EMBO/soda-nel huggingface dataset:
Respiratory supercomplexes act as a platform for complex III-mediated maturation of human mitochondrial complexes I and IV (2019)
Figure 6

Figure 6: Complex I assembly kinetics in ∆4-CYB cells.

(A) SDS-PAGE resolving the radioactively labeled mitochondrial translation products after a 2-hour pulse (P). The 35S-Met and the cycloheximide were removed from the medium and cells were collected at the indicated chase times (2, 5 and 24 hours).

(B) First dimension (1D) BNGE analysis of the same cells as in (A), prepared with digitonin.

(C) Denaturing second dimension (2D) BNGE analysis of the same samples allowed following the incorporation of the individual labeled subunits inside their corresponding complex and supercomplex species.

(D) Complex I IGA (cI-IGA) analysis of digitonin-solubilized samples after inhibiting mitochondrial translation with doxycycline for 6 days (0h). After removing the drug and restoring synthesis of the mtDNA-encoded subunits, the cells were collected at the indicated times to follow the appearance of cI reactivity with time. The gels were incubated in the reaction mixture for 24 hours. SS = steady state. The asterisk indicates the presence of a high molecular weight cI-containing band of unknown nature (see main text).

(E) 2D BNGE, Western blot and immunodetection analysis of WT and ∆4-CYB mitochondria from cells collected at the same times after doxycycline treatment to follow the incorporation kinetics of the indicated cI nuclear-encoded subunits, belonging to different structural modules. The blots shown were either exposed for 16 sec. (low exposures) or 160 sec. (high exposures) in order to visualize the qualitative signals in the ∆4-CYB samples.
Respiratory supercomplexes act as a platform for complex III-mediated maturation of human mitochondrial complexes I and IV (2019)
Figure 7

Figure 7: Alternative oxidase (AOX) expression and function in WT and ∆4-CYB cells.

(A) SDS-PAGE, Western blot and immunodetection of AOXHA expression in whole-cell lysates from WT and ∆4-CYB cells transduced with the AOXHA/pWPXLd-ires-HygroR lentiviral vector. The transduction and selection controls were the same cell lines transfected with an empty pWPXLd-ires-HygroR vector (EV).

(B) Growth curves of the AOXHA expressing cell lines and their corresponding EV controls. Cell growth was monitored every six hours after substituting the medium in two replicate 24-well plates, one plate with medium without uridine (Uridine-), and the second plate with medium supplemented with 50 µg/ml uridine (Uridine+). The graphs show the average confluence ± SD at each time point (n=6 wells per cell line).

(C) Immunodetection of cI structural subunits and NDUFAF2 in the same samples as in panel (A).

(D) 1D BNGE, Western blot and immunodetection analyses of digitonin-solubilized mitochondria from WT and ∆4-CYB cybrids expressing AOXHA and their corresponding EV controls.

(E) Complex I in gel activity assays (IGA) after BNGE as in panel (D). The gels were incubated in the IGA reaction mixture for 5 hours. The asterisk indicates the presence of a high molecular weight cI-containing band of unknown nature (see main text).

(F) Spectrophotometric kinetic measurements of cI activity in WT and ∆4-CYB AOXHA and EV samples, normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD (n=5 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test **p=0.0077 (WT AOXHA vs. ∆4-CYB AOXHA); **p=0.0044 (∆4-CYB EV vs. ∆4-CYB AOXHA); ***p=0.0003; ****p<0.0001.

(G) High resolution respirometry analyses performed in intact cells in an Oroboros instrument. ROUTINE: cellular basal oxygen consumption rate (in pmol O2/sec) per million cells in DMEM medium. LEAK is the non-phosphorylating respiration in the presence of the ATP synthase inhibitor oligomycin. ETS: Maximal respiration rate in the presence of the uncoupler CCCP. AOX: oxygen consumption in the presence of antimycin A, inhibiting cIII2 activity but not that of AOX. ROTENONE: Oxygen consumption in the presence of the cI inhibitor rotenone. In all cases this was equal to the background. Results are expressed as mean ± SD (n=2 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test ****p<0.0001.

