EMBO/soda-nel huggingface dataset:
LRRK2 localizes to endosomes and interacts with clathrin-light chains to limit Rac1 activation (2015)
Figure 2

Figure 2. Endogenous genome-edited LRRK2 localizes to endosomes

PCR results of LRRK2-WT from clone E1 (1) using primers that detect endogenous LRRK2. Clone E1 is positive for 3xHA-LRRK2 (2) using a primer pair with the antisense in the 3xHA insert and the sense primer in endogenous LRRK2. (3) Control unedited COS-7 cells using the same primer combination as in (2). (4) 1Kb marker.
Schematic diagram of the oligonucleotide used to direct insertion of the 3xHA tag into the 5' end of the human LRRK2 coding sequence and the corresponding coding sequence in the same colors (the LRRK2 start codon is underlined, as is the GGGGS linker).
COS-7 clone E1 cells were serum starved followed by 20 min incubation with Alexa488-EGF, after which the cells were fixed and processed for immunofluorescence using HA antibody. Scale bar = 10 m for bottom 6 panels and 25 m for top 3 panels.
COS-7 clone E1 cells were fixed and processed for immunofluorescence using HA and CLC antibodies. Scale bar = 10 m.
COS-7 clone E1 cells were fixed an processed for immunofluorescence using HA and EEA1 antibodies. Scale bar = 10 m for bottom 6 panels and 25 m for top 3 panels.
LRRK2 localizes to endosomes and interacts with clathrin-light chains to limit Rac1 activation (2015)
Figure 4

Fig. 4. KD of dlrrk or Clc enhances eye morphogenetic defects caused by Rac1 overexpression in the Drosophila eye.

Ai-x. Lateral view of the adult fly head. Scale bar = 100 μm.

i. longGMR,UAS-Rac1W/TM6B, Hu, Tb causes a mild rough eye phenotype with fused (arrows) and disorganized but still separated ommatidia (arrowheads).

ii/iii. Rac1 overexpression with KD of dlrrk (UAS-dlrrk-RNAi (v22139 or v22140)/longGMR-GAL4, UAS-Rac1W), results in smaller eyes where most ommatidia are fused.

iv/v. Rac1 overexpression with KD of Clc (UAS-Clc-RNAi (v22318 or v106632)/+;longGMR-GAL4,UAS-Rac1W/+), results in smaller eyes with some ommatidia fused.

vi. Rac1 overexpression with KD of the inwardly rectifying potassium channel 3 (irk3), a non-relevant RNAi control (UAS-irk3-RNAi(v101174)/+; longGMR-GAL4,UAS-Rac1W/+), has no effect.

vii/viii. KD of dlrrk with GMR-GAL4/+; UAS-dlrrk-RNAi(v22139 or 22140)/+, respectively as control have no effect on eye morphology.

ix/x. KD of Clc with GMR-GAL4/UAS-Clc-RNAi (v22318 or v106632), respectively as control have no effect on eye morphology.

Bi-ii. Lateral view of the adult fly head. Scale bar: 100 μm.

i. Rac1 overexpression with removing one copy of dlrrk (dlrrke03680/longGMR-GAL4,UAS-Rac1W), results in smaller eyes where most ommatidia are fused together.

ii. Removing one copy of dlrrke03680 in GMR-GAL4 background, as control has no effect on eye morphology (GMR-GAL4/+;dlrrke03680/+).

Ci-viii. Lateral view of the adult fly head. Scale bar: 100 μm.

i. longGMR,UAS-Rac1W/TM6B, Hu, Tb causes a mild rough eye phenotype with fused and disorganized but still separated ommatidia (as explained in Ai).

ii. Rac1 co-expression with dlrrk-WT using UAS-dlrrk-WT/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.

iii. Rac1 co-expression with Clc using UAS-GFP-Clc/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.

iv. Rac1 co-expression with dlrrk-WT-3KD using UAS-dlrrk-3KD/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.

v. A wild-type eye of the longGMR-GAL4 promoter as a control.

vi-viii. Overexpression of dlrrk-WT, dlrrk-3KD, or Clc alone with GMR-GAL4 as control, have no effect on eye morphology.
Actin filament oxidation by MICAL1 suppresses protections from cofilin-induced disassembly (2020)
Figure 1

FIGURE 1: Cofilin at low concentration quickly binds and severs oxidized actin filament.

