follicle dpc growth medium  (PromoCell)

Journal: EMBO Reports

Article Title: Cyst-independent oocyte phagocytosis builds the female reproductive reserve in mice

doi: 10.1038/s44319-025-00663-7

Figure Lengend Snippet: ( A ) A representative time-lapse image showing the oocyte developmental dynamics in live ovaries in vitro over a period of 162 h, from c-17.5 dpc to c-PD4. Scale bar: 20 μm. ( B ) Tracing the developmental dynamics of oocytes in the time-lapse image. All oocytes were inverted to black/white (b/w), and a survival oocyte was colored cyan, while a sacrificed oocyte was colored orange to highlight their developmental dynamics. Scale bar: 20 μm. All images of the surviving oocyte development are shown in Appendix Fig. . ( C ) Representative images showing the typical behaviors of oocytes during development, including growth (arrows in lane 1), shrinkage (arrows in lane 2), degradation and deformation. Scale bar: 10 μm. ( D ) Representative images showing the filopodia formation (arrows) of oocytes during development. Green, oocytes; Red, somatic cells. Scale bar: 10 μm. ( E ) 3D static images showing that oocytes are connected to cyst-like structures at any detected time points. Green, oocyte; Red, somatic cells. Scale bar: 10 μm. ( F ) Tracing the oocyte movement in the 4D tracing images (from the time point 1 to point 2 of Fig. 2E). Showing connected oocytes moved in different directions, and most of the cyst-like structures were actually assembled by separated single oocytes in the ovaries after c-17.5 dpc. Cyan arrows: oocyte movement direction. Scale bar: 10 μm. ( G ) Quantifying the ratio of separated oocytes in total oocytes from c-17.5 dpc to c-PD1. By continuously tracing the oocyte movement in the 4D time-lapse imaging, the ratio of oocytes with separation events in total oocytes was counted every 24 h. Data were presented as the mean ± SD. n = 6 ovaries ( n > 40 oocytes per ovary). c-18.5 dpc vs. c-17.5 dpc: p value = 0.213; c-19.5 dpc vs. c-17.5 dpc: p value = 0.0003; c-PD1 vs. c-17.5 dpc: p value = 5.20E-06; c-19.5 dpc vs. c-18.5 dpc: p value = 0.0033; c-PD1 vs. c-18.5 dpc: p value = 7.60E-05; c-PD1 vs. c-19.5 dpc: p value = 0.011. ( H ) Identifying the movement of labeled oocytes in the Oct4-CreER T2 ;mTmG ovaries after low dosage of tamoxifen treatment. Only a small portion of cysts were labeled in the ovaries, with most of them breaking down into single oocytes at c-17.5 dpc. Green, oocytes; Red, somatic cells. Scale bar: 20 μm. ( I ) The ratio of single oocytes in total labeled oocytes from c-17.5 dpc to c-PD1 in ovaries with low labeling density of oocytes. Data were presented as the mean ± SD. n ≥ 6 ovaries ( n > 15 oocytes per ovary). c-18.5 dpc vs. c-17.5 dpc: p value = 3.01E-05; c-19.5 dpc vs. c-17.5 dpc: p value = 0.0001; c-PD1 vs. c-17.5 dpc: p value = 7.64E-08; c-19.5 dpc vs. c-18.5 dpc: p value = 0.0208; c-PD1 vs. c-18.5 dpc: p value = 0.0007; c-PD1 vs. c-19.5 dpc: p value = 0.608. Statistical significance was determined by unpaired one-way ANOVA tests. P (a, b) < 0.05, P (a, c) < 0.05, P (b, c) < 0.05. .

Article Snippet: In the autophagy inhibitors or Formin inhibitor treatment experiments, after 4 days of adherent culture, the Oct4-CreER T2 ;mTmG or Oct4-CreER T2 ;Rainbow ovaries at c-17.5 dpc were treated with or without autophagy signaling cascade inhibitors (5 mM 3-MA, M9281, Sigma-Aldrich; 400 nM MRT68921, S7949, Selleckchem; 0.2 nM Bafilomycin A1, HY-100558, MedChemExpress; 4 μM Chloroquine, HY-17589A, MedChemExpress) or Formin inhibitor (20 μM SMIFH2, HY-16931, MedChemExpress) for 3 or 7 days, which media was changed every other day.