(H) Respirometry analyses, performed as in panel (G), in WT EV controls and AOXHA- expressing cells untreated or treated with 2.5 µM antimycin A for 7 days (+AA). Results are expressed as mean ± SD (n=4 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test ****p<0.0001.

(I) 1D BNGE followed by cI-IGA (right) or Western blot and immunodetection of cIII2 subunit CYC1 (left) in digitonin-solubilized samples from WT EV controls and AOXHA- expressing cells untreated (-) or treated (+) with 2.5 µM antimycin A for 7 days.
Respiratory supercomplexes act as a platform for complex III-mediated maturation of human mitochondrial complexes I and IV (2019)
Figure 8

Figure 8: Assembly state of cV, cII and cIV in ∆4-CYB cells.

(A) Complexome profiles of the two cV structural and assembly modules. The graphs were generated as in Figure 3 but in this case, the peptide intensity values for the individual subunits belonging to each module (He et al., 2018) were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments.

(B) Complexome profiles of the three detected cII subunits. The graphs were generated as in Figure 3. The represented values are the mean ± SEM of the two reciprocal labeling experiments.

(C) Complexome profiles of the different cIV assembly modules and of the last subunit to be incorporated (NDUFA4). The graphs were generated as in Figure 3 but in this case, the peptide intensity values for the individual subunits belonging to each assembly module (Vidoni et al., 2017) were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments. The bar graph represents the quantification of the total area under the curve (calculated as in Figure 3C) in the profiles corresponding to each cIV module. The plotted values are mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test **p=0.0088 (Early); **p=0.0063 (MT-CO2); **p=0.0040 (NDUFA4); *p=0.0264.

(D) 1D BNGE, Western blot and immunodetection of two cIV subunits (MT-CO1 and COX7B) in samples from WT and ∆4-CYB cells solubilized with DDM and digitonin (Dig).
MEX3A regulates Lgr5+ stem cell maintenance in the developing intestinal epithelium (2020)
Figure 1

Figure 1. Mex3a knockout mice exhibit smaller size and postnatal lethality

A. Scheme of the targeting vector for intragenic deletion of the mouse Mex3a gene. The insertion of the Velocigene cassette ZEN-Ub1 created a deletion of 1125bp in exon 2 of the Mex3a locus.

B. Representative images of the size of Mex3a mutant mice and control littermates at postnatal day (P)15. Scale bar, 1 cm. Genotypes were confirmed by Mex3a mRNA ISH in intestinal tissue (right panels). Scale bars, 50 μm. The offspring number (n) and observed genotype frequencies (%) resulting from heterozygous crosses are indicated below.

C. Absolute weight of Mex3a KO mice and control littermates at different ages. Data is represented in a box and whiskers plot as mean (middle line) with the minimum and maximum distribution values. Each point depicts one animal (WT: P1, n = 15; P8, n = 36; P15, n = 43; P22, n = 14; Adult, n = 14; Mex3a+/-: P1, n = 24; P8, n = 90; P15, n = 90; P22, n = 37; Adult, n = 21; Mex3a-/-: P1, n = 9; P8, n = 24; P15, n = 26; P22, n = 14; Adult, n = 8) ***P = 0.0003, ****P < 0.0001, two-way ANOVA test.

D. Kaplan-Meier survival curves for the different Mex3a genotypes (n = 80 for each genotype) ****P < 0.0001, Log-rank (Mantel-Cox) test.
MEX3A regulates Lgr5+ stem cell maintenance in the developing intestinal epithelium (2020)
Figure 2

Figure 2. Mex3a deletion leads to loss of Lgr5+ stem cells and impairs normal intestinal crypt development

A. Macroscopic assessment of the gastrointestinal tract of Mex3a KO and WT mice. These images are representative of the phenotype observed in mutant animals euthanized at different postnatal days. Boxed areas depict the distal small intestinal section (ileum) used for subsequent immunohistochemical analyses.

B. H&E staining of a Mex3a KO and WT littermate at P19. Inserts depict high magnification of the boxed areas.

C and D. Average crypt depth (C) of WT animals (n = 3, > 40 crypts counted per animal) and Mex3a mutants (n = 6, > 20 crypts counted per animal). Also shown is the average villi height (D) of WT animals (n = 3, > 25 villi counted per animal) and Mex3a mutants (n = 6, > 15 villi counted per animal). Data is represented as mean ± standard error between P16 and P18. *P = 0.03, ***P = 0.0006, Student`s t test.

