EMBO/soda-nel huggingface dataset:
Polarization of Myosin II refines tissue material properties to buffer mechanical stress. (2018)
Figure 1

Figure 1: Myosin RNAi clones are softer and less elastic.

(A) Stretching/compression device; 1: clamping mechanism, 2: arms, 3: stage insert, 4: drive mechanism, 5: media-filled Polydimethylsiloxane (PDMS) chamber, 6: two layers of stretchable elastomer (PDMS), one of which is pre- patterned with microchannels.

(B) Scheme of PDMS prepatterning; the dimension of microchannels are 80- 120 μm in width and 50 μm in depth. (C) Cross-sectional schematic view of the stretching device; wing disc (in red) is positioned over the microchannel with sides clamped by the two PDMS layers. The central portion of the tissue is submerged in the microchannel, perfused with ex vivo culture media (Mao et al., 2013; Mao et al., 2011).

(D) Stretching of the PDMS sandwich concomitantly stretches the wing disc inside the microchannel (top-down view); wing disc resting on a stretching device (anchor) and 10 min after stretch; M=microchannel. (E) zipper RNAi clones (zip iR, in red) in Arm::GFP expressing anchored and stretched third instar wing disc; lower panel shows color coded cell aspect ratio in anchored and stretched discs (bright colors indicate high elongation); zip RNAi clone is outlined in white.

(F) Box plot showing distribution of cell aspect ratio for control (wild-type) and zipper RNAi cells in anchored and stretched (20 min) wing disc; median represented by horizontal line, 75th and 25th centiles are represented by top and bottom of the boxes respectively; n=8 wing discs.

(G) Color-coded aspect ratio in control and zip RNAi cells (zip RNAi clone outlined in white) subjected to anchoring, stretching (20 min) and relaxation (10 min, post stretch). (H) Box plot showing distribution of cell aspect ratio in anchored, stretched (20 min) and relaxed (10 min) wing disc for control (wild-type) and zipper RNAi cells; median represented by horizontal line; 75th and 25th centiles are represented by top and bottom of the boxes respectively, n=3 wing discs.

(I) zip-RNAi clone in Arm::GFP wing disc at the beginning (5 min) and at the end (40 min) of stretching experiment. Yellow and red arrows indicate the control and zip RNAi cells respectively. (J) Change of zip-RNAi aspect ratio (a.r.) relative to change in control cells aspect ratio (a.r.) in the course of stretch, blue line shows fitted regression; dotted line represents 95% confidence bounds of the best fit line; n=3 wing discs.

(K) Schematics describing behavior of zip-RNAi cells; spring represents control cells which contract and pull on less elastic zip-RNAi clone (dashpot) during 40 min stretch. Red horizontal arrows indicate direction of stretch. * p<0.05, ****p<0.0001 with t-test. n.s. = non significant. Scale bars, 5 cm (A), 50 μm (D), 10 μm (E,G), 5 μm (I).
Polarization of Myosin II refines tissue material properties to buffer mechanical stress. (2018)
Figure 2

Figure 2: Myosin polarizes with mechanical stretch.

(A) Wing disc expressing E-cad::GFP and Sqh::mCherry prior to stretch (anchor) and after stretch (15 min). (A"), Insets of discs shown in (A). (B) Schematics demonstrating change in Sqh concentration before and after stretch on vertical (V) and horizontal (H) junctions

(C) Quantification of mean fluorescent intensity of E-cad::GFP and Sqh::mCherry on horizontal (H) junctions relative to the intensity on vertical (V) junctions (referred to as "polarity") prior to and after 15 min stretch. The detailed rationale of intensity determination is described in Fig. S3 and Experimental Procedures.

(D-E), Quantification of E-cad::GFP (D) and Sqh::mCherry (E) concentration (i.e. mean intensity per junctional unit area) prior and after stretch on both horizontal and vertical junctions normalized to starting concentration on unstretched (anchored) vertical junctions.