Techniques: In Vitro, Imaging, Labeling

Journal: EMBO Reports

Article Title: Cyst-independent oocyte phagocytosis builds the female reproductive reserve in mice

doi: 10.1038/s44319-025-00663-7

Figure Lengend Snippet: ( A ) Evaluation of the labeling efficiency in oocytes within Oct4-CreER T2 ;mTmG ovaries at 11.5 dpc. Pregnant females carrying Oct4-CreER T2 ;mTmG fetus were treated with tamoxifen (20 mg·kg −1 BW) at 10.5 dpc (left upper). Immunofluorescent staining of oocytes using DDX4 antibody in the same sections (left bottom). Statistical analysis of oocyte labeling efficiency, determined by the ratio of GFP-positive oocytes to the total number of DDX4-positive cells. Data collected from 13 sections across four ovaries are presented as mean ± SD (right). ( B ) Showing the labeling efficiency of oocytes in the Oct4-CreER T2 ;mTmG ovaries. Showing the labeled single germ cells at 11.5 dpc and separated cysts at 13.5 dpc in the ovaries after a low dosage of Tam treatment. Scale bar: 50 μm. ( C ) Tracing the movement of labeled oocytes demonstrated majority of oocytes moving as single cells from c-17.5 dpc to c-PD1. Oocytes were displayed in inverted black/white (b/w) to highlight. Scale bar: 20 μm. ( D ) Showing the criteria for identifying single oocytes. Oocytes with any potential connections (arrows) were excluded from the counting of single cells. Scale bar: 5 μm.

Article Snippet: In the autophagy inhibitors or Formin inhibitor treatment experiments, after 4 days of adherent culture, the Oct4-CreER T2 ;mTmG or Oct4-CreER T2 ;Rainbow ovaries at c-17.5 dpc were treated with or without autophagy signaling cascade inhibitors (5 mM 3-MA, M9281, Sigma-Aldrich; 400 nM MRT68921, S7949, Selleckchem; 0.2 nM Bafilomycin A1, HY-100558, MedChemExpress; 4 μM Chloroquine, HY-17589A, MedChemExpress) or Formin inhibitor (20 μM SMIFH2, HY-16931, MedChemExpress) for 3 or 7 days, which media was changed every other day.

Techniques: Labeling, Staining

Journal: EMBO Reports

Article Title: Cyst-independent oocyte phagocytosis builds the female reproductive reserve in mice

doi: 10.1038/s44319-025-00663-7

Figure Lengend Snippet: ( A ) 3D reconstructed images of Oct4-CreER T2 ;Rainbow ovaries are shown, displaying random expressions of RFP (red) or CFP (blue) in oocyte cytoplasm. Cytoplasm exchange leads to a mixture of fluorescence in the oocyte (purple, arrow). Scale bar: 10 μm. ( B ) Analysis of oocyte fluorescence reveals an increased number of oocytes with mixed fluorescent cytoplasm (purple, arrows) from c-17.5 dpc to c-PD3. Scale bar: 10 μm. ( C ) The quantification of the ratio of purple oocytes in total oocytes from c-17.5 dpc to c-PD4, showing that the peak of cytoplasm exchanges occurs between c-PD1 to c-PD3. Data were presented as the mean ± SD. n = 4 ovaries. More than 200 oocytes were measured at each time point. ( D ) Tracing the attachment between oocytes with different fluorescence in the time-lapse imaging. No cytoplasm exchange occurs after attachments (arrowheads) between oocytes. Scale bar: 5 μm. ( E ) The reconstructed 3D images demonstrate the existence of ODs (arrowheads) surrounding the oocyte (arrow) in the Oct4-CreER T2 ;Rainbow ovaries. ( F ) The time-lapse images trace the progress of a blue surviving oocyte (arrow) absorbing red ODs (arrowheads), leading to a gradual change of cytoplasm color from blue to purple. The bottom line highlights the color change of the surviving oocyte cytoplasm. Scale bar: 10 μm. ( G ) Analyzing the absolute area of mitochondria in oocytes (red: mitochondria, green: oocytes). Showing a significantly increased average mitochondria area in oocytes at c-PD2 compared to that in oocytes at c-19.5 dpc. Scale bar: 10 μm. More than 50 oocytes were measured at each time point. Data were presented as the mean ± SD. p value = 3.21E-11. *** P < 0.001, by two-tailed unpaired Student’s t -test. ( H ) Comparing the relative density of mitochondria in oocytes and ODs (red: mitochondria, green: oocytes or ODs) at c-PD1 (left). A great variation of mitochondria density was observed in ODs but not in oocytes (right). Scale bar: 10 μm. Oocytes: n = 23; ODs: n = 107. Data were presented as the mean ± SD. .