E-G. Immunohistochemistry staining for (E) the tuft cell marker DCLK1 (black arrowheads indicate positive cells), (F) the Paneth cell marker LYZ1 and (G) the proliferation marker KI67.

H-K. mRNA ISH staining for the stem cell markers (H) Lgr5, (I) Olfm4, (J) Lrig1, and (K) Hopx in ileal sections of Mex3a mutants and littermate controls at P19. Inserts depict high magnification of the boxed areas.

L. qPCR analysis of the expression level of the indicated stem cell markers in freshly isolated crypt fractions from Mex3a KO and WT animals (n = 3 of each genotype). Data is represented as mean fold-change plus standard error in Mex3a KO mice relatively to WT animals (dashed line). *P = 0.0208, **P < 0.01, ***P = 0.0007, Student`s t test.

Data information: Scale bar (A) 1 cm, all other scale bars 50 μm.
MEX3A regulates Lgr5+ stem cell maintenance in the developing intestinal epithelium (2020)
Figure 3

Figure 3. Intestinal epithelial turnover and organoid maturation is delayed in Mex3a KO mice

A. Quantification of the size (diameter length) of Mex3a KO and WT organoid-initiating structures 2 days after crypt isolation (n = 3 for each genotype, > 150 structures counted in total). Data is represented in a box and whiskers plot as mean (middle line) with the minimum and maximum distribution values. Each point depicts one organoid-initiating structure. ****P < 0.0001, Student`s t test. Phase contrast microscopy images of intestinal organoids generated by Mex3a-/- and WT crypts 6 days after crypt isolation. Scale bars, 100 μm.

B. Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids at days 2 and 4 of culture after 10 weekly passages. Scale bars, 100 μm.

C. qPCR analysis of crypt lineage marker genes in organoid cultures at day 2. Data is presented as the mean fold-change plus standard error (n = 4 for each genotype) for target gene expression in Mex3a KO intestinal organoids relative to WT (dashed line). *P < 0.05, Student`s t test.

D. Immunohistochemistry staining for the Paneth cell marker LYZ1 in Mex3a KO and WT intestinal organoid sections at day 2 of culture. Scale bars, 50 μm.

E. qPCR analysis of crypt lineage marker genes in organoid cultures at day 4. Data is presented as the mean fold-change plus standard error (n = 4 for each genotype) for target gene expression in Mex3a KO intestinal organoids relative to WT (dashed line).

F. mRNA ISH staining for the stem cell markers Mex3a, Lgr5, Hopx and Lrig1 in Mex3a KO and WT intestinal organoid sections at day 2 of culture. Scale bars, 50 μm.

G. Representative images of immunofluorescence staining for the proliferation marker KI67 and BrdU incorporation in Mex3a KO and WT animals. Mice were injected at P17 and sacrificed at P20 (n = 3 for each genotype). Scale bars, 50 μm.
MEX3A regulates Lgr5+ stem cell maintenance in the developing intestinal epithelium (2020)
Figure 4

Figure 4. Mex3a deletion leads to activation of the PPAR pathway at the crypt level

A. Hierarchical clustering of samples (n = 3 for each genotype) and heatmap of differentially expressed genes between the Mex3a-/- and WT mice. The red colour represents over-expression, while the blue colour under-expression.

B and C. GO analysis of differentially expressed genes concerning (B) upregulated (red bars) and (C) downregulated (blue bars) biological processes. The 10 most significant terms in each class are depicted according to their P value.

D. KEGG pathway analysis of differentially expressed genes. The 10 most significant terms in each class are depicted according to their P value.

E-G. GSEA pre-ranked analysis comparing the Mex3a-/- transcriptional profile against (E) the Mex3ahigh, (F) the Lgr5high and (G) the Wnt signalling pathway gene signatures.

