EMBO/soda-nel huggingface dataset:
Polarized Cortical Tension drives Zebrafish Epiboly Movements (2016)
Figure 4

Figure 4. Epiboly biomechanics

(a) Yolk microrheology. Mean squared displacement (MSD) of thermal fluctuations of fluorescent nanoparticles embedded in the yolk of zebrafish embryos. Solid and dashed black lines are Mean ± Standard Error computed from 99 particles. Red line is the fit of the Stokes-Einstein equation. MSD of nanoparticles embedded in the yolk exhibits a proportional dependence on time lag, Δt, consistent with a Newtonian liquid behavior with a viscosity of 129 mPa·s. A linear time dependence of MSD with similar values of yolk viscosity has been recently reported in Drosophila (He et al., 2014). Interestingly, the viscosity of the zebrafish yolk is two orders of magnitude lower than that of C. elegans embryos (Daniels et al., 2006) and of the cytoplasm of several cell types (Wirtz, 2009). (b) Viscoelasticity of the yolk cortex. Effective complex modulus (g*) measured by indenting the surface of zebrafish embryos with AFM. Red symbols are the elastic modulus (g’) and yellow symbols are the viscous modulus (g’’), respectively. Standard Errors are displayed. In all the measured frequencies cortex rheology is dominated by a solid-like behavior with the viscous modulus 5 times lower that the elastic modulus. No significant frequency dependence was found in the elastic (ANOVA, p = 0.123) and viscous modulus (ANOVA, p = 0.719). (c) Flow lines from snapshots of the PIV measurements (at 40, 60 and 80 % epiboly) of a two-photon video time-lapse (from Movie EV4) of a Tg (β-actin:m-GFP) embryo (top). Fitted flow lines using HR ar shown at the bottom. The relative mean square errors (RMSE) of the fitting are displayed as a percentage at the bottom left of each panel. Scale bar 100 μm. (d) Mechanical power maps over time obtained by HR of the same embryos as in (c) (from Movie EV5). The RMSE of the power is shown as a percentage at the bottom left of each panel. The mechanical work to produce doming (40 % epiboly) arises mainly at the blastoderm (left); at 60 % epiboly (middle) DCs undergo ingression (red) while the E-YSL resists constriction (blue). At 80 % epiboly (right), the active work leading to the active movement of the EVL / yolk margin mainly maps at the E-YSL (red) while the blastoderm undergoes elastic resistance (blue). White arrows point to the main observed events. Scale bar 100 μm. (e) AV (red) and CC (green) stresses and their differences (blue) normalized values in arbitrary units from the regression analysis (Movie EV6) were plotted as a function of the φ angle (0-1) along the cortex from animal to vegetal. The equator - dotted yellow line -, yolk surface - purple shadow - and the RMSE of the mechanical pressure as a percentage for each time point are displayed (40 %; 60 %; 80 % epiboly). The CC stress steeps up from animal to vegetal and the AV shows a more complex profile. Their difference changes senses around the EVL / yolk margin.
Polarized Cortical Tension drives Zebrafish Epiboly Movements (2016)
Figure 6

Figure 6. Epiboly is driven by E-YSL contractility and differential surface tensions

(a) Kymograph showing the deformation of the cortex along a segment perpendicular to a laser cut (red line time point) in the E-YSL of a Myosin-GFP transgenic embryo [Tg (β-actin:myl 12.1-e-GFP)]. After a rapid opening, an active contraction displaces the cut centroid vegetalward indicating that the EVL is more easily stretched (elastic) than the vegetal yolk cell membrane/cortex. When the gap is closed, the cut center returns to its initial position and the elastic equilibrium is restored. Scale bar 10 μm. (b) Proposed model of epiboly progression. The contractile E-YSL and the imbalance of stiffness between the EVL and the yolk surface account for epiboly progression. The vegetal boundary of the E-YSL is not mechanically free but linked to the tense yolk membrane. The tension originated by constriction of the actomyosin ring (E-YSL) is thus conveyed by the yolk membrane towards the vegetal pole leading to the progression of the less stiff EVL. Passive movements within the yolk arise as a result of force transmission from the surface. (c) Dynamic simulation of epiboly based on a single spherical cortex model (SC) considering a gradient of elastic tension from animal to vegetal and a constricting area at the E-YSL (from Movie EV8 - 40 %; 60 %; 80% epiboly). Color-coded mechanical power map normalized values in arbitrary units (positive - red, negative - blue) (left). Flow lines (right). The mechanical power is supplied at the surface and internal vortices and their shift from animal to vegetal are precisely simulated. (d) Dynamic simulation of AV and CC stresses normalized values in arbitrary units (from Movie EV9 – 40 %; 65 %; 80 % epiboly) represented as in Figure 1a. The AV and CC stresses are equal at the poles, as the epiboly simulation progresses, the CC stress steeps up from animal to vegetal and the difference between CC and AV stresses change sense at the EVL / yolk margin.
Polarized Cortical Tension drives Zebrafish Epiboly Movements (2016)
Figure 7

