EMBO/soda-nel huggingface dataset:
Mfn2 localization in the ER is necessary for its bioenergetic function and neuritic development (2021)
Figure 2

Figure 2. Mfn2 KO cells show alterations in ER and mitochondrial Ca2+ that may mediate impaired bioenergetic function.

(A, B) (A) Representative images of WT and Mfn2 KO MEFs co-transfected with plasmids encoding mitochondria-targeted RFP and ER-targeted GFP. Scale bar: 5 μm. (B) After 48 h, the cells were fixed and the degree of colocalization between ER and mitochondria based on red and green fluorescent pixel overlap was analyzed using Mander's coefficient (n= 30 cells analyzed in 3 independent experiments). Data are presented as mean ± SEM. *p<0.05, two-tailed Student's t-test.

(C-E) (C) Representative EM micrographs of WT and Mfn2 KO cells. Scale bar: 250 nm. (D) Average of ER-mitochondria distance (n= 111-168 mitochondria). *p<0.05, Mann-Whitney test. Data are presented as mean ± SEM. (E) Quantification of the percentage of mitochondria in contact with the ER distributed at the indicated distance range (n= 3 independent experiments in which were analyzed 627-779 mitochondria from 68-98 cells). Data are presented as mean ± SEM. *p<0.05, two-tailed Student's t-test.

F) WT and Mfn2 KO MEFs were co-transfected with GFP and ERMITO-RLuc plasmids. After 24 h, the transfected cells were sorted and plated for another 24 h, after which RLuc was analyzed, the activity of which requires the proper apposition of the ER and mitochondria to reconstitute the split RLuc (n= 3 independent experiments). Data are presented as mean ± SEM. *p<0.05, two-tailed Student's t-test.

G) Determination of mitochondrial Ca2+. Fluorescence was recorded every second in WT and Mfn2 KO MEFs that were loaded with Rhod2. Mitochondrial Ca2+ was released with the addition of the uncoupler CCCP (10 μM) as indicated (n= 40 cells from 4 independent experiments).Data are presented as mean ± SEM.

H) Determination of ER Ca2+. WT and Mfn2 KO MEFs were transfected with plasmid encoding the ER-located Ca2+ sensor LAR-GECO1. After 48 h, fluorescence was recorded every 3 seconds. ER Ca2+ release was induced with caffeine (20 mM) as indicated (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM.

I-K) Mitochondrial Ca2+ was determined in WT and Mfn2 KO MEFs loaded with Rhod2. The effect of (I) caffeine-induced (20 mM), (J) IP3R activation with ATP-induced (100 μM) and (K) histamine-induced (100 μM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM.

(L, M) (L) MMP potential was measured in WT and Mfn2 KO cells treated with vehicle or methyl-pyruvate (5 mM) for 24 h (n= 3 independent experiments). *p<0.05, one-way ANOVA followed by Tukey's post hoc test. (M) Effect of methyl-pyruvate in mitochondrial Ca2+ uptake after caffeine stimulation (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM.

Data information: Note that experiments in figures 2D and 3C, and 2E and 3B were performed at the same time, so the values of WT and Mfn2 KO are the same but the figures have been split in two for the sake of linearity.
Mfn2 localization in the ER is necessary for its bioenergetic function and neuritic development (2021)
Figure 4

Figure 4. ER located Mfn2 is necessary to maintain mitochondrial bioenergetics.

WT and Mfn2 KO MEFs were co-transfected with plasmids encoding:

A) mt-RFP and ER-GFP and ER-targeted Mfn2 (ER-Mfn2) or a control plasmid (CT). Mitochondria-ER colocalization was analyzed using Mander's coefficient (n= 15 cells analyzed in 3 independent experiments). Data are presented as mean ± SEM.

B) GFP and ERMITO-RLuc and ER-Mfn2 or control plasmid. After 24 h, the transfected cells were sorted and plated for another 24 h, after which RLuc was analyzed (n= 4 independent experiments). Data are presented as mean ± SEM.

C-F) mt-RFP and ER-Mfn2 or control (CT) plasmid. After 48 h, the cells were fixed. (C) Representative images. Scale bar= 5 μm. (D) Mitochondrial morphology was analysed (n=113-422 cells analyzed in 3 independent experiments) and (E) aspect ratio and (F) circularity calculated (n=360 mitochondria analyzed in 3 independent experiments). Data are presented as mean ± SEM.