A. Three steps of a typical experiment (see also Materials and Methods). Filaments are polymerized with 0.6-1 µM ATP-G-actin and aged for 15 minutes with ATP-G-actin at critical concentration (0.1 µM) to maintain the filament length. This solution is supplemented with MICAL1 and NADPH to oxidize filaments. Tpm can also be added at this step to fully decorate filaments (Figure 3 and 4).

B. Fraction (1/17th) of a typical field of view. In a microfluidic chamber, actin filaments (yellow) are anchored by their pointed ends and align with the flow.

C. Time-lapse images showing the assembly of cofilin-1 domains (blue) and subsequent disassembly of actin filaments (yellow). The time-lapse of an oxidized filament shows filament severing at 6s; the first cofilin domain nucleation at 2s; growth of 4 individual domains as their fluorescence intensity increases; and severing (lightning symbol) at the top domain, 2s after its nucleation.

D. Global measurement of the severing of filaments exposed to 20 nM mCherry-cofilin-1 from time t=0 onwards. N = 20 (from 1 representative experiment) and 50 (1 experiment) for non-oxidized and oxidized actin filaments, respectively. P-value = 8.10-8 (log-rank test).

E. Nucleation of the first cofilin domain onto 5 µm-long actin segments. Filaments are exposed to 20 nM mCherry-cofilin-1 from time t=0 onwards. N = 60 filaments, from 1 experiment for each condition. P-value = 4.10-15 (log-rank test).

F. Growth rate of single cofilin domains, normalized by the cofilin concentration. N = 50 (from 3 independent experiments) and 20 (1 experiment) domains on non-oxidized and oxidized filaments, respectively. Measurements were obtained using 20 and 100 nM cofilin (non-oxidized actin) and 10 nM cofilin (oxidized actin). Note that this normalized growth rate does not depend on the cofilin concentration (Appendix Fig S4A). Bars: mean and S.D. P-value = 7.10-8 (t-test).

G. Filament severing rate at single cofilin domains. Time t=0 is defined for every domain as the frame on which they nucleate. N = 163 (from 2 independent experiments) and 203 (3 experiments) cofilin domains on non-oxidized and oxidized actin respectively. Measurements were performed at 100 nM cofilin (non-oxidized actin) and 10, 20 or 30 nM cofilin (oxidized actin). Since the severing rate does not depend on the cofilin concentration (Appendix Fig S4B and (Wioland et al., 2017, 2019a)) data for 10, 20 and 30 nM cofilin on oxidized filaments were pooled. Measurements on non-oxidized filaments were done at 100 nM mCherry-cofilin-1. P-value = 9.10-41 (log-rank test).

Data information: (D, E, G) Thick solid and dashed lines are survival fractions calculated from the experimental data. Thin grey lines are single exponential fits. 95% confidence intervals are shown as shaded surfaces.
Actin filament oxidation by MICAL1 suppresses protections from cofilin-induced disassembly (2020)
Figure 2

FIGURE 2: Phospho-mimic (S3D) cofilin efficiently binds and severs oxidized F-actin.

A. Time-lapse images of eGFP-S3D-cofilin-1 (blue) binding and severing of an oxidized actin filament (yellow).

B. Global quantification of the severing events on oxidized and non-oxidized filaments exposed to unlabelled S3D-cofilin1 from time t=0 onwards. From top to bottom, N = 50 (from 1 experiment), 50 (1 experiment), 100 (2 experiments) filaments.

