P < 0.01,*** P < 0.001 (Aligned Rank Transformation ANOVA, Group factor: F(1, 77) = 11.3, P = 0.0012; Region factor: F(1, 77) = 6.96, P = 0.0102; Interaction, F(1, 77) = 9.53, P = 0.0028; Bonferroni post-hoc test, P non-engram_syn– vs. non-engram_syn+ > 0.9999; P engram_syn– vs. engram_syn+ < 0.001; P engram_syn+ vs. non-engram_syn+ = 0.0034). h Proposed molecular model of ATP5a reorganization in dendritic spines of memory engram cells during learning-related processes.

Article Snippet: Recent advancements in single-molecule localization microscopy techniques, such as MINFLUX, offer high-resolution insights into cellular structures and molecular dynamics , .

Techniques: Imaging, Labeling, Staining, Transformation Assay

Journal: Communications Biology

Article Title: Polarized ATP synthase in synaptic mitochondria induced by learning and plasticity signals

doi: 10.1038/s42003-025-08963-3

Figure Lengend Snippet: a Representative images of DIV21 cultured primary cortical neurons expressing mGFP. Scale bar: 50 μm. b Mitochondria were labeled with anti-ATP5a and AF647-conjugated secondary antibodies. Presynaptic sites were labeled with anti-synaptophysin and AF555-conjugated secondary antibodies. Red: ATP5a, Green: Syn, Gray: mGFP. c Representative images showing postsynaptic mitochondria in spine synapses and shaft synapses. Line profiles show ATP5a and Syn confocal signals across the postsynaptic sites in spine and shaft synapses. Scale bar: 1 μm. d – f Reorganization of mitochondrial ATP5a in dendritic spines under Control ( d ) and cLTP ( e ) conditions. Confocal images show mitochondrial ATP5a (red) and Syn (green) in dendritic spines of cultured neurons. The white dashed line indicates the mitochondria area. The bottom panel shows z-projection of MINFLUX data for ATP5a localization. Confocal scale bar: 400 nm; MINFLUX scale bar: 200 nm. f Quantification of ATP5a local density enrichment near synaptic contact regions in Control and cLTP conditions. (n-cLTP = 51 spine mitochondria from 34 neurons/4 cultures, n-Control = 47 spine mitochondria from 32 neurons/ 5 cultures). Significance: # P < 0.05,** P < 0.01. (ART ANOVA, Group factor: F(1, 195) = 1.14, P = 0.2873; Region factor: F(1, 195) = 0.55, P = 0.4573; Interaction, F(1, 195) = 6.60, P = 0.0110; Bonferroni post hoc test, P Control_syn– vs. Control_syn+ = 0.5784; P cLTP_syn– vs. cLTP_syn+ = 0.0047; P cLTP_syn+ vs. Control_syn+ = 0.0159). g , h Distribution of mitochondrial ATP5a protein in dendritic spines at different time points after cLTP induction. g Top: confocal images showing mitochondrial ATP5a (red) and adjacent Syn (green) in dendritic spines. Bottom: z projection of MINFLUX data showing ATP5a localization in the same dendritic spines. Confocal scale bar: 400 nm; MINFLUX scale bar: 200 nm. h Quantification of local density enrichment of ATP5a near synaptic contact regions. (n-Control = 40 spine mitochondria from 27 neurons/3 cultures, n-cLTP = 22 spine mitochondria from 15 neurons/3 cultures, n-30 min = 40 spine mitochondria from 20 neurons/4 cultures, n-12 h = 28 spine mitochondria from 19 neurons/3 cultures and n-3days = 38 spine mitochondria from 25 neurons/3 cultures). Significance: * P , # P < 0.05, ** P , ## P < 0.01. (ART ANOVA, Group factor: F(4, 339) = 4.08, P = 0.0031; Region factor: F(1, 339) = 0.24, P = 0.6227; interaction, F(4, 339) = 4.57, P = 0.0013; Bonferroni post hoc test: P Ctrl_syn–vs. Ctrl_syn+ > 0.9999; P cLTP_syn–vs. cLTP_syn+ = 0.0084; P 30min_syn–vs. 30 min_syn+ = 0.0212; P 12hrs_syn–vs. 12 h_syn+ = 0.0025; P 3days_syn–vs. 3days syn+ > 0.9999; P Ctrl_syn+ vs. cLTP_syn+ = 0.0032; P Ctrl_syn+ vs. 30 min_syn+ = 0.0426; P Ctrl_syn+ vs. 12 h_syn+ = 0.0056; P Ctrl_syn+ vs. 3days_syn+ > 0.9999;).

Article Snippet: Recent advancements in single-molecule localization microscopy techniques, such as MINFLUX, offer high-resolution insights into cellular structures and molecular dynamics , .

Techniques: Cell Culture, Expressing, Labeling, Control

Journal: Communications Biology

Article Title: Polarized ATP synthase in synaptic mitochondria induced by learning and plasticity signals

doi: 10.1038/s42003-025-08963-3

Figure Lengend Snippet: a Representative 3D dual-color MINFLUX imaging of ATP5a and TOMM20 in dendritic spines under Control and cLTP conditions. Confocal images show the inner mitochondrial membrane protein ATP5a (red), outer membrane protein TOMM20 (green), and Syn (blue). MINFLUX imaging reveals the distribution of TOMM20 and ATP5a. Maximum z-projection (z-proj.) and 3D reconstructions were generated using ImageJ. Pseudo-coloring is consistent across all modalities. b , c Quantification of local density enrichment of TOMM20 ( b ) and ATP5a ( c ) near synaptic contact regions in the Control and cLTP groups. Local density calculations for dual-color imaging are provided in the Supplementary Fig. . (n-cLTP: 20 spine mitochondria from 14 neurons/3 cultures, n-Control: n = 16 spine mitochondria from 11 neurons/3 cultures) Significance: ** P , ## P < 0.01. (TOMM20:ART ANOVA, Group factor: F(1, 71) = 0.7498, P = 0.3896; Region factor: F(1, 71) = 0.9172, P = 0.3416; Interaction (Group×Region): F(1, 71) = 1.3784, P = 0.2445. Bonferroni post hoc test: P Control_syn–vs. Control_syn+ <0.9999; P cLTP_syn– vs. cLTP_syn+ = 0.5494; P cLTP_syn+ vs. Control_syn+ = 0.3472; ATP5a:ART ANOVA, Group factor: F(1, 71) = 10.0817, P = 0.0023; Region factor: F(1, 71) = 1.9934, P = 0.1625; Interaction F(1, 71) = 5.9228, P = 0.0176. Bonferroni post hoc test: P Control_syn-vs. Control_syn+ > 0.9999; P cLTP_syn–vs. cLTP_syn+ = 0.0098; P cLTP_syn+ vs. Control_syn+ = 0.0066). d Proposed model showing that mitochondrial inner membrane protein ATP5a, but not outer membrane protein TOMM20, relocates within synapses in response to cLTP induction.

Article Snippet: Recent advancements in single-molecule localization microscopy techniques, such as MINFLUX, offer high-resolution insights into cellular structures and molecular dynamics , .

Techniques: Imaging, Control, Membrane, Generated