(F) Quantification of total amount of Sqh::mCherry and E-cad::GFP fluorescence on vertical and horizontal junctions in anchored and stretched discs.

(G) Tissue expressing E-cad::GFP and Sqh::mCherry subjected to increasing levels of stretch. (H) Quantification of Sqh::mCherry polarity (as in c) of experiment described in (G). The level of stretch is measured as a change in tissue strain (deformation relative to unstretched tissue).

(I-K) Sqh::mCherry polarity emergence as a function of cell aspect ratio change (I), stress (J), stress applied on average cell area of 10 μm2 (K). Yellow asterisks indicate equivalent regions in the tissue. N=8 wing discs (A- F), N=3 wing discs (G-K); all experiments are plotted as mean ± S.E.M., * p<0.05 with t-test. Scale bars, 5 μm (A), 3 μm (A"), 10 μm (G).
Polarization of Myosin II refines tissue material properties to buffer mechanical stress. (2018)
Figure 3

Figure 3: Uncoupling of tissue shape and MyoII polarity after prolonged stretch. (A) Short (15 min) stretch and relaxation (10 min) of E-cad::GFP and Sqh::mCherry expressing discs.

(B) Quantification of Sqh::mCherry polarity, E-cad::GFP polarity and aspect ratio change of experiment described in (A); schematics below the graph illustrates quantified changes in cell aspect ratio (yellow) and Sqh polarity (red). Statistical test compares Sqh polarity between anchored and stretched or stretched and relaxed disc.

(C) Long (200 min) stretch and relaxation (10 min) of E-cad::GFP and Sqh::mCherry expressing discs. (D) Quantification of Sqh::mCherry polarity, E-cad::GFP polarity and aspect ratio change of experiment described in (C); schematics below the graph illustrates quantified changes in aspect ratio (yellow) and Sqh polarity (red). Statistical test compares changes with respect to initial (anchored) state. Yellow asterisks indicate equivalent regions in the tissue. N=3 wing discs; all experiments are plotted as mean ± S.E.M., * p<0.05 with t-test. Scale bars, 5 μm.
Polarization of Myosin II refines tissue material properties to buffer mechanical stress. (2018)
Figure 4

Figure 4: Phosphorylated MyoII polarizes in mechanically stretched cells. (A) Overview of the E-cad::GFP disc prior to (anchor) and after compression. (B) E-cad::GFP and Sqh::mCherry localization in anchored and compressed wing disc.

(C) Laser cuts (perpendicular to the line of stretch) across circa 10 cells in E- cad::GFP expressing wing disc tissue subjected to anchoring, short (<20 min) stretching and short (<20 min) compression. Plot shows increase in distance between recoiling short axis [μm] of the fitted ellipse (see Fig. S5A for details) against time [sec.]; data is plotted as mean ± S.E.M, n= 7-10 discs/condition. (D) Activated Myosin (anti-phosphorylated-Sqh; in red) and anti-E-cad (green) staining in compressed discs.

(D") Close up of compression experiment described in (D); yellow arrow points towards an example of polarized phosphorylated-Sqh cable in compressed discs. Scale bars, 5μm (B, D"), 10μm (D), 30μm (A).
Polarization of Myosin II refines tissue material properties to buffer mechanical stress. (2018)
Figure 5

Figure 5: Emergent tension patterns reveal tissue stiffening upon mechanical stretch. (A) Laser cuts (perpendicular to the line of stretch) across circa 10 cells in E- cad::GFP expressing wing disc tissue subjected to anchoring, short (<20 min) and long (>120 min) stretching and stretch retraction (tissue forced back to the original, pre-stretch shape.

(B) Quantification of the experiment described in (A). Plot shows increase in distance between recoiling short axis [μm] of the fitted ellipse (see Fig S4A-B for details) against time [sec.]; data is plotted as mean ± S.E.M; anchor and short stretch data plotted here is the same as in Fig. 4B, n=3-11 wing discs per condition.