Article Snippet: In the autophagy inhibitors or Formin inhibitor treatment experiments, after 4 days of adherent culture, the Oct4-CreER T2 ;mTmG or Oct4-CreER T2 ;Rainbow ovaries at c-17.5 dpc were treated with or without autophagy signaling cascade inhibitors (5 mM 3-MA, M9281, Sigma-Aldrich; 400 nM MRT68921, S7949, Selleckchem; 0.2 nM Bafilomycin A1, HY-100558, MedChemExpress; 4 μM Chloroquine, HY-17589A, MedChemExpress) or Formin inhibitor (20 μM SMIFH2, HY-16931, MedChemExpress) for 3 or 7 days, which media was changed every other day.

Techniques: Fluorescence, Imaging, Two Tailed Test

Journal: EMBO Reports

Article Title: Cyst-independent oocyte phagocytosis builds the female reproductive reserve in mice

doi: 10.1038/s44319-025-00663-7

Figure Lengend Snippet: ( A ) Tracing the oocyte development with or without 3-MA treatment. Normal oocyte phagocytosis and selection were observed in control ovaries, but not in the 3-MA-treated ovaries. More oocytes with a dramatically decreased size survived at the end of culture at c-PD4. Scale bar: 50 μm. ( B ) Representative images display the morphology of oocytes in 3-MA-treated ovaries and control ovaries at c-PD1. Surviving oocytes with FLs (arrows) and sacrificed oocyte-forming ODs (arrowheads) were observed in the control ovaries but not in 3-MA-treated ovaries. Scale bar: 10 μm. ( C ) Statistical analysis of the oocytes with FLs shows that only a few oocytes formed FLs in 3-MA-treated ovaries. More than 100 oocytes were measured at each time point. c-18.5 dpc: p value = 0.0007; c-19.5 dpc: p value = 0.001; c-PD1: p value = 5.8E-06; c-PD2: p value = 2.78E-06; c-PD3: p value = 2.81E-07; c-PD4: p value = 5.7E-09. ( D ) Quantification of oocyte average diameter shows significant growth retardation in oocytes from 3-MA-treated ovaries compared to those from control ovaries. More than 50 oocytes were measured at each time point. c-17.5 dpc: p value = 0.5; c-18.5 dpc: p value = 1.28E-06; c-19.5 dpc: p value = 2.37E-11; c-PD1: p value = 9.59E-24; c-PD2: p value = 2.02E-27; c-PD3: p value = 1.99E-18; c-PD4: p value = 5.79E-20. ( E ) Statistical analysis demonstrates a dramatically decreased frequency of oocyte death in 3-MA-treated ovaries compared to that in control ovaries from c-19.5 dpc to c-PD2. n = 4 ovaries in every group. c-19.5 dpc: p value = 0.001; c-PD1: p value = 4.01E-07; c-PD2: p value = 6.35E-07. ( F ) Immunofluorescent staining displays ovarian morphology at c-PD4. More oocytes survived in 3-MA-treated ovaries compared to those in control ovaries. Magnified images show ovarian follicles formed in both the 3-MA group and the control group. Red: DDX4, Green: FOXL2, Blue: Hoechst. Scale bar (top): 100 μm; Scale bar (bottom): 20 μm. ( G ) Investigation of the growth capability of oocytes derived from ovaries with or without 3-MA treatment. Although 3-MA was removed, oocytes in 3-MA-treated ovaries were unable to fully grow. Scale bar: 10 μm. ( H ) Histological analysis of ovarian development after 3-MA treatment. Normal follicle development and corpus luteum formation were observed in control ovaries during 4 weeks of in vivo development. In the 3-MA group, only a few oocytes survived after 2 weeks of transplantation, and no healthy follicles were found in the ovaries at 4 weeks after surgery. Scale bar: 200 μm. ( I ) The model of two-step oocyte selection in mammals. In the first round of cyst-dependent selection, orderly cytoplasm exchange occurs in cysts between intercellular bridges to improve the quality of selected germ cells. After cyst breakdown, the second round of selection, which is cyst-independent, occurs through intense oocyte phagocytosis. The surviving oocytes form FLs to capture organelles enriched ODs, which are derived from sacrificed oocytes, to enhance their quality. The best oocytes survive through two-step oocyte selection, constructing the ovarian reserve that supports female fertility throughout their life. The colors were inverted to black/white (b/w) in ( D ) and ( G ) to highlight oocyte morphology. Data were presented as the mean ± SD. *** P < 0.001, by two-tailed unpaired Student’s t -test. .

