Journal: bioRxiv

Article Title: The endo-lysosomal system drives lumen formation in a human epiblast model

doi: 10.1101/2025.08.04.668503

Figure Lengend Snippet: A) A schematic overview of apicosome formation in hPSC and classification of stages. Pre-apicosome stage occurs first as multiple PODXL vesicles (green) accumulates peri-nuclearly. PODXL vesicle traffic and fusion lead to the recruitment of EZRIN and aPKCσ (red circle) and proto-apicosome formation. Mature apicosome is formed as the proto-apicosome grows in size and acquire additional apical characteristics (e.g., microvilli (green projections), primary cilium (yellow projection), high Ca 2+ concentration (not shown)). B-E) Representative confocal images of H9 hESC harvested at 3-(B,D) and 6-(C,E) hr timepoints, which were stained with indicated markers (B and C, EE and RE components; D and E, LE/lysosome components). Two additional representative cells are shown in (EE/RE) and S3A-D (LE/lysosome). Arrowheads indicate PODXL/WGA structures that contain RAB7; arrows indicate structures that contain PODXL, RAB7 and lysotracker; asterisks indicate highly enlarged LE/lysosome compartments labeled by RAB7, LAMP1 and/or lysotracker, which lack PODXL/WGA. F) A schematic summarizing molecular characteristics of the endo-lysosomal system during the earliest stages of apicosome formation. Scale = 20µm. Blue pseudocolor indicates DNA in all images.

Article Snippet: Generation of the PODXL-APEX2 and APEX2-NES constructs has been previously described ( ). piggyBac-based DOX inducible RAB35 (Addgene#47424, 47425, 47426 (gift of Peter McPherson ( ))) and RAB7 (Addgene#12605 (gift of Richard Pagano ( )), 28048, 28049 (gift of Qing Zhong ( ))) constructs were generated by PCR amplification (forward primer: Clo-dTOPO-EGFP-fw; reverse primers: Clo_dTOPO_hRab35_rv (RAB35), Clo_dTOPO_EGFP-RAB7A-rv (RAB7)) and by subcloning the PCR products into the pENTR-dTOPO (Thermo), followed by Gateway cloning (Thermo) into the PB-TA-ERN (Addgene#80474, gift of Knut Woltjen ( )).

Techniques: Concentration Assay, Staining, Labeling

Journal: bioRxiv

Article Title: The endo-lysosomal system drives lumen formation in a human epiblast model

doi: 10.1101/2025.08.04.668503

Figure Lengend Snippet: Stained with indicated markers. Selected markers are shown. Insets indicate magnified regions in the merged images. Scale = 20µm. Blue pseudocolor indicates DNA in all images. Arrowheads indicate RAB7-labeled PODXL/WGA vesicles; asterisks indicate highly enlarged LE/lysosome compartments labeled by RAB7 and/or LAMP1. Scale = 20µm. Blue pseudocolor indicates DNA in all images.

Article Snippet: Generation of the PODXL-APEX2 and APEX2-NES constructs has been previously described ( ). piggyBac-based DOX inducible RAB35 (Addgene#47424, 47425, 47426 (gift of Peter McPherson ( ))) and RAB7 (Addgene#12605 (gift of Richard Pagano ( )), 28048, 28049 (gift of Qing Zhong ( ))) constructs were generated by PCR amplification (forward primer: Clo-dTOPO-EGFP-fw; reverse primers: Clo_dTOPO_hRab35_rv (RAB35), Clo_dTOPO_EGFP-RAB7A-rv (RAB7)) and by subcloning the PCR products into the pENTR-dTOPO (Thermo), followed by Gateway cloning (Thermo) into the PB-TA-ERN (Addgene#80474, gift of Knut Woltjen ( )).

Techniques: Staining, Labeling

Journal: bioRxiv

Article Title: The endo-lysosomal system drives lumen formation in a human epiblast model

doi: 10.1101/2025.08.04.668503

Figure Lengend Snippet: A-C) H9 hESC carrying DOX-inducible GFP-RAB35-WT transgenic construct were treated with DOX starting at 0-hr, harvested at indicated timepoints (3-hr, (A); 6-hr, (B), 20-hr, (C)), and were stained with indicated markers. Representative confocal images of these samples are shown. Arrowheads indicate RAB7-labeled PODXL vesicles. Asterisks in (A) indicate highly enlarged LE/lysosome compartments that lack PODXL. Insets indicate magnified regions in the merged images. Scale = 20µm. Blue pseudocolor indicates DNA in all images.