(C) An overlay of a junction prior to (red) and 15.44 sec. after ablation (green). (D) Plot showing relative initial recoil velocity (velocity on horizontal, stretched junctions normalized to velocity on vertical, unstretched junctions) for individual junctions ablated in anchored or stretched (<30 min, >100 min) E-cad::GFP expressing discs. Data is plotted as a mean ± S.E.M, n=17-24 cuts per condition. (E) Laser cuts (perpendicular or parallel to the line of stretch) across circa 10 cells in E-cad::GFP expressing wing disc tissue subjected to anchoring or short stretching (< 30 min)..

(F) Quantification of experiment described in (E). Data is quantified and presented as in (B). N=4-7 wing discs per condition. (G) Image depicting injury site (yellow asterisk) and selection of cell rows used for tissue recoil propagation measurement; r: row.

(H) An overlay of anchored and stretched discs prior to ablation (red) and 15.44 seconds post ablation (green). Inset zooms in on the area next to ablation site (marked with yellow asterisk).

(I) Recoil area (% increase from pre-ablation area) for row 0, row 1, row 2 and row 3 in stretched and anchored discs. Data is plotted as mean ± S.E.M, n=8- 13 discs/condition.

Error bars indicate S.E.M.; * p<0.05 and **p<0.01 with t-test. Scale bars, 5 μm (A,E,G), 3 μm (C,H), 2 μm (H: inset).
Polarization of Myosin II refines tissue material properties to buffer mechanical stress. (2018)
Figure 6

Figure 6: Myosin polarity is Rok independent.

(A) RokK116a::Venus localization in anchored, stretched, relaxed third instar imaginal discs. (B) Drok2 mitotic clones (RFP-) in Sqh::GFP third instar imaginal disc. Inset demonstrates Sqh::GFP polarity inside Drok2 clone.

(C) Sqh::GFP, enGal4>UAS-rok RNAi, UAS-Act5CRFP discs subjected to stretching; inset demonstrates Sqh::GFP polarity in rok RNAi cells (marked by red. (D) zip RNAi clones (labeled as zip iR, indicated in red) in stretched third instar RokK116a::Venus discs.

Error bars indicate S.E.M.; * p<0.05 and **p<0.01 with t-test. Scale bars, 2 μm (D: inset), 5 μm (A,D, B: inset, C: inset), 10 μm (B,C).
Polarization of Myosin II refines tissue material properties to buffer mechanical stress. (2018)
Figure 7

Figure 7: MyoII polarity is required for wing disc shape maintenance via Dia-dependent actin polymerization. (A) Discs expressing Sqh::GFP and enGal4>UAS-dia RNAi, UAS-Act5c-RFP that were stretched; UAS-Act5C::RFP marks dia iR (posterior side) in red.

(A") Insets comparing Sqh::GFP polarity in stretched enGal4>dia iR, UAS- Act5c-RFP discs; panel 1 refers to anterior (ctrl) side; panel 2 refers to posterior (dia iR) side. (B) Tissue marked with E-cad::GFP and Sqh::mCherry, expressing dia-RNAi (dia iR) with hhGal4 driver and subjected to stretching. Dotted line indicates anterior-posterior (A-P) compartment boundary.