H. qPCR analysis of selected genes in a cohort of independent mice. Data is presented as the mean fold-change of target gene expression in 2 biological replicates (Mex3a-/- Vs WT #1 and Mex3a-/- Vs WT #2) of Mex3a KO intestinal crypts relative to the respective WT controls (dashed line).
MEX3A regulates Lgr5+ stem cell maintenance in the developing intestinal epithelium (2020)
Figure 5

Figure 5. MEX3A regulates PPARγ signalling

A. Immunohistochemistry staining for PPARγ in Mex3a KO and WT mice ileum. Inserts depict high magnification of the boxed areas. Scale bars, 50 μm.

B. Pparg mRNA ISH in ileal sections of Mex3a mutants and littermate controls at P18. Inserts depict high magnification of the boxed areas. Scale bars, 50 μm.

C. qPCR analysis of PPARγ transcriptional targets in organoid cultures at day 4. Data is presented as the mean fold-change plus standard error (n = 3 for each genotype) for target gene expression in Mex3a KO intestinal organoids relative to WT (dashed line).

D. Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids after 5 days of treatment with 50 μM Pioglitazone or vehicle (DMSO). Scale bars, 100 μm.

E. qPCR analysis of Lgr5, Pcna and Angptl4 expression in organoid cultures after 5 days of treatment with 50 μM Pioglitazone. Data is presented as the mean fold-change plus standard error (n = 4 for each genotype) for target gene expression in Pioglitazone-treated organoids relative to the DMSO vehicle (dashed line). * P = 0.02, Student`s t test.

F. Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids at day 1 after passaging Pioglitazone- or vehicle (DMSO)-treated organoids. Scale bars, 100 μm.

EXPANDED VIEW FIGURES
A missense change in the ATG4D gene links aberrant autophagy to a neurodegenerative vacuolar storage disease. (2015)
Fig 4

Immunohistochemistry indicates disturbed autophagic flow in neurons.

(A) Axonal spheroids stain diffusely positive for LC3B. IHC LC3B, scale bar 100 μm. (B,C) The granular cores of the spheroids are positive for (B) ubiquitin (arrow) and (C) p62. IHC ubiquitin and p62, scale bars 20 and 100 μm, respectively. (D) Smooth axonal swellings in the cerebellar cortex contain ATG4D. Inset: control. IHC ATG4D, scale bar 100 μm. (E) Affected neurons show increased perinuclear granular LC3B positivity. Inset: control. IHC LC3B, scale bar 100 μm. (F) Neuronal vacuoles are partially LAMP2 positive (arrow) and partially negative (arrow head). Inset: control. IHC LAMP2, scale bar 20 μm.
A missense change in the ATG4D gene links aberrant autophagy to a neurodegenerative vacuolar storage disease. (2015)
Fig 5

Phenotype of <italic>atg4da</italic> morphant zebrafish embryos.

(A-F) Lateral views of control and morphant zebrafish embryos. (A,B) Control embryos appear normal at 1 dpf. (C,D) Morphant embryos show severe abnormalities in different regions of the brain at 1 dpf. Black arrowheads denote the developing cerebellum. Purple arrows indicate the hindbrain irregularities. (E) Control embryo at 2 dpf. (F) A morphant embryo at 2 dpf displaying hydrocephalus (yellow arrow) and small head and eye. (G) RT-PCR assay showing the efficiency of the atg4daSMO.
A missense change in the ATG4D gene links aberrant autophagy to a neurodegenerative vacuolar storage disease. (2015)
Fig 6

Suppression of <italic>atg4da</italic> in zebrafish leads to loss of cerebellar neurons.

Immunostaining with (A-C) anti-Pvalb7 and (D-F) anti-zebrinII antibody show loss of cerebellar Purkinje cells in atg4da morphants. (A,D) Control embryos at 4.5 dpf. (B,E) Morphants with mild phenotype show partial loss of cerebellar Purkinje cells. (C,F) Morphants with strong phenotype show either total loss of Purkinje cells or presence of few differentiated neurons, which are laterally located in the cerebellum. (G-I) Labeling of cerebellar granule cells with anti-Vglut1 antibody in control and morphant embryos. (H) Mildly affected morphants show reduced expression of Vglut1 in the cerebellum. (I) Vglut1 expression is strongly reduced in embryos showing severe phenotype. White arrowheads indicate the region of cerebellum. Abbreviations: hb, hindbrain.
The stress granule protein G3BP1 promotes pre-condensation of cGAS to allow rapid responses to DNA (2021)
Figure 1

Figure 1. G3BP1 engages cGAS in a condensed state.