Article Snippet: Generation of the PODXL-APEX2 and APEX2-NES constructs has been previously described ( ). piggyBac-based DOX inducible RAB35 (Addgene#47424, 47425, 47426 (gift of Peter McPherson ( ))) and RAB7 (Addgene#12605 (gift of Richard Pagano ( )), 28048, 28049 (gift of Qing Zhong ( ))) constructs were generated by PCR amplification (forward primer: Clo-dTOPO-EGFP-fw; reverse primers: Clo_dTOPO_hRab35_rv (RAB35), Clo_dTOPO_EGFP-RAB7A-rv (RAB7)) and by subcloning the PCR products into the pENTR-dTOPO (Thermo), followed by Gateway cloning (Thermo) into the PB-TA-ERN (Addgene#80474, gift of Knut Woltjen ( )).

Techniques: Transgenic Assay, Construct, Staining, Labeling

Journal: bioRxiv

Article Title: The endo-lysosomal system drives lumen formation in a human epiblast model

doi: 10.1101/2025.08.04.668503

Figure Lengend Snippet: (A) Quantitations for 20-hr cells with no apicosome (left, pre-apicosome stage), single apicosome (middle), or more than one apicosomes (right, same as the graph shown in ) in control (unmodified H9 hESC, black), RAB35 -KO (red), and RAB35 -KO + GFP-RAB35-WT without (green) or with (purple) DOX treatment (n = 100 cells in each of the three independent experiments in each background, statistical significance based on one-way ANOVA (p < 0.05) as well as by Tuckey’s test (asterisks indicate statistical significance of selected pairwise comparisons, p < 0.05)). (B) Quantitation for the diameter at the widest point of PODXL negative LAMP1 + LE/lysosome vesicles in control (blue) as well as in RAB35 -KO (red) cells at 3-hr. Twenty pre-apicosome cells were counted for each background. Only nine PODXL negative LE/lysosome compartments were found in the KO background (among 20 total cells); twenty nine LE/lysosome compartments were found among 20 cells in the control background. Statistical significance (asterisk, p < 0.05) is based on Student’s t-test. (C,D) Representative confocal images of 20-hr control and RAB35 -KO cells stained with selected apical (PODXL, WGA, pERM), EE (RAB5, EEA1), RE (RAB11), LE/lysosome (RAB7, LAMP1) markers. Insets indicate magnified regions in the merged images. Scale = 20µm. Blue pseudocolor indicates DNA in all images.

Article Snippet: Generation of the PODXL-APEX2 and APEX2-NES constructs has been previously described ( ). piggyBac-based DOX inducible RAB35 (Addgene#47424, 47425, 47426 (gift of Peter McPherson ( ))) and RAB7 (Addgene#12605 (gift of Richard Pagano ( )), 28048, 28049 (gift of Qing Zhong ( ))) constructs were generated by PCR amplification (forward primer: Clo-dTOPO-EGFP-fw; reverse primers: Clo_dTOPO_hRab35_rv (RAB35), Clo_dTOPO_EGFP-RAB7A-rv (RAB7)) and by subcloning the PCR products into the pENTR-dTOPO (Thermo), followed by Gateway cloning (Thermo) into the PB-TA-ERN (Addgene#80474, gift of Knut Woltjen ( )).

Techniques: Control, Quantitation Assay, Staining

Journal: bioRxiv

Article Title: The endo-lysosomal system drives lumen formation in a human epiblast model