C. Growth rate of single cofilin domains, normalized by the cofilin concentration. N = 20 domains for WT mCherry-cofilin-1 (1 experiment) and eGFP-S3D-cofilin-1 (2 experiments). The growth rate was measured at 10 nM WT cofilin-1, and 500 nM and 1 µM S3D-cofilin-1 (Appendix Fig S4C). Bars: mean and S.D. P-value = 8.10-9 (t-test).

D. Filament severing at single cofilin domains. Time t=0 is defined for every domain as the frame on which they nucleate. N = 60 (1 experiment) and 75 (2 experiments) domains of WT and S3D-cofilin-1,respectively. The severing dynamic was measured at 10 nM WT eGFP-cofilin-1 and 1 µM eGFP-S3D-cofilin-1. P-value = 0.98 (log-rank test).

Data information: (B, D) All lines are survival fractions calculated from the experimental data 95% confidence intervals are shown as shaded surfaces.
Actin filament oxidation by MICAL1 suppresses protections from cofilin-induced disassembly (2020)
Figure 3

FIGURE 3: MICAL1 can oxidize Tpm-saturated filaments and does not affect Tpm unbinding.

A. Time-lapse images of Tpm-saturated actin filament depolymerizing from their barbed end (BE), with or without MICAL1. Depolymerization is initiated at time t=0 from a non-oxidized actin filament. The increase in the depolymerization rate reflects the oxidation of actin with time. Tpm is labeled with neonGreen (nGr), and actin is unlabeled.

B. Barbed end depolymerization rate as a function of the exposure time to either 100 nM nGr-Tpm1.8 alone ("no MICAL1"), or supplemented with 100 nM MICAL1 and 12 µM NADPH. N = 31 (1 experiment) and 29 (1 experiment) filaments, without and with MICAL1, respectively. Lines: mean velocity. Shaded surfaces: S.D.

C. Time-lapse images showing the disassembly of Tpm domains. Filaments were saturated with 100 nM nGr-Tpm1.8 before the experiment and Tpm was removed from solution at time t=0 (the solution contains 0.6 µM unlabeled G-actin to prevent filament depolymerization). PE and BE indicate F-actin pointed and barbed end, respectively.

D. Disassembly rate of single Tpm domains, at boundaries located towards the pointed end (PE) and towards the barbed end (BE) of actin filaments. N = 100 (non-oxidized F-actin, 4 experiments) and 50 (oxidized F-actin, 2 experiments) domains per direction. Bars: mean and S.D. P-value (t-test) = 0.27 (BE) and 0.12 (PE).
Actin filament oxidation by MICAL1 suppresses protections from cofilin-induced disassembly (2020)
Figure 4

FIGURE 4: Tpm protects non-oxidized but not oxidized F-actin from cofilin-induced severing.

A. Time-lapse images of filaments, oxidized or not, bare or Tpm-saturated, exposed to 1 µM mCherry-cofilin-1, from time t=0 onwards. Other examples are shown in Appendix Fig S6.

B. Nucleation of the first cofilin domain onto 5 µm-long Tpm-saturated actin filaments. Filaments are exposed to 1 µM mCherry-cofilin from time t=0 onwards. N = 50 filaments from 1 experiment for each condition. P-value = 4.10-15 (log-rank test).

C. Growth rate of single cofilin domains, normalized by the cofilin concentration. Filaments saturated by Tpm during aging (Figure 1A) were then exposed to cofilin, along with 0-100 nM nGr-Tpm1.8 to test the competition between the two soluble proteins. Note the scale difference for oxidized filaments. Bars: Mean and S.D. Number of experiments, first to last condition: 2, 1, 1, 3 and 1.

D,E. Filament severing rate at single cofilin domains. Time t=0 is defined for every domain as the frame on which they nucleate. N = 163 (2 experiments), 180 (4 experiments), 116 (3 experiments), 203 (3 experiments) domains for bare actin (100 nM mCherry-cofilin-1), non-oxidized Tpm-saturated (1 µM mCherry-cofilin-1), oxidized Tpm-saturated filaments (100 nM mCherry-cofilin-1), and bare oxidized actin (10, 20, 30 nM mCherry-cofilin-1), respectively.