Article Snippet: In the autophagy inhibitors or Formin inhibitor treatment experiments, after 4 days of adherent culture, the Oct4-CreER T2 ;mTmG or Oct4-CreER T2 ;Rainbow ovaries at c-17.5 dpc were treated with or without autophagy signaling cascade inhibitors (5 mM 3-MA, M9281, Sigma-Aldrich; 400 nM MRT68921, S7949, Selleckchem; 0.2 nM Bafilomycin A1, HY-100558, MedChemExpress; 4 μM Chloroquine, HY-17589A, MedChemExpress) or Formin inhibitor (20 μM SMIFH2, HY-16931, MedChemExpress) for 3 or 7 days, which media was changed every other day.

Techniques: Selection, Control, Staining, Derivative Assay, In Vivo, Transplantation Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: Cyst-independent oocyte phagocytosis builds the female reproductive reserve in mice

doi: 10.1038/s44319-025-00663-7

Figure Lengend Snippet: ( A ) The analysis of oocyte phagocytosis and development in cultured ovaries treated from c-17.5 to c-PD4 with autophagy inhibitors (3MA, MRT68921, BafA1, and CQ). Autophagy inhibition consistently reduced sacrifice and phagocytosis at c-PD1 and increased the survival of oocytes by c-PD4. Scale bar: 20 μm. ( B ) Statistical evaluations showed that suppression of autophagy significantly diminishes oocyte sacrifice and phagocytosis at c-PD1, demonstrating a sharp decline in the ratio of surviving oocytes with FLs and a decrease in the formation of ODs from sacrificed oocytes. The analysis included four ovaries per group, evaluating more than 70 oocytes per ovary. FL: control vs. 3-MA: p value = 5.9E-09; control vs. MRT: p value = 6.7E-08; control vs. BafA1: p value = 1.9E-08; control vs. CQ: p value = 1.1E-06; 3-MA vs. MRT: p value = 0.21; 3-MA vs. BafA1: p value = 0.79; 3-MA vs. CQ: p value = 0.003; MRT vs. BafA1: p value = 0.80; MRT vs. CQ: p value = 0.22; BafA1 vs. CQ: p value = 0.05. OD: control vs. 3-MA: p value = 2.3E-10; control vs. MRT: p value = 4.1E-10; control vs. BafA1: p value = 7.2E-09; control vs. CQ: p value = 7.8E-08; 3-MA vs. MRT: p value = 0.96; 3-MA vs. BafA1: p value = 0.54; 3-MA vs. CQ: p value = 0.66; MRT vs. BafA1: p value = 0.23; MRT vs. CQ: p value = 0.94; BafA1 vs. CQ: p value = 0.07. ( C , D ) Immunofluorescent staining of the ovarian morphology ( C ) and oocyte number counting ( D ) at c-PD4 showed an increased number of surviving oocytes in ovaries treated with autophagy inhibitors. Red - DDX4; Green - FOXL2; Blue - Hoechst. Scale bar: 100 μm. Data were from at least three ovaries per group. Data were presented as the mean ± SD. control vs. 3-MA: p value = 7.25E-05; control vs. MRT: p value = 0.034; control vs. BafA1: p value = 0.011; control vs. CQ: p value = 0.0054; 3-MA vs. MRT: p value = 0.012; 3-MA vs. BafA1: p value = 0.035; 3-MA vs. CQ: p value = 0.155; MRT vs. BafA1: p value = 0.96; MRT vs. CQ: p value = 0.71; BafA1 vs. CQ: p value = 0.95. Statistical significance was determined by ANOVA tests. P (a, b) < 0.05, P (a, c) < 0.05, P (b, c) < 0.05.

Article Snippet: In the autophagy inhibitors or Formin inhibitor treatment experiments, after 4 days of adherent culture, the Oct4-CreER T2 ;mTmG or Oct4-CreER T2 ;Rainbow ovaries at c-17.5 dpc were treated with or without autophagy signaling cascade inhibitors (5 mM 3-MA, M9281, Sigma-Aldrich; 400 nM MRT68921, S7949, Selleckchem; 0.2 nM Bafilomycin A1, HY-100558, MedChemExpress; 4 μM Chloroquine, HY-17589A, MedChemExpress) or Formin inhibitor (20 μM SMIFH2, HY-16931, MedChemExpress) for 3 or 7 days, which media was changed every other day.