doi: 10.1101/2025.08.04.668503

Figure Lengend Snippet: A,B) Representative confocal images of 3-hr control and RAB35 -KO cells stained with indicated markers (EE and RE compartments (A); LE/lysosome compartments (B)). Asterisks in (B) indicate highly enlarged LE/lysosomes that are labeled by RAB7, LAMP1 and/or lysotracker. C) Western blot analysis of 3-hr control, RAB35 -KO #1 and RAB35 -KO #2 cells, blotted for PODXL, RAB7 and β-actin. Three independent replicates are shown for each sample group. D,E) Quantitations for normalized levels of PODXL (D) and RAB7 (E), normalized against total protein, using samples in (C). One-way ANOVA analysis showed a statistical significance in D (PODXL, p < 0.05), but not in E (RAB7, p > 0.05); asterisks indicate a statistical significance based on Tuckey’s test (p < 0.05). F) Quantitation for the number of PODXL vesicles (widest diameter larger than 2µm) in 3-hr control and RAB35 -KO cells (n = 18 cells per background, asterisk indicates a statistically significant difference based on Student’s t-test (p < 0.05)). Insets indicate magnified regions shown as merged images. Scale = 20µm. Blue pseudocolor indicates DNA in all images.

Article Snippet: Generation of the PODXL-APEX2 and APEX2-NES constructs has been previously described ( ). piggyBac-based DOX inducible RAB35 (Addgene#47424, 47425, 47426 (gift of Peter McPherson ( ))) and RAB7 (Addgene#12605 (gift of Richard Pagano ( )), 28048, 28049 (gift of Qing Zhong ( ))) constructs were generated by PCR amplification (forward primer: Clo-dTOPO-EGFP-fw; reverse primers: Clo_dTOPO_hRab35_rv (RAB35), Clo_dTOPO_EGFP-RAB7A-rv (RAB7)) and by subcloning the PCR products into the pENTR-dTOPO (Thermo), followed by Gateway cloning (Thermo) into the PB-TA-ERN (Addgene#80474, gift of Knut Woltjen ( )).

Techniques: Control, Staining, Labeling, Western Blot, Quantitation Assay

Journal: bioRxiv

Article Title: The endo-lysosomal system drives lumen formation in a human epiblast model

doi: 10.1101/2025.08.04.668503

Figure Lengend Snippet: A) Representative confocal images of RAB35 -KO cells carrying DOX-inducible GFP fused RAB35-WT,-CA (constitutively active, Q67L) and -DN (dominant negative, S22N) transgenic constructs treated with DOX for 20 hours, stained with pERM and PODXL. In the “no DOX” condition (top), DOX-untreated 20-hr RAB35 -KO + RAB35-WT cells were harvested at the 20-hr timepoint. Note that the expression of the RAB35-DN construct does not lead to single apicosome formation. B) Quantitations for 20-hr cells with more than one apicosome for indicated background (n = 100 cells in each of the three independent experiments in each background. A statistical significance of the sample group was seen based on one-way ANOVA (p < 0.05) as well as by Tuckey’s test (asterisks indicate statistical significance of selected pairwise comparisons, p < 0.05)). C-F) Representative optical section of RAB35 -KO cells carrying inducible GFP-RAB35-WT (DOX-untreated in (C); DOX-treated in (D)), -CA (E) and -DN (F) constructs, harvested at the 3-hr timepoint (with 3-hr DOX treatment) and stained with indicated markers. Note that, while seen with the expression of RAB35-WT or -CA, the expression of the RAB35-DN construct does not lead to the formation of highly enlarged RAB7/LAMP1 double positive LE/lysosomes. Asterisks indicate the highly enlarged LE/lysosome compartments (D,E). G) Quantitation for the number of PODXL vesicles (widest diameter larger than 2µm) in 3-hr cells from indicated sample group (n = 18 cells per background; numbers indicate total counted vesicles). Number of the PODXL vesicles is reduced to levels similar to controls when the RAB35-WT or -CA construct is expressed in the RAB35 -KO background, which is not seen with the expression of the RAB35-DN. A statistical significance of the sample group was seen based on one-way ANOVA (p < 0.05) as well as by Fisher’s LSD test; selected pairwise comparisons are shown (asterisks, p < 0.05; ns, p > 0.05). Insets indicate magnified regions in the merged images. Scale = 20µm. Blue pseudocolor indicates DNA in all images.

Article Snippet: Generation of the PODXL-APEX2 and APEX2-NES constructs has been previously described ( ). piggyBac-based DOX inducible RAB35 (Addgene#47424, 47425, 47426 (gift of Peter McPherson ( ))) and RAB7 (Addgene#12605 (gift of Richard Pagano ( )), 28048, 28049 (gift of Qing Zhong ( ))) constructs were generated by PCR amplification (forward primer: Clo-dTOPO-EGFP-fw; reverse primers: Clo_dTOPO_hRab35_rv (RAB35), Clo_dTOPO_EGFP-RAB7A-rv (RAB7)) and by subcloning the PCR products into the pENTR-dTOPO (Thermo), followed by Gateway cloning (Thermo) into the PB-TA-ERN (Addgene#80474, gift of Knut Woltjen ( )).