(B") Inset demonstrates lack of Sqh::GFP polarity in dia-RNAi cells. (C-D), Quantification of E-cad::GFP polarity (C) and Sqh::mCherry polarity (D) in experiment described in (B); polarity is measured as mean fluorescent intensity on horizontal junctions relative to vertical junctions (see Experimental Procedures for details); n=4 wing discs. (E) Stretched discs expressing Sqh::GFP and enGal4>UAS-cofilin (upper panel) or UAS-AIP1 (lower panel) RNAi and UAS-Act5c-RFP (marks posterior side in red). (E"), Insets comparing Sqh::GFP polarity in stretched discs described in (E); panel 1 refers to anterior (ctrl) side; panel 2 refers to posterior (dia iR) side. (F) Sqh::GFP polarity in enGal4> dia iR, UAS-Act5C::RFP discs labeled with Phalloidin-647; Inset (Phall-647) zooms in on cell shape in anterior and posterior disc compartments. (G) Discs expressing nubGal4 and Cd8::GFP (control) or dia iR (KK) labeled with DAPI; ellipse is fitted into the pouch region (red dotted line) and aspect ratio determined as long ellipse axis (b) divided by short ellipse axis (a) (white lines). (H) Box plot showing distribution of wing disc pouch aspect ratios in conditions described in (G), n=19 (ctrl) and 16 (dia iR) wing pouches; horizontal line indicates median. (I) Model of Diaphanous-Myosin force buffering pathway. Tissue (E-cad marks cell junctions in green) subjected to mechanical stretching polarizes MyoII and Actin (in red) in Dia-dependent manner. This mechanism is required to limit tension induced cell deformation and thus maintain wing disc pouch shape. Error bars indicate S.E.M.; * p<0.05 and **p<0.01 with t-test. Scale bars, 5 μm (A",B",E", F: inset), 10 μm (A,B,E), 30 μm (F, G).
A Zn-dependent structural transition of SOD1 modulates its ability to undergo phase separation (2022)
Figure 2

Figure 2. ApoSOD12SH undergoes liquid-liquid phase separation.

A. SOD12SH undergoes LLPS on the removal of metal ion cofactors.

B. DIC microscopic image of WTSOD12SH diluted to 100 µM in HEPES buffer, pH 7.4 and 100 mM NaCl and incubated 37 °C, 180 RPM for 30 min shows no droplet formation; scale bar: 100 µm; inset shows Alexa Fluor 488 maleimide labelled WTSOD12SH; scale bar: 5 µm

C. Solution turbidity plot (absorbance at 600 nm) shows that WTSOD12SH (at concentrations ranging from 25 to 200 µM does not undergo LLPS in presence or absence of heparin, LLPS inducer; data is represented as mean ± SD (from 3 biological replicates).

D. DIC microscopic image of ApoSOD12SH under condensate inducing conditions (37 C, 180 RPM) at a concentration of 100 µM incubated 100 mM NaCl; scale bar:100 µm (inset on bottom left shows Alexa Fluor 488 maleimide labeled protein condensates; scale bar: 5 µm.

E. Fluorescent microscopic images of Alexa Fluor 488 maleimide labeled ApoSOD12SH condensates diluted in HEPES buffer, pH 7.4 and 100 mM NaCl and incubated (180 RPM, 37 C) in absence (left) and presence (right) of heparin at 30 min time-point; scale bar: 100 µm.

F. Solution turbidity measurements with ApoSOD12SH (absorbance at 600 nm) shows that while in the absence of heparin the turbidity increases with time, the presence of heparin results in faster LLPS; data is represented as mean ± SD (from 3 biological replicates).

G. Comparison of ApoSOD12SH droplet numbers per microscopic field of view (view area 0.001963 mm2) calculated from 5 different images of droplets incubated with and without heparin at 2 h timepoint. Data is represented as mean ±SD. Statistical significance was established using unpaired non-parametric t-test (****p< 0.0001).

H. Liquid nature of ApoSOD12SH showing droplet fusion and surface wetting; scale bar: 5 µm. I. Amplitude normalized FCS curves of ApoSOD12SH (in blue) of dilute phase (DP) and condensed phase (CP). Intense color indicates CP and light variant indicates DP. The increase in τD (as shown by the arrow) translates to a decrease in D (please refer to the Equation 2 in Methods section)

J. Scheme depicting that presence of Zn and not Cu in pre-incubation mixture inhibits LLPS.

K. DIC microscopic images of ApoSOD12SH in presence of Zn (top) showing no droplet formation and Cu (below) showing the presence of droplets; scale bar: 100 µm.