A Turbidity and bright-field microscope images of indicated groups (left) and the quantitative analysis of turbidity of samples were measured by absorbance at 600 nm (right). 10 μM cGAS, 5 μM G3BP1, 5 μM BSA and 100 nM dsDNA (60 bp) were used in these assays. n = 3 biological replicates.

B cGAMP production assays. Recombinant cGAS (10 μM) and G3BP1 (10 μM) were incubated with 100 nM dsDNA. The produced cGAMP was quantitatively analyzed by Liquid Chromatography-Mass Spectrometry/Multiple Reaction Monitoring (LC-MS/MRM). ND, not detected. n = 3 technical replicates.

C Bright-field microscope images of cGAS (10 μM) with indicated concentrations of BSA or G3BP1 (left) and a quantitative analysis of the total area of condensates (right). n = 3 biological replicates.

D Time-lapse imaging of liquid droplets fusion after mixing 10 μM cGAS-mCherry with 1 μM FAM-labeled dsDNA.

E Time-lapse imaging of liquid droplets fusion after mixing 40 μM cGAS-mCherry with 10 μM G3BP1-mEGFP.

F FRAP analysis of DNA-induced droplets of cGAS (1 μM dsDNA, 10 μM cGAS-mCherry), the yellow dotted circle indicated the region of photobleaching.

G FRAP curve of (F). n = 3 biological replicates.

H FRAP analysis of cGAS-G3BP1 droplets. 10 μM cGAS-mCherry and 5 μM G3BP1 were used. The yellow dotted circle indicated the region of photobleaching.

I FRAP curve of (H). n = 3 biological replicates.

Data information: Representative images are shown (A, C-F and H). Error bars, mean with s.d. (A-C, G and I), *P < 0.05, ****P < 0.0001, two-tailed t-test. NS, non-significant. FRAP, Fluorescence recovery after photobleaching. Scale bars, 10 μm (A and C), 5 μm (D), 3 μm (E), 2 μm (F and H). See also Figure EV1.
The stress granule protein G3BP1 promotes pre-condensation of cGAS to allow rapid responses to DNA (2021)
Figure 2

Figure 2. G3BP1 engages cGAS in a condensed state in vivo.

A Immunofluorescent staining of cGAS in both wild-type (WT) and G3BP1-/- U937 cells. Leica TCS SP8 Confocal Microscopy was used to acquire images. The white arrows indicate the cGAS-G3BP1 condensates.

B Immunofluorescent staining of cGAS in both WT and G3BP1-/- U937 cells. 3D images were reconstituted with Leica LAS X software.

C Quantitative analysis of total cGAS puncta number (left) and volume (right) per cell of (B). n = 51 cells.

D Immunoblotting of fractionated lysates from WT and G3BP1-/- U937 cells. GAPDH and Lamin B1 blots were used as controls for cytoplasmic and nuclear fractions, respectively.

E qPCR analysis of IFNB mRNA levels in HT-DNA-treated WT and the indicated rescued cells (U937) (top). Immunoblotting analysis of indicated proteins (bottom). n = 3 biological replicates.

F ELISA analysis of the secreted IFN-β in HT-DNA-treated WT or G3BP1-/- U937 cells. n = 3 biological replicates.

G cGAMP production in ISD-treated WT or G3BP1-/- U937 cells were analyzed by LC-MS/MRM. ND, not detected. n = 3 technical replicates.

Data information: Representative images are shown (A and B). Scale bars, 5 μm. Hoechst (blue), nuclear staining. Error bars, mean with s.d (C, E-G), *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type. IB, immunoblotting. HT-DNA, Herring testes DNA. ISD, interferon stimulatory DNA. See also Figure EV1 and 2.
The stress granule protein G3BP1 promotes pre-condensation of cGAS to allow rapid responses to DNA (2021)
Figure 3

Figure 3. The engagement with DNA, but not with RNA, leads to the dissociation of G3BP1 from cGAS.