Techniques: Dominant Negative Mutation, Transgenic Assay, Construct, Staining, Expressing, Quantitation Assay

Journal: bioRxiv

Article Title: The endo-lysosomal system drives lumen formation in a human epiblast model

doi: 10.1101/2025.08.04.668503

Figure Lengend Snippet: (A) Quantitations for 20-hr cells with no apicosome (left, pre-apicosome stage), single apicosome (middle), or more than one apicosomes (right, same as the graph shown in ) in control (unmodified H9 hESC), RAB35 -KO, RAB35 -KO + GFP-RAB35-WT without DOX treatment, as well as DOX-treated RAB35 -KO + GFP-RAB35-WT, RAB35 -KO + GFP-RAB35-CA and RAB35 -KO + GFP-RAB35-DN cells (n = 100 cells in each of the three independent experiments in each background). A statistical significance of the sample group was seen based on one-way ANOVA (p < 0.05) as well as by Fisher’s LSD test; selected pairwise comparisons are shown (asterisks, p < 0.05; ns, p > 0.05)). (B) Quantitations for 20-hr cells with no apicosome (left, pre-apicosome stage), single apicosome (middle), or more than one apicosomes (right, same as the graph shown in ) in control (unmodified H9 hESC), RAB35 -KO, RAB35 -KO + GFP-RAB7-WT without DOX treatment, as well as DOX-treated RAB35 -KO + GFP-RAB7-WT, RAB35 -KO + GFP-RAB7-CA and RAB35 -KO + GFP-RAB7-DN cells (n = 100 cells in each of the three independent experiments in each background). A statistical significance of the sample group was seen based on one-way ANOVA (p < 0.05) as well as by Fisher’s LSD test; selected pairwise comparisons are shown (asterisks, p < 0.05; ns, p > 0.05)).

Article Snippet: Generation of the PODXL-APEX2 and APEX2-NES constructs has been previously described ( ). piggyBac-based DOX inducible RAB35 (Addgene#47424, 47425, 47426 (gift of Peter McPherson ( ))) and RAB7 (Addgene#12605 (gift of Richard Pagano ( )), 28048, 28049 (gift of Qing Zhong ( ))) constructs were generated by PCR amplification (forward primer: Clo-dTOPO-EGFP-fw; reverse primers: Clo_dTOPO_hRab35_rv (RAB35), Clo_dTOPO_EGFP-RAB7A-rv (RAB7)) and by subcloning the PCR products into the pENTR-dTOPO (Thermo), followed by Gateway cloning (Thermo) into the PB-TA-ERN (Addgene#80474, gift of Knut Woltjen ( )).

Techniques: Control

Journal: bioRxiv

Article Title: The endo-lysosomal system drives lumen formation in a human epiblast model