Figure 7. Experimental validation of the differential surface tension model

(a) Speed of the EVL advancing front at different epiboly stages (ϕ angle) for two distinct simulated conditions: 1) a difference of elastic tension between the EVL and the yolk associated to a two-phase reduction of the contractile E-YSL width (from 20º to 5º) as epiboly advances (from 50º to 155º) (wild type - blue). The contractile E-YSL width decreases at a slower rate until 80º and shrinks afterwards at a rate 2.25 times faster, and 2) Same assumptions as in 1 but with no shrinkage of the E-YSL (constant 20º) (contractileless - red). (b) Experimental values for the EVL advancing front speeds at different epiboly stages (ϕ angle - top) and at different times (bottom) for wild type controls (blue) and Msn1 yolk morphants with reduced E-YSL contractility (red). Experimental profiles closely approach to those inferred in the simulations (compare to (a)). (c) Timeline showing medial sections and power isolines for wild type (top) and Msn1 yolk morphants (bottom) along epiboly. Mechanical parameters were inferred by HR from experimental velocity fields (Movie EV10). The RMSE of power is shown as a percentage at each time point. DCs in the Msn1 yolk morphants undergo ingression on schedule at 50 % epiboly (yellow arrow). Importantly, as in wild type controls, the active work in morphants is detected at the E-YSL (red arrow). This power supply fades away as epiboly slows down and eventually fails. Scale bar 100 μm. (d) Tensional profiles normalized values in arbitrary units inferred by HR for wild type at 80 % epiboly and its temporal equivalent (9 hours after egg laying) for Msn1 yolk morphants. The characteristic profiles of longitudinal and latitudinal stresses and the animal to vegetal stress gradient that are built up during epiboly are never properly developed in morphants.
Endosome and Golgi-associated degradation (EGAD) of membrane proteins regulates sphingolipid metabolism (2019)
Figure 1

Figure 1: ESCRT-independent functions of the Dsc complex in membrane proteostasis.

See also Figure EV1 and Appendix Tables S1-S3.

(A) Synthetic genetic array technology of S.cerevisiae was used to cross the ESCRT mutant (query mutation vps4::NatR) with all non-essential genes (xxx::KanR). The growth of double mutants (vps4::NatR xxx::KanR) was scored in two independent replicates for normal growth, synthetic sick or synthetic lethal phenotypes. 119 genes (Appendix Table S1) showed synthetic sickness in one replicate and synthetic lethality in the other replicate or synthetic lethality in both replicates.

(B) Gene Ontology (GO) analysis for macromolecular complexes of the 119 genes that showed synthetic sickness in one replicate and synthetic lethality in the other replicate or synthetic lethality in both replicates (Appendix Table S1, 2). A hypergeometric test was used to estimate if the mapped GO term is significantly enriched with the selected genes. Significant GO terms (p < 0.05) and their associated genes identified in the screen are shown with a cut-off for 15-fold enrichment over the genome frequency.

(C) Equal amounts of WT cells and indicated single or double mutants in serial dilutions were incubated on agar plates at the indicated temperatures. The synthetic growth defects of vps4∆ tul1∆ or vps4∆ ubx3∆ double mutants at 26°C and 37°C were complemented with plasmids encoding VPS4 or TUL1 or UBX3. The dashed boxes indicate the non-complemented double mutants.