Data information: (B, D, E) Thick solid and dashed lines are survival fractions calculated from the experimental data. Thin grey lines are single exponential fits. 95% confidence intervals are shown as shaded surfaces.
Actin filament oxidation by MICAL1 suppresses protections from cofilin-induced disassembly (2020)
Figure 5

FIGURE 5: Actin filament oxidation removes their protections from cofilin-induced disassembly.

A. Time-lapse of actin filaments constantly exposed to nGr-Tpm1.8 (yellow), WT mCh-cofilin-1 (blue) and S3D-cofilin-1, and to MICAL1 from time t=100s onwards. Here we mixed WT with an excess of S3D-cofilin-1 to mimic cellular conditions in which most of cofilin-1 is inhibited by phosphorylation. While Tpm prevents cofilin from binding to non-oxidized filaments, MICAL1 rapidly oxidizes actin, allowing cofilin to bind and sever filaments. See also Supp Movie.

B. Summary of the results. Different mechanisms can simultaneously down regulate the activity of cofilin (here cofilin phosphorylation and protection by Tpm). Actin oxidation is a rapid means to cancel these protections without, for example, the need to activate a large concentration of cofilin.
Asymmetry in the frequency and position of mitosis in the mouse embryo epiblast at gastrulation (2020)
Figure 4

Figure 4: Mitosis is more frequent at the PS, and PS mitotic rounding is not always apical.

(a) Graphic representation of a transverse section from an E6.5 embryo. The epiblast is divided in three distinct regions: anterior (red), posterior (green) and PS (blue). The PS region is defined as the area where the basement membrane (yellow) is degraded.

(b) Representation of cell and mitosis disposition at the PS. Apical mitoses are in red, non-apical mitoses in cyan and mesenchymal mitoses in green. Mitosis is considered non-apical when occurring away from the epiblast apical border at a minimum distance corresponding to the size of one nucleus (± 10μm), in a cell that has not crossed the perforated basement membrane, retains its apical attachment and does not display a mesenchymal phenotype.

(c) Z-projections of transverse sections from E6.5 (top), E6.75 (mid) and E7 (bottom) embryos. Samples were stained for F-actin (Phalloidin, grey) and mitosis (Phh3, magenta) on the left and nuclei (DAPI, cyan) and basement membrane (collagen IV, yellow) on the right. Yellow arrowheads show non-apical mitosis. White arrows delimitate borders of the basement membrane degradation region. Scale bars: 25 μm. A: anterior, P: posterior.

(d) Mitotic Index (Phh3/DAPI) in percentage at the anterior, posterior, and PS regions of E6.5 (top), E6.75 (mid) and E7 (bottom) embryos. The posterior region quantification excludes counts from the PS region. Normality was assessed using a Shapiro-Wilk test followed by either two-tail t-test or Mann-Whitney test. ns: non-significant, *: P-value≤0.05, **: P-value≤0.01 and ***: P-value≤0.001.

(e) Ratio of non-apical mitosis (non-apical Phh3/ Phh3, normalized by number of nuclei) in percentage at the anterior, posterior, and PS regions of E6.5 (top), E6.75 (mid) and E7 (bottom) embryos. Normality was assessed using a Shapiro-Wilk test followed by Mann-Whitney tests. ns: non-significant, *: P-value≤0.05, **: P-value≤0.01 and ***: P-value≤0.001.

Data information (d, e): Values are shown as Mean ± SEM. E6.5: n=18 sections from 6 embryos, E6.75: n=11 sections from 5 embryos, E7: n=18 sections from 4 embryos. E6.5 n cells: anterior=730, posterior=445, PS=221. E6.75 n cells: anterior=518, posterior=259, PS=115. E7 n cells: anterior=1324, posterior=712, PS=296.