Techniques: Cell Culture, Inhibition, Control, Staining

Journal: Nature Communications

Article Title: Actomyosin forces trigger a conformational change in desmoplakin within desmosomes

doi: 10.1038/s41467-025-64124-4

Figure Lengend Snippet: A Schematic of desmosome structure. Extracellular regions of desmoglein-2 (Dsg2) interact to mediate adhesion, while their cytoplasmic tails bind to the plaque protein, plakoglobin (PG), plakophilin (PKP), and desmoplakin (DP). B Representative confocal image of desmosome puncta along cell-cell borders in WT cells. The desmosomes were immunolabeled for DPC (green) and Dsg2 (red). Scale bar: 50 μm. Images were taken across three biological replicates. C Representative STED image of desmosomes showing a characteristic ‘railroad track’ pattern. Scale bar: 10 μm. Images were taken across three biological replicates. Representative close-up STED images of desmosomes exhibiting the signature ‘railroad track’ pattern in D MCF7 WT cells, E MCF7 K19-KO cells, and F MCF7 K19-GFP cells. Scale bar: 500 nm. G The boxed area from the representative STED images of WT cells shows a closer look at an individual DP railroad track with Dsg2 between the two parallel DP plaques. Scale bar: 200 nm. H Line-scan analysis of DPC and Dsg2 fluorescence intensity (indicated by the dashed line in G ). I Quantification of DPC-DPC distance from WT, K19-GFP, and K19-KO cells shows that desmosomes in WT and K19-GFP cells are wider than in K19-KO cells. Number of data points ( n ) = 630 (WT), 505 (K19-KO), 716 (K19-GFP); Number of replicates ( N ) = 3. Group differences were assessed with a two-sided Kruskal–Wallis test. Post-hoc pairwise comparisons used Dunn’s test with Holm adjustment for multiple comparisons; adjusted P = 1.03E-12 (WT vs. KO), P = 1.55E-09 (K19-Rescue vs. KO), P = 0.158 (WT vs. K19-Rescue); *** P < 0.001; ns, P > 0.05. Boxplots show median, 25th and 75th percentile with whiskers reaching the last data point within 1.5× interquartile range. Data points outside this range are plotted individually as outliers. The number of data points n represents the number of line scans across the desmosomes.

Article Snippet: The following primary antibodies were used: human anti-Dsg2 (MAB947, R&D systems), rabbit anti-Dsg2 (21880-1-AP, Proteintech), rabbit anti-DPC antibody (A303-356A, Bethyl Lab), rabbit anti-DPN antibody (25318-1-AP, Proteintech), chicken anti-GFP (600-901-215, Rockland), mouse anti-K19 antibody (A53-B/A2, Santa Cruz Biotechnology), mouse anti-K8 antibody (MA1-06318, ThermoFisher), mouse anti-K18 antibody (MA1-19047, ThermoFisher), control mouse IgG (sc-2025, Santa Cruz Biotechnology), and mouse anti-actin antibody (66009-1-Ig, Proteintech).

Techniques: Immunolabeling, Fluorescence

Journal: Nature Communications

Article Title: Actomyosin forces trigger a conformational change in desmoplakin within desmosomes