G) GFP and ER-Mfn2 or control (CT) plasmid. Mitochondrial Ca2+ was determined. Mitochondria were uncoupled with CCCP (10 μM) as indicated (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM

H) LAR-GECO1 and ER-Mfn2 or control (CT) plasmid. ER Ca2+ levels were determined. ER Ca2+ release was induced with caffeine (20 mM) as indicated (n= 35 cells from 4 independent experiments). Data are presented as mean ± SEM

I) GFP and ER-Mfn2 or control (CT) plasmid. The effect of caffeine-induced (20 mM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM

J-N) GFP and ER-Mfn2 or control (CT) plasmid. (J) MMP was determined. The values were normalized to surrounding untransfected cells. (n= 90 cells analyzed in 3 independent experiments). (K) After 24 h, the transfected cells were sorted and plated for another 24 h, after which cells were treated with 2-DG (10 mM) for 6 h, and ATP levels were measured (n= 3 independent experiments). (L) Oxygen consumption was measured and (M) RCR and (N) SRC was calculated (n= 4 independent experiments). Data are presented as mean ± SEM.

Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. Note that experiments in figures 4A and Appendix Fig. S4E, 4D and Appendix Fig. S4B, 4K and 3I, 4L and 3J, 4M and 3K, 4N and 3L, 4G and Appendix Fig. S4G, 4H and Appendix Fig. S4H, and 4J and Appendix Fig. S4J were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.
Mfn2 localization in the ER is necessary for its bioenergetic function and neuritic development (2021)
Figure 5

Figure 5. ER located Mfn2 and mitochondrial Mfns are necessary to maintain mitochondrial bioenergetics.

WT and Mfn1/2 double KO were transfected with:

A) mt-RFP, ER-GFP and plasmids expressing the indicated proteins. Mitochondria-ER colocalization was analyzed using Mander's coefficient (n= 15 cells analyzed in 3 independent experiments). Data are presented as mean ± SEM.

B) GFP and ERMITO-RLuc and indicated expression vectors. After 24 h, the transfected cells were sorted and plated for another 24 h, after which RLuc was analyzed (n= 3 independent experiments). Data are presented as mean ± SEM.

C) GFP and the indicated plasmids. After 48 h, mitochondrial Ca2+ was determined (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM.

D) LAR-GECO1 and the indicated plasmids. After 48 h ER Ca2+ was released with caffeine (20 mM) as indicated (n= 25-27 cells from 3 independent experiments). Data are presented as mean ± SEM.

E) GFP and the indicated plasmid. After 48 h, the effect of caffeine-induced (20 mM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 25 cells from 3 independent experiments). Data are presented as mean ± SEM.

F-J) GFP and the indicated plasmids. (F) After 48 h, mitochondrial membrane potential was determined by measuring TMRM fluorescence. The values were normalized to surrounding untransfected cells. (n= 90 cells analyzed in 3 independent experiments). (G-I) After 24 h, the transfected cells were sorted and plated for another 24 h. (G) Cells were treated with 2-DG (10 mM) for 6 h, and ATP levels were measured (n= 3 independent experiments). (H) Oxygen consumption was measured and (I) RCR and (J) SRC were calculated (n= 3 independent experiments). Data are presented as mean ± SEM.

Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments in Figures 5A and Appendix S5B, 5C and Appendix S5G, and 5D and Appendix Fig S5H were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.
Mfn2 localization in the ER is necessary for its bioenergetic function and neuritic development (2021)
Figure 6

Figure 6. Mfn2 mediated ER-mitochondria tethering is necessary for neurite outgrowth.

A) Primary cortical neurons were transfected 48 h before the indicated day in vitro (DIV) with mt-RFP and ER-GFP, fixed at the indicated DIV, and mitochondria-ER colocalization was analyzed using Mander's coefficient (n= 21 neurons were analyzed in 3 independent experiments). Data are presented as mean ± SEM.

B) Representative Western blots of the indicated proteins during the neuronal maturation in vitro (n= 3 independent experiments).

C) Representative Western blots of the indicated proteins of WT and Mfn2 KO neurons at DIV7 (n= 3 independent experiments).