(f) Violin plots representing the distribution of cells in G2/M phase in cells with high, compared to low, Brachyury expression, among cells annotated as primitive streak and mesoderm harvested from E6.5, E7, and E7.25 embryos. Violin plots are overlaid with white box plots marking 25th percentile, median and 75th percentile. Data information (from Pijuan-Sala et al., 2019): E6.5: 3 biological and 2 technical replicates; E7.0: 6 biological and 3 technical replicates; E7.25: 3 biological replicates. *: P-value≤0.05, **: P-value≤0.01 and ***: P-value≤0.001 using student's T test.
Asymmetry in the frequency and position of mitosis in the mouse embryo epiblast at gastrulation (2020)
Figure 5

Figure 5: Non-apical divisions occur in the posterior epiblast before and at the onset of gastrulation.

(a) 3D reconstruction from wholemount lightsheet imaging of an E5.75 Hex-GFP (green, anterior marker) embryo, stained for nuclei (DAPI, grey) on the left, mitosis (Phh3, magenta) and F-actin (Phalloidin, grey) on the right. Scale bar: 50µm.

(b) Z-slice (left) and 3D reconstruction (right) from wholemount lightsheet imaging of an E6.25 embryo stained for a posterior marker (Eomesodermin, cyan), mitosis (Phh3, magenta) and F-actin (Phalloidin, grey). Scale bar: 50 μm.

(a', b') Mitotic Index (Phh3/DAPI) in percentage at the anterior and posterior regions of E5.75 (a') and (b') E6.25 embryos. Posterior region includes the PS region, as it could not be precisely discriminated. Normality was assessed using a Shapiro-Wilk test followed by two-tail t-test or Mann-Whitney test. ns: non-significant.

(a", b") Ratio of non-apical mitosis (non-apical Phh3 over total Phh3, normalized by number of nuclei) in percentage in the anterior and posterior region of E5.75 (a") and E6.25 (b") embryos. Normality was assessed using a Shapiro-Wilk test followed by Mann-Whitney tests. **: P-value≤0.01 and ***: P-value≤0.001.

Data information (a', b', a", b"): Values are shown as Mean ± SEM. E5.75: n embryos=11; n cells: anterior=1491, posterior=1548. E6.75: n embryos=7; n cells: anterior=1602, posterior=1629.
Asymmetry in the frequency and position of mitosis in the mouse embryo epiblast at gastrulation (2020)
Figure 6

Figure 6: Non-apical mitosis is a feature associated with the primitive streak.

(a) Representation of the position of the AVE-opposed area (green) and the AVE-adjacent area (pink) in WT (left) and RhoAVE-deleted (right) embryos.

(b, c) 3D reconstruction (b) and confocal Z-slice (c) of E6.25 WT (left) and RhoAVE-deleted (right) embryos stained for an AVE marker (Cerberus 1, yellow), F-actin (Phalloidin, grey) and mitosis (Phh3, magenta). Scale bar: 25 μm. Blue dotted line delimitates the pro-amniotic cavity (apical side of the epiblast). Red arrowheads point to non-apical mitosis. White lines delimitates the boundary between extraembryonic (up) and embryonic (down) regions of the embryo.

(d) Percentage of AVE migration in WT and RhoAVE-deleted mutants. Data information: Values are shown as Mean ± SEM. WT: n=31 embryos; RhoAVE-deleted: n=17 embryos. Normality was assessed using a Shapiro-Wilk test followed by a two-tail t-test. ***: P-value≤0.001.

(e) Ratio of non-apical mitosis (non-apical Phh3 over total Phh3, normalised by the volume in μm3) in percentage, in AVE-adjacent and AVE-opposed regions from WT and RhoAVE-deleted embryos. Normality was assessed using a Shapiro-Wilk test followed by Kruskall-Wallis test and Mann-Whitney tests. *: P-value≤0.05, ***: P-value≤0.001.