L. Solution turbidity plot (absorbance at 600 nm) of ApoSOD12SH subjected to increasing concentrations of Cu and Zn; turbidity decreases in a dose dependent manner for Zn while no significant change is observed in presence of Cu; data is represented as mean ± SD (from 3 biological replicates).
A Zn-dependent structural transition of SOD1 modulates its ability to undergo phase separation (2022)
Figure 3

Figure 3. ALS mutants with compromised Zn binding undergo LLPS.

A. DIC microscopic images of I113T SOD12SH (top) and G85R SOD12SH (bottom) droplets incubated in the absence and presence of Zn and Cu respectively (scale bar: 100 µm); insets on bottom left show Alexa Fluor 488 maleimide labeled protein condensates for I113T SOD12SH and G85R SOD12SH respectively; scale bar: 5 µm. Droplet dissolution was observed for I113T SOD12SH on addition of Zn while droplets persisted on addition of Cu. Red arrowhead indicates fusion of liquid droplets.

B. Amplitude normalized FCS curves of I113T SOD12SH (in green) and G85R SOD12SH (in grey) of dilute phase (DP) and condensed phase (CP) showing an increase in diffusion time from DP to CP. The intense color indicates CP and light variant indicates DP.

C. On metal cofactor removal, ApoG37R SOD12SH formed condensates which ceased to exist when protein was incubated with Zn, while condensates persisted on Cu addition (left to right); scale bar: 100 µm.
A Zn-dependent structural transition of SOD1 modulates its ability to undergo phase separation (2022)
Figure 4

Figure 4. Zn drives disorder to order transition in SOD1.

A. Scheme shows Zn dependent disorder to order transition in monomeric SOD1.

B. Plot of the loge of the hydrodynamic radius (rH) versus the log e of the number of residues in the polypeptide chain. The line fitted to these data for the native folded proteins (yellow dashed) has a slope of 0.29 ± 0.02 and a y-axis intercept of 1.56 ± 0.1, while the other fitted to the chemically denatured protein (grey dashed) data has a slope of 0.57 ± 0.02 and a y-axis intercept of 0.79 ± 0.07. Literature data have been used for folded and chemically denatured proteins while we employed FCS to calculate the rH of all SOD12SH variants with (solid shapes) and without Zn (hollow shapes). The hollow grey circle of G85R SOD12SH is directly under hollow green I113T SOD12SH in the log-log plot.

C. Variation of diffusion coefficients of de-metalated protein variants determined from FCS measurement with increasing Zn concentration (inset shows a comparison of binding constants between mutants and Zn; ApoSOD12SH and ApoG37R SOD12SH has a higher binding affinity to Zn than ApoI113T SOD12SH and ApoG85R SOD12SH). Data is represented as mean ± SD (from 3 biological replicates).

D. Disordered/ extended conformation content decreases in ApoSOD12SH and ApoI113T SOD12SH with the addition of Zn calculated from FTIR spectra; data is represented as mean ± SD (from 3 biological replicates).
A Zn-dependent structural transition of SOD1 modulates its ability to undergo phase separation (2022)
Figure 5

Figure 5. Characterization of SOD12SH variants and molecular interactions in the condensed phase using molecular dynamics simulations.

A, B. (A) Per-residue RMSF profiles of ApoSOD12SH & its mutants computed from 5 µs simulations performed for each variant. (B) Per-residue RMSF profiles of ApoSOD12SH, ApoSOD1S-S, ZnSOD1S-S and ZnSOD12SH computed from 5 µs simulations performed for each variant. Error bars in A and B correspond to standard errors which are estimated by block averaging over 10 (500 ns each) blocks.

C. Salt-bridge analysis of ApoG85R SOD12SH (dist. cutoff <0.5 nm = salt-bridge).

D. Snapshots from the ApoG85R SOD12SH trajectory showing the formation of non-native salt-bridges which are likely to be detrimental for Zn-binding.