D) Primary neurons of tamoxifen-inducible Mfn2 KO mice were transfected at DIV3 with ER-GFP, mt-RFP plus the indicated plasmids. Tamoxifen-treated (for 72 h) and vehicle-treated neurons (WT; red dashed line) were fixed and mitochondria-ER co-localization was analyzed using Mander's coefficient (n= 15 neurons analyzed in 3 independent experiments). Data are presented as mean ± SEM.

(E-G) E) Sholl analysis and (F) neurite length measurement and representative tracings (G) of primary cortical neurons of WT and Mfn2 KO mice co-transfected at DIV4 with GFP and the indicated plasmids. After 72 h, the neurons were fixed, immunofluorescence with anti-GFP antibodies was performed and neurite length was measured (n= 30 neurons from 3 independent experiments). Data are presented as mean ± SEM.

Data information: *p<0.05 versus WT; #p<0.05 versus Mfn2 KO neurons, one-way ANOVA followed by Tukey's post-hoc test. Scale bar: 500 μm.

Expanded View Figure legends
Myf6/MRF4 is a Myogenic Niche Regulator Required for the Maintenance of the Muscle Stem Cell Pool (2020)
Figure 1

Figure 1. Genome-Wide Binding Sites of Myf6/MRF4 in Primary Myotubes. A Heat map from RNA-Seq data from FACS-isolated muscle stem cells and single myofibers showing relative expression of Pax7, Myf5, MyoD, Myog and Myf6. B Pie chart showing the distribution of Myf6 ChIP-Seq peaks within various genomic features. C Motif analysis using MEME suite (Bailey et al., 2009) identified canonical E-box motif as the most highly enriched sequence among all sequences under Myf6 peaks, followed by the Mef2a DNA binding motif. D Spatial distribution of transcription factor binding motifs within Myf6 ChIP-Seq peaks using CentriMo available via MEME suite (Bailey et al., 2009). E Normalized distribution of E-box motifs that occur within Myf6 peaks compared to their occurrences in the genome normalized per kilo base of DNA (two-tailed t-test). F Gene Ontology (GO Molecular Function) analysis of genes associated with Myf6 peaks based on association by proximity (single gene within 100kb of peaks) analyzed by Genomic Regions Enrichment of Annotations Tool (GREAT) (McLean et al., 2010). G Gene Ontology (GO Biological Process) analysis of genes associated with Myf6 peaks based on proximity similar to the analysis in "f". H Colormap of Myf5, MyoD and Myf6 peaks within 100kb of the Transcription Start Sites (TSS) of myokines (supplemental table EV3) ranging from zero (black) to six peaks (red) occupancy. The onset of differentiation coincides with increased binding of MRFs to the regulatory domains of the myokines genes.
Myf6/MRF4 is a Myogenic Niche Regulator Required for the Maintenance of the Muscle Stem Cell Pool (2020)
Figure 4

Figure 4. Genetic Deletion of Myf6 Leads to Depletion of the Muscle Stem Cell Pool. A Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at postnatal day 7. Arrowheads indicate PAX7+ satellite cells. Scale bar=200μm B Quantification of the number of PAX7+ cells per unit area (mm2) in TA muscle cross-sections from Myf6-KO and WT counterparts at postnatal day 7. (n=30 fields of view from n=3 animals), two-tailed t-test, error bars = +/- SD. C Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at postnatal day 21. Arrowheads indicate PAX7+ satellite cells. Scale bar=200μm D Quantification of the number of PAX7+ cells per unit area (mm2) in TA muscle cross-sections from Myf6-KO and WT counterparts at postnatal day 21. (n=27 fields of view from n=3 animals) , two tailed t-test, error bars = +/- SD). E Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at 3 months of age. Arrowheads indicate PAX7+ satellite cells. Scale bar=200μm F Quantification of the number of PAX7+ cells per unit area (mm2) in TA muscle cross-sections from Myf6-KO and WT counterparts at 3 months of age. (n=52 fields of view from n=5 animals), two tailed t-test, error bars = +/- SD. G Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at 10 months of age. Arrowheads indicate PAX7+ satellite cells. Scale bar=200μm H Quantification of the number of PAX7+ cells per unit area (mm2) in TA muscle cross-sections from Myf6-KO and WT counterparts at 10 months of age. (n=60 fields of view from n=3 animals), two tailed t-test, error bars = +/- SD. I Immunofluorescent analysis of myofiber-associated MuSCs isolated from the EDL muscles of 3 month-old Myf6-KO and WT counterparts. Fibers were stained with PAX7 antibody and counterstained with DAPI at time T=0 hours post fiber isolation. Scale bar=25μm J Quantification of the number of MuSCs per fiber between 3 month-old Myf6-KO (n=41 fibers from n=3 mice) and WT (n=31 fibers from n=3 mice) counterparts. Two-tailed t-test, error bars = +/- SD K Immunofluorescent analysis of myofiber-associated MuSCs isolated from the EDL muscles of 10 month-old Myf6-KO and WT counterparts. Fibers were stained with PAX7 antibody and counterstained with DAPI at time T=0 hours post fiber isolation. Scale bar=25μm L Quantification of the number of MuSCs per fiber between 10 month-old Myf6-KO (n=38 fibers from n=2 mice) and WT (n=67 fibers from n=3 mice) counterparts. Two-tailed t-test, error bars = +/- SD
LRRK2 and Rab10 coordinate macropinocytosis to mediate immunological responses in phagocytes (2020)
Figure 2