doi: 10.1038/s41467-025-64124-4

Figure Lengend Snippet: A Schematic of the interactions between the desmosome keratin intermediate filaments, and actin filaments. Keratin filaments bind to the C-terminal of DP and interact with actin filaments in the cytoplasm. B Representative STED images of K19-GFP rescue cells immunolabeled for K19 (magenta), DP (green), and actin (cyan). The individual components are shown in grayscale to highlight the structural features. The merged images between K19 and DP or actin are shown at the right. Scale bar: 500 nm. The images show that K19 filaments colocalize with the DP “railroad” tracks and with cortical actin belts. Images were taken across three biological replicates. C Co-IP in WT cells performed with anti-K19 antibody or IgG control. The co-IPs show that K19 interacts with both DP and actin. Experiments were done across three biological replicates. D Representative STED images of DPC (green) at the cell border of WT cells and Blebbstatin-treated WT cells (WT+Blebb). E Quantification of desmosome widths from WT, K19-KO, and WT+Blebb cells showing that Blebbistatin treatment reduces the desmosome width to a similar level as in the K19-KO cell line. n = 630 (WT), 505 (K19-KO), 650 (WT+Blebb); N = 3. Group differences were assessed with a two-sided Kruskal–Wallis test. Post-hoc pairwise comparisons used Dunn’s test with Holm adjustment for multiple comparisons; adjusted P = 7.37E–11 (WT vs. KO), P = 1.05E-12 (WT+Blebb vs. WT), P = 0.847 (WT+Blebb vs. KO); *** P < 0.001; ns, P > 0.05. F Representative STED images of DPC (green) at the cell border of K19-KO cells and Calyculin A-treated K19-KO cells (KO+Caly). G Quantification of desmosome widths from WT, K19-KO, and KO+Caly cells showing that Calyculin A increases the desmosome width of K19-KO cells to a similar level as in the WT cell line. n = 630 (WT), 505 (K19-KO), 353 (KO+Caly); N = 3. Group differences were assessed with a two-sided Kruskal–Wallis test. Post-hoc pairwise comparisons used Dunn’s test with Holm adjustment for multiple comparisons; adjusted P = 1.08E–07 (WT vs. KO), P = 2.01E–05 (KO+Caly vs. KO), P = 0.736 (WT vs. KO+Caly); *** P < 0.001; ns, P > 0.05. All boxplots show median, 25th and 75th percentile with whiskers reaching the last data point within 1.5× interquartile range. Data points outside this range are plotted individually as outliers. The number of data points n represents the number of line scans across the desmosomes.

Article Snippet: The following primary antibodies were used: human anti-Dsg2 (MAB947, R&D systems), rabbit anti-Dsg2 (21880-1-AP, Proteintech), rabbit anti-DPC antibody (A303-356A, Bethyl Lab), rabbit anti-DPN antibody (25318-1-AP, Proteintech), chicken anti-GFP (600-901-215, Rockland), mouse anti-K19 antibody (A53-B/A2, Santa Cruz Biotechnology), mouse anti-K8 antibody (MA1-06318, ThermoFisher), mouse anti-K18 antibody (MA1-19047, ThermoFisher), control mouse IgG (sc-2025, Santa Cruz Biotechnology), and mouse anti-actin antibody (66009-1-Ig, Proteintech).

Techniques: Immunolabeling, Co-Immunoprecipitation Assay, Control

Journal: Nature Communications

Article Title: Actomyosin forces trigger a conformational change in desmoplakin within desmosomes

doi: 10.1038/s41467-025-64124-4

Figure Lengend Snippet: A Schematic of the desmosome under tension. The N-terminal region of DP transitions from a closed conformation to an open conformation. B Representative STED images of DPC (green) and Dsg2 (red) at the cell border for WT and K19-KO cells. Scale bar: 500 nm. C Representative STED images of DPN (green) and Dsg2 (red) at the cell border for WT and K19-KO cells. Scale bar: 500 nm. D Quantification of desmosome half-unit widths (Dsg2-DPC distance) from WT and K19-KO cells. n = 1260 (WT), 1010 (K19-KO); N = 3. Two-sided Mann–Whitney’s U test; P = 1.542E–66, *** P < 0.001. The distances between Dsg2 and DPC are significantly greater in the WT compared to the K19-KO cells. E Quantification of desmosome half-unit widths (Dsg2-DPN distance) from WT and K19-KO cells. n = 692 (WT), 826 (K19-KO); N = 3. Two-sided Mann–Whitney’s U test; ns, P > 0.05. The Dsg2-DPN distance is similar in both cell lines. F The mean values from the results in D and E are summarized in the bar chart to compare the DP length (DPN - DPC distance) between the WT and K19-KO cells. The DP length is 66 nm for the WT and 33 nm for the K19-KO, indicating that DP extends 33 nm in the WT cells. G The entire plakin domain structure predicted by AlphaFold2 suggests a U-shape conformation in the absence of force. Two common electrostatic interactions (R592-N744 and K444-E858) observed in all 3 MD simulations (below) are shown. H During the MD simulations, 10 to 15 hydrogen bonds were formed between the plakin domain’s long arm (SR3-4 and SR5-6) and short arm (SR7-8 and SR8-CT). I During the SMD simulations, the distance between the plakin domain’s C-terminus and N-terminus increased by 30~33 nm. This elongation correlates with the distance change experimentally measured in Fig. . J Comparison of the plakin domain structure at the start (left) and at the end (right) of the SMD simulation. The structures show that the plakin domain was elongated by the pulling force without significant secondary structure unfolding. All boxplots show median, 25th and 75th percentile with whiskers reaching the last data point within 1.5× interquartile range. Data points outside this range are plotted individually as outliers. The number of data points n represents the number of line scans across the desmosomes.