doi: 10.1101/2025.08.04.668503

Figure Lengend Snippet: A) Representative optical section of RAB35 -KO cells inducibly expressing GFP-RAB7-WT, -CA (Q67L) or -DN (S22N) construct at 20-hr (with 20-hr DOX treatment), stained with indicated markers. In the “no DOX” condition (top), DOX-untreated 20-hr RAB35 -KO + RAB7-WT cells were harvested at the 20-hr timepoint. RAB7-WT or -CA expression leads to single apicosome formation while several apicosomes are still seen with RAB7-DN expression. B) Quantitation for the percentage of cells with more than one apicosome in the indicated genetic background (n = 100 cells in each of the three independent experiments in each background). Increase in RAB7 level or activity leads to reduced multi-apicosome formation. A statistical significance of the sample group was seen based on one-way ANOVA (p < 0.05) as well as by Tuckey’s test; selected pairwise comparisons are shown (asterisks, p < 0.05; ns, p > 0.05). C-F) Representative confocal images of RAB35 -KO cells inducibly expressing GFP-RAB7-WT (minus DOX in (C), plus DOX in (D)), -CA (Q67L, (E)) or -DN (T22N, (F)) construct at 3-hr (with or without 3-hr DOX treatment), stained with indicated markers. Formation of then highly enlarged LE/lysosomal compartments (asterisks) is seen with RAB7-WT or -CA expression (D,E). G) Quantitation for the number of PODXL vesicles (widest diameter larger than 2µm) in 3-hr cells from indicated sample group (n = 18 cells per background; numbers indicate total counted vesicles). Number of the PODXL vesicles is reduced to levels similar to controls when the RAB7-WT or -CA construct (but not -DN) is expressed in the RAB35 -KO background. A statistical significance of the sample group was seen based on one-way ANOVA (p < 0.05) as well as by Fisher’s LSD test; selected pairwise comparisons are shown (asterisks, p < 0.05; ns, p > 0.05). H,I) Proposed model of apicosome formation at whole cell (H) and vesicle (I) levels. RAB35-RAB7 machinery promotes removal of excess PODXL vesicles, and contributes to single apicosome formation (H). At the earliest stages of apicosome formation (I), an EE/LE hybrid vesicle population is formed from a group of PODXL vesicles labeled with RAB35 and RAB5 by recruiting RAB7 and LAMP1. Subsequently, these hybrid vesicles shed RAB35 and RAB5, and are trafficked to an acidic LE/lysosomal compartment in a RAB35-RAB7-dependent manner, where PODXL is degraded. Much of the highly enlarged LE/lysosome compartments are resolved by the time the proto-apicosome is formed (top). Another group of RAB35/RAB5 PODXL vesicles removes RAB5 and recruits ERM proteins, which contribute to the formation of the proto-apicosome (bottom). Insets indicate magnified regions in the merged images. * = statistically significant; ns = statistically insignificant. Scale = 20µm. Blue pseudocolor indicates DNA in all images.

Article Snippet: Generation of the PODXL-APEX2 and APEX2-NES constructs has been previously described ( ). piggyBac-based DOX inducible RAB35 (Addgene#47424, 47425, 47426 (gift of Peter McPherson ( ))) and RAB7 (Addgene#12605 (gift of Richard Pagano ( )), 28048, 28049 (gift of Qing Zhong ( ))) constructs were generated by PCR amplification (forward primer: Clo-dTOPO-EGFP-fw; reverse primers: Clo_dTOPO_hRab35_rv (RAB35), Clo_dTOPO_EGFP-RAB7A-rv (RAB7)) and by subcloning the PCR products into the pENTR-dTOPO (Thermo), followed by Gateway cloning (Thermo) into the PB-TA-ERN (Addgene#80474, gift of Knut Woltjen ( )).

Techniques: Expressing, Construct, Staining, Quantitation Assay, Activity Assay, Labeling

Journal: bioRxiv

Article Title: Auto-inhibition of myoblast fusion by cyclic receptor signalling

doi: 10.1101/553420

Figure Lengend Snippet: a-b, d-e , DML-derived myocytes electroporated with MLC promoter driving expression of dominant negative (DN) variants of RAB11 (b) and RAB7 (e), together with membrane GFP (green) and nuclear mCherry (red). c,f , Column graph for a-b and d-e showing the population of electroporated myocytes containing the indicated number of nuclei relative to their controls (in %). g , A model describing the receptor complementation mechanism: prior to fusion, myocytes originating from the DML (in orange) express the ligand TGFB3, the effectors SMAD 3, & 4 and the receptor TGFBR1. They are competent to fuse. Myoblasts originating from the posterior or anterior borders of the somite (in blue) express TGFBR2. Upon fusion of myoblasts to DML-derived myocytes, membrane merging of the fusion partners allow the activation of the TGFβ pathway by a receptor heterodimer complementation mechanism (in purple), ultimately leading to a temporary inhibition of fusion of another myoblast. Return to a fusion-competent state is dependent upon RAB-mediated receptor degradation.***P<0.0001. Scale bars: 50μm.

Article Snippet: A dominant negative form of the human Rab7A (T22N) was obtained from plasmid DsRed-rab7 DN (a gift from Richard Pagano, Addgene plasmid #12662 ).

Techniques: Derivative Assay, Expressing, Dominant Negative Mutation, Activation Assay, Inhibition