Figure 2. Rab10 is recruited to early plasma-membrane PIP3/ PI(3,4)P2 macropinsomes.

A. Raw 264.7 macrophage cells were transfected with FLAG(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain (PI(3,4,5)P3 marker), or eGFP(C-term)-SARA-Fyve domain (PI3P marker), or eGFP(C-term)-PLC-PH domain (PI(4,5)P2 marker), or mkate2(N-term)-2xTAPP1-PH domain (PI(3,4)P2 marker). Representative photomicrographs (from 25 images analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells immuno-stained for FLAG-tag (Rab10, shown as green) together with eGFP epifluorescence (shown as red). White bounding boxes show "High Mag" panels that magnify representative individual vesicles. The colocalization of Rab10 and indicated markers in the bounding boxes were analyzed using Image J as described in the method and shown in the "CoLoc" panels. The colocalized pixels are shown in white with the same intensity. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate representative Rab10 vesicles positively colocalized with the indicated marker.

B. At least 25 cells across three independent experiments were analyzed for each condition (see Materials and Methods section). % of Rab10 vesicles positive with the indicated marker within each cell was quantified. Each dot represents the mean value from one cell analyzed. Group means are given, with error bars representing ± SEM.

C. Raw 264.7 macrophage cells were transfected with FLAG(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain (PI(3,4,5)P3 marker) and treated with wortmannin (1 μM) for one hour. Representative photomicrographs (from 25 cells analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells immuno-stained for FLAG-tag (Rab10, shown as green) together with eGFP epifluorescence (shown as red). Scale bars are 10 μm, or 1 μm in "Hig Mag" panel.

D. Percent of cells treated with or without wortmannin harboring Rab10 vesicles was calculated from >30 cells across five independent experiments. Each dot on the plot represents mean values from one experiment. Error bars represent ± SEM and significance was assessed by Mann-Whitney test (owing to non-normal distributions) with ** indicating P<0.01.

E. Raw 264.7 cells were co-transfected with eGFP(N-term)-Rab10 and mRuby(N-term)-Rab5 for 48 hours, with live-cell recordings for ~10 minutes. A representative vesicle is shown over the presented time-lapse. Scale bar represents 1 μm.

F. Fluorescence intensities of individual vesicles are calculated over time. Dots show mean values calculated from n=8 recorded vesicles from cells from n=3 biologically independent experiments. Error bars show ± SEM.
LRRK2 and Rab10 coordinate macropinocytosis to mediate immunological responses in phagocytes (2020)
Figure 3

Figure 3. A GTP-locking mutation in Rab10 stalls macropinosome recycling.

A-D Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). 24-hours later, cells were incubated with (A) TRITC-dextran (70 kDa, shown as red signal), or (B) DQ-ovalbumin, for 30 min before washing and fixation. (C, D) Cells were further stained for (C) EEA1, or (D) Lamp1, which was detected with Cy5 dye (show as red signal). Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as "CoLoc." Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker.

E) 25 cells across three independent experiments were analyzed for each condition. % of Rab10 vesicles positive with the indicated marker in each cell were quantified. Each dot represents the mean results from vesicles analyze in one cell. The group means of % Rab10 vesicles positive with the marker are shown, with error bars representing ± SEM. Significance was assessed by Mann-Whitney test for each marker, where ** representing p<0.01, **** p<0.0001 and N.S p>0.05.
LRRK2 and Rab10 coordinate macropinocytosis to mediate immunological responses in phagocytes (2020)
Figure 4

Figure 4. LRRK2 phosphorylates PI(3,4,5)P3-positive Rab10 macropinosomes in macrophage cells.