A Time-lapse imaging of cGAS-G3BP1 condensates.

B Fluorescence intensity distribution on cGAS-G3BP1 droplets. A quantitative analysis of a representative cGAS-G3BP1 droplet is shown. Along the white line on the merged image, the fluorescence intensity of both G3BP1 and cGAS channels were recorded.

C Time-lapse imaging of cGAS-DNA droplets in the presence of G3BP1-mEGFP.

D cGAS, G3BP1 and Cy5-dsDNA were incubated. A quantitative analysis of a representative cGAS-DNA droplet is shown. Along the white line on the merged image, the fluorescence intensity of G3BP1, cGAS and dsDNA channels were recorded.

E Time-lapse imaging of condensates formed by cGAS, G3BP1 and dsRNA.

F cGAS, G3BP1 and Cy5-dsRNA were incubated. Similar quantification analysis as in D was performed.

Data information: Representative images are shown (A-F). Scale bars, 10 μm (A-E), 5 μm (F). 45 μM cGAS-mCherry, 10 μM G3BP1-mEGFP, 2 μM dsDNA and 2 μM dsRNA were used in the assays. See also Figure EV3.
The stress granule protein G3BP1 promotes pre-condensation of cGAS to allow rapid responses to DNA (2021)
Figure 4

Figure 4. G3BP1 promotes DNA-induced LLPS and activation of cGAS.

A, B Recombinant cGAS (10 μM) and the indicated amounts of Cy5-labeled dsDNA were incubated, with or without G3BP1 (2 μM). The DNA-induced cGAS-DNA liquid droplets were analyzed with time-lapse imaging.

C Quantitative analysis of (A). The partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background. n = 3 biological replicates.

D cGAMP production assays. Recombinant cGAS (10 μM) was incubated with the indicated amounts of dsDNA, with or without G3BP1 (10 μM). The produced cGAMP was quantitatively analyzed by LC-MS/MRM. n = 3 technical replicates.

E, F Immunofluorescent staining of cGAS in WT and G3BP1-/- U937 cells were transfected with ISD (2 μg ml-1) for 3 h (E). The percentage of cells with cGAS foci was quantified, n = 3 biological replicates (F). Hoechst (blue), nuclear staining.

G qPCR analysis of IFNB mRNA levels in WT and G3BP1-/- U937 cells transfected with dsDNA as indicated. n = 3 technical replicates.

Data information: Representative images are shown (A, B and E). Error bars, mean with s.d. (C, D, F and G), **P < 0.01, two-tailed t-test. Scale bars, 10 μm (A and B), 20 μm (E). ISD, interferon stimulatory DNA. See also Figure EV4.
The stress granule protein G3BP1 promotes pre-condensation of cGAS to allow rapid responses to DNA (2021)
Figure 5

Figure 5. Full-length G3BP1 binds to cGAS and promotes LLPS of cGAS.

A Schematic drawing of domain architecture and truncations of human G3BP1.

B Coomassie blue staining of purified cGAS, full length and truncated G3BP1 recombinant proteins.

C cGAS-G3BP1 interaction analysis. Full-length and truncated G3BP1 was pulled-down using anti-Flag M2 beads. The cGAS-G3BP1 interaction was further analyzed with immunoblotting (IB). 2 μg recombinant full-length cGAS and 2 μg each of truncated G3BP1 were used in the assay.

D, E Time-lapse imaging of DNA-induced LLPS of cGAS, in the presence of full length or truncated G3BP1 (2 μM) (D). Partition coefficient of D was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background (E). n = 3 biological replicates.10 μM cGAS, 100 nM dsDNA were used in the assay

F cGAMP production assays. Recombinant cGAS (10 μM) and full length or truncated G3BP1 (10 μM each) were incubated, in the presence or absence of dsDNA (200 nM). The produced cGAMP was quantitatively analyzed by LC-MS/MRM. n = 3 technical replicates.

Data information: Representative images are shown (D). Error bars, mean with s.d. (E and F). Scale bars, 10 μm (D). See also Figure EV5.