Article Snippet: The following primary antibodies were used: human anti-Dsg2 (MAB947, R&D systems), rabbit anti-Dsg2 (21880-1-AP, Proteintech), rabbit anti-DPC antibody (A303-356A, Bethyl Lab), rabbit anti-DPN antibody (25318-1-AP, Proteintech), chicken anti-GFP (600-901-215, Rockland), mouse anti-K19 antibody (A53-B/A2, Santa Cruz Biotechnology), mouse anti-K8 antibody (MA1-06318, ThermoFisher), mouse anti-K18 antibody (MA1-19047, ThermoFisher), control mouse IgG (sc-2025, Santa Cruz Biotechnology), and mouse anti-actin antibody (66009-1-Ig, Proteintech).

Techniques: Comparison

Journal: Nature Communications

Article Title: Actomyosin forces trigger a conformational change in desmoplakin within desmosomes

doi: 10.1038/s41467-025-64124-4

Figure Lengend Snippet: A Schematic of polarized CMs. Myofibrils terminate at the axial membrane. The lateral membrane runs parallel to the myofibrils. B Representative STED images of axially aligned desmosomes in CMs. The CMs were treated with DMSO (CM, i) or Blebbistatin (CM+Blebb, ii), and immunolabeled for DPC (red), Dsg2 (yellow), and F-actin (cyan). Scale bar: 3 µm. Insets display characteristic DP “railroad track” patterns at the junction. C Representative STED images of laterally aligned desmosomes in CMs treated with DMSO (CM, i) or Blebbistatin (CM+Blebb, ii). Scale bar: 3 µm. Insets display DP “railroad track” patterns. D Quantification of axially aligned desmosome widths (DPC-DPC distance) in CM and CM+Blebb cells. n = 384 (CM), 330 (CM+Blebb). Two-sided Mann–Whitney’s U test; P = 9.813E–28, *** P < 0.001. E Quantification of laterally aligned desmosome widths (DPC-DPC distance) in CM and CM+Blebb cells. n = 239 (CM), 297 (CM+Blebb). Two-sided Mann–Whitney’s U test; ns, P > 0.05. F Quantification of axially aligned desmosome widths (DPN-DPN distance) in CM and CM+Blebb cells. n = 498 (CM), 476 (CM+Blebb). Two-sided Mann–Whitney’s U test; ns, P > 0.05. G Quantification of laterally aligned desmosome widths (DPN-DPN distance) in CM and CM+Blebb cells. n = 236 (CM), 187 (CM+Blebb). Two-sided Mann–Whitney’s U test; ns, P > 0.05. Desmosomes in ( D – G ) were analyzed from CMs isolated from 20 to 24 pups total from two separate preps. H Model for force-induced DP conformational change in desmosomes. When desmosomes experience no tension, DP adopts a folded (closed) conformation. Under mechanical stress, the flexible DP plakin domain unfolds to an extended (open) conformation. All boxplots show median, 25th and 75th percentile with whiskers reaching the last data point within 1.5× interquartile range. Data points outside this range are plotted individually as outliers. The number of data points n represents the number of line scans across the desmosomes.

Article Snippet: The following primary antibodies were used: human anti-Dsg2 (MAB947, R&D systems), rabbit anti-Dsg2 (21880-1-AP, Proteintech), rabbit anti-DPC antibody (A303-356A, Bethyl Lab), rabbit anti-DPN antibody (25318-1-AP, Proteintech), chicken anti-GFP (600-901-215, Rockland), mouse anti-K19 antibody (A53-B/A2, Santa Cruz Biotechnology), mouse anti-K8 antibody (MA1-06318, ThermoFisher), mouse anti-K18 antibody (MA1-19047, ThermoFisher), control mouse IgG (sc-2025, Santa Cruz Biotechnology), and mouse anti-actin antibody (66009-1-Ig, Proteintech).

Techniques: Membrane, Immunolabeling, Isolation