A. Raw 264.7 macrophage cells were transfected with Flag(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain(PI(3,4,5)P3 marker), or eGFP(C-term)-SARA-Fyve domain((PI3P marker), or eGFP(C-term)-PLC-PH domain (PI(4,5)P2 marker, epifluorescence, shown in blue) followed by anti-Flag immunofluorescence (shown in green) and pT73-Rab10 antibody (shown in red). To evaluate colocalization of phospho-Rab10 with early endosomes or late endosome/lysosome markers, immunofluorescence detection was performed for endogenous EEA1 or Lamp1 (shown in blue). Raw 264.7 macrophage cells were further fed 0.05 mg mL-1 of 70 kDa TRITC-conjugated dextran (epifluorescence, shown in blue) or 0.05 mg mL-1 BODIPY conjugated ovalbumin for 30min prior to immunofluorescence analysis. Representative images were selected from 25 cells analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Scale bars represent 10 μm and 1 μm for "High Mag". Positive colocalization signal (depicted as white) in the "CoLoc (pRab10/Rab10)' and 'CoLoc (pRab10/Marker)' columns are indicated. White arrow heads highlight vesicles scored as co-positive between pT73-Rab10 and Rab10 or the indicated marker.

B. 25 cells across three independent experiments were analyzed for each condition, with each dot representing the mean of the vesicle analysis across individual cells. The group means of %pT73-Rab10 vesicles positive for the indicated marker are given, with error bars showing ± SEM.
LRRK2 and Rab10 coordinate macropinocytosis to mediate immunological responses in phagocytes (2020)
Figure 8

Figure 8. LRRK2 inhibits the fast recycling of receptors internalized through macropinocytosis.

A-D. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-mLRRK2 BAC transgenic mice were immunostained with anti-mouse CCR5, or CD11b, or MHC II antibody (shown as green signal) and (A) total Rab10 antibody (red signal), or (B) pT73-Rab10 antibody (red signal) as indicated. Representative photomicrographs (from >20 images analyzed for each condition from n=3 biologically independent experiments) are shown. (C) Photomicrographs showing the different cargo with respect to Rab10 tubular structures. (D) EEA1 (shown as green signal) with total Rab10 or pT73-Rab10 (shown as red signals) as indicated. Bounding boxes in panels A-D are magnified in "High Mag" panels that show individual vesicles. Scale bars represent 10 μm and 2 μm for "High Mag" images.

E. Calculated percentage of vesicles positive with tubular structures (n>30 vesicles analyzed from cells from n=3 biologically independent experiments) are shown. Each dot represents one vesicle, with significance assessed by Mann-Whitney test. ** represents P<0.01. Error bars represent ± SEM.

F. BMDM cells were treated with 0.004% DMSO or 100 nM MLi2 prior to live cell surface staining with Alexa-594 conjugated rat anti-mouse CD11b antibody on ice for ten minutes. Three wells of cells from each condition were fixed and imaged for initial surface CD11b binding (shown in red as indicated). Six wells of cells from each condition were further incubated at 37°C to allow rapid internalization of CD11b-antibody complexes for ten minutes prior to striping of surface CD11b antibody using 0.5% acetic acid on ice for one min. Three of the six wells were fixed and surface-stained with Alexa-488 conjugated goat anti-rat antibody to show CD11b-antibody complexes remaining on the cell surface (shown in green as indicated). Internalized CD11b-antibody complexes are shown in red. The other three wells of cells were further incubated at 37°C again for ten min to allow recycling of internalized CD11b-antibody complex followed by surface staining with Alexa-488 conjugated goat anti-rat antibody to show CD11b-antibody complexes recycled back to the surface. Representative images from n=3 biologically independent experiments are shown. Scale bars represent 30 μm.

G-I. (G) The initial surface CD11b, or (H) internalized CD11b, or (I) recycled CD11b were calculated from >20 images analyzed for each condition from n=3 biologically independent experiments. Each dot represents the mean value from one experiment. Error bars show ± SEM with significance assessed by Mann-Whitney test. * represents P<0.05.