E. Density profile of ApoSOD12SH and its loop-deletion variants computed from the CG slab simulations. The condensed phase is centered in the middle of the simulation box.

F. Slab snapshots from CG simulations of ApoSOD12SH and its variants at 275 K (blue beads represent the loop IV, red beads represent loop VII, orange beads represent the other domains.)

G. Pairwise intermolecular contact formation (normalized by maximum probability) in the condensed phase as a function of residue index.

H. Saturation concentration measured in the CG phase coexistence simulations for ApoSOD12SH and its variants including ALS mutants at 275 K. The error bars correspond to SD, which are estimated from block averages over 4 blocks.
A Zn-dependent structural transition of SOD1 modulates its ability to undergo phase separation (2022)
Figure 6

Figure 6. Maturation of liquid droplets precedes aggregation.

A. Cartoon scheme showing the maturation of protein droplet with time (region1: diffused region outside droplet, region 2: low intensity portion inside the droplet, region 3: highly intense portion inside droplet appears during maturation) at different time points.

B. Diffusion coefficient of region 1 and 2 (within droplet) was measured using point FCS as shown in the scheme.

C. Upper panel shows fluorescence images of the maturation of Alexa Fluor 488 labeled ApoSOD12SH droplets with time, samples were incubated at 37 C up to ~25 hours. Images are taken at indicated time point: 0* hour (the time after incubation required for droplet formation), 12 hours and >24 hours; images below show magnified scale bar: 5 µm. The middle panel shows magnified view of the marked region from corresponding images on the upper panel. Red arrowhead indicates matured (solid) portion inside the droplet. Lower panel shows coexistence of liquid and solid phase inside droplet; scale bar: 10 µm. Intensity plot (bottom right) shows the distribution of high intensity region within matured droplet.

D. Diffusion coefficient obtained from point FCS measurement at different regions of the droplet during maturation course; 0* (x-axis label) indicates the time after incubation required for droplet formation. Data is shown mean ± SD (from 3 biological replicates). Statistical significance was measured using paired t-test; ns denotes non-significant; **p< 0.01; ***p< 0.001

E. ThT fluorescence assay plot showing aggregation kinetics of all SOD12SH variants. ApoSOD12SH, I113T SOD12SH and G85R SOD12SH show high ThT fluorescence.

F. AFM micrographs of ApoSOD12SH, I113T SOD12SH and G85R SOD12SH show fibrillar aggregates after prolonged incubation at 37 ℃, 180 RPM in absence of Zn (left panel); AFM micrographs of ApoSOD12SH, I113T SOD12SH and G85R SOD12SH incubated with Zn (at 1:5 protein: Zn molar ratio) show presence of oligomers for ApoSOD12SH, I113T SOD12SH and short fibrils for G85R SOD12SH (right panel); scale bar: 200 nm.

G. Cell proliferation of SHSY5Y human neuroblastoma cells assessed by MTT assay post 12 h treatment with 5 µM ApoSOD12SH, I113T SOD12SH and G85R SOD12SH condensates and aggregates (fibrils). Cell culture medium (DMEM) was used as negative control and untreated SHSY5Y cells were used as positive control. Data are shown as mean ± SEM (from 3 biological replicates). P values were determined by two tailed unpaired t tests; *p<0.05, **p< 0.01; ***p< 0.001, ****p< 0.0001.

H. Dot plots showing flow cytometric analysis of annexin V and propidium iodide (PI) staining of apoptotic SHSY5Y neuroblastoma cells following a 12 h treatment with 5 µM ApoSOD12SH condensates, I113T SOD12SH condensates, G85R SOD12SH condensates, ApoSOD12SH fibrils, I113T SOD12SH fibrils and G85R SOD12SH fibrils respectively (see Methods for details). High cytotoxicity was observed for SOD12SH condensates.