EXPANDED VIEW FIGURES LEGENDS
Protein translocation by the SecA ATPase occurs by a power-stroke mechanism (2019)
Figure 1

Figure 1. SecA's two-helix finger makes large movements during the ATP hydrolysis cycle.

(A) Experimental setup to measure single-molecule FRET in translocation complexes immobilized on a surface.

(B) Cy5 and Cy3 fluorophores were introduced into the two-helix finger of SecA (PDB 3DIN; red space filling model; helices highlighted) at position 809 and into SecY (blue) at position 394, respectively.

(C) Representative traces obtained with ADP•BeFx. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event.

(D) Distribution of FRET values determined from 97 traces as in (C) fit with a Gaussian model (black curve).

(E) As in (C), but in the presence of ATP. Periods in which a fluorescently labeled SecA molecule is bound are indicated by grey shading.

(F) As in (D), but with ATP (257 traces).
Protein translocation by the SecA ATPase occurs by a power-stroke mechanism (2019)
Figure 3

Figure 3. SecA's clamp opens and closes during the ATPase cycle.

(A) Cy5 and Cy3 fluorophores were introduced into the clamp of SecA (PDB 3DIN; red space filling model) at position 233 and into the N-terminal half of SecY (blue) at position 103, respectively. The PPXD and NBD2 making up the clamp are shown as a violet and magenta ribbon models, respectively.

(B) Rotated view of (A) with SecY masked except for the labeled residue 103.

(C) Representative traces obtained with ADP•BeFx. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event.

(D) Distribution of FRET values determined from 197 traces as in (B) fit with a Gaussian model (black curve).

(E) As in (C), but with ATP. Periods in which a fluorescently labeled SecA molecule is bound are indicated by grey shading.

(F) As in (D), but with ATP (228 traces).
GSDMD drives canonical inflammasome-induced neutrophil pyroptosis and is dispensable for NETosis (2022)
Figure 2

Figure 2. Canonical inflammasomes driving IL-1β maturation and secretion in neutrophils. (A-H) LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or stimulated with LeTx (3 hr) (A, B), TcdA (3 hr) (C, D), infected with F. tularensis (18 hr) (E, F) or stimulated with Flatox (3 hr) (G, H). IL-1β secretion levels were determined in culture media (A, C, E, G); and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) (B, D, F, H). Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).
GSDMD drives canonical inflammasome-induced neutrophil pyroptosis and is dispensable for NETosis (2022)
Figure 3

Figure 3. Canonical inflammasomes promote plasma membrane permeabilization (PMP) in neutrophils.

(A-D) LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with LeTx (A), TcdA (B), ATP (C) or FlaTox (D). PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.

(E) LPS-primed B6 BMNs were left untreated (LPS) or subsequently stimulated with FlaTox (LPS+FlaTox) for 3 hr. Data are representative of n=3 biological repeats.

(F) B6 BMNs were left untreated (Mock) or infected with S. Typhimurium at MOI 25. Culture media (Sup) and whole cell lysates (Lys) were immunoblotted for release of cytosolic GAPDH into culture media. Data are representative of n=3 biological repeats.
GSDMD drives canonical inflammasome-induced neutrophil pyroptosis and is dispensable for NETosis (2022)
Figure 4

Figure 4. GSDMD drives canonical inflammasome-induced PMP and IL-1β secretion in neutrophils.

(A-I) LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with TcdA (3 hr) (A, D, G), FlaTox (3 hr) (B, E, H) or nigericin (100 mins) (C, F, I).

(A-C) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted separately for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD (p30). * with Western blots indicates non-specific cross-reactivity of the antibody. All data are representative of n=3 biological repeats.

(D-F) PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).

(G-I) IL-1β secretion levels were determined by Luminex assay. Values represent mean ± SEM of n=3 (G, I) or n=4 (H) biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01.