(D) Cartoon presentation of Dsc complex members and evolutionary conserved functional domains with human candidate genes that show the highest homology in the indicated domains.
Endosome and Golgi-associated degradation (EGAD) of membrane proteins regulates sphingolipid metabolism (2019)
Figure 2

Figure 2: Proteome analysis reveals Orm2 as an endogenous Dsc complex substrate.

See also Figure EV2 and Appendix Tables S4 and S5.

(A) Vacuolar proteolysis-deficient pep4∆ and tul1∆ pep4∆ cells were labeled to saturation with heavy 13C6,15C2-L-lysine and then chased for 3 hours in presence of light L-lysine, following quantitative proteome analysis by LC-MS (two biological replicates). To analyze Dsc complex-dependent protein turnover, the ratio of H/L ratios of tul1∆ pep4∆ over pep4∆ cells of 2511 proteins quantified in both strains was plotted, and proteins with a ratio of H/L ratios >2 were selected. Red squares: regulated proteins with at least one predicted transmembrane (TM) domain; black triangles: regulated proteins without TM domains.

(B) Schematic representation of the Dsc ubiquitin ligase complex (a top hit from the SGA screen) and the SPOTS complex (a putative Dsc substrate). SPT, serine-palmitoyltransferase; LCB, long chain bases.

(C-F) SDS-PAGE and Western blot analysis with the indicated antibodies (C,D) of total yeast lysates from WT cells and the indicated mutants; (E) of input and elution (with FLAG peptide) from denaturing FLAG-Orm2 immunoprecipitations (IP) from WT cells and the indicated mutants. Control cells expressed untagged Orm2; (F) of input and elution (with FLAG peptide) from native FLAG-Orm2 co-immunoprecipitations from WT cells and the indicated mutants. The control strain expresses untagged Orm2.
Endosome and Golgi-associated degradation (EGAD) of membrane proteins regulates sphingolipid metabolism (2019)
Figure 3

Figure 3: Non-ubiquitinated Orm2 accumulates in Golgi and endosome structures.

See also Figure EV3.

(A) Epifluorescence and phase contrast microscopy of living WT yeast cells and the indicated mutants expressing GFP-Orm2 or GFP-Orm2-K25,33R (green) with dsRed-HDEL (red). Arrowheads point to GFP-Orm2 accumulations outside the endoplasmic reticulum (ER).

(B) Epifluorescence and phase contrast microscopy of tul1∆ mutants expressing GFP-Orm2 (green) with Vps4-mCherry (red, endosomes) or with Sec7-mCherry (red, Golgi) or Vps4-eGFP (green) with Sec7-mCherry.

(C) Left panel: Epifluorescence and phase contrast microscopy of the indicated mutants expressing GFP-Orm2 (green) with mCherry-Cps1 (red, MVB cargo). E indicates class E compartments; Right panel: Equal amounts of the indicated mutants in serial dilutions were incubated on agar plates at the indicated temperatures.

Data information: Scale bars = 5 µm.
Endosome and Golgi-associated degradation (EGAD) of membrane proteins regulates sphingolipid metabolism (2019)
Figure 4

Figure 4: The Dsc complex mediates degradation of Orm2 at cytosolic proteasomes.

See also Figure EV4.

(A-C) WT cells and the indicated mutants were left untreated (0 min) or were treated with cycloheximide (CHX) to block protein synthesis for 90 min and 180 min at 26°C or at non-permissive temperature (37°C) where indicated. Total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. (A) Graphs display the FLAG-Orm2 protein levels determined by densitometric quantification of Western blots from cell lysates of WT cells and the indicated mutants 0, 90 and 180 min after the addition of CHX. Each experiment was repeated at least three times, FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each time-point was related to t=0 min (set to 1). Data are presented as mean ±standard deviation. (C) SEC13 WT and sec13-4 cells were preincubated for 30 minutes at 37°C before CHX was added.

(D) Epifluorescence and phase contrast microscopy of living WT cells and cdc48-3 mutants expressing GFP-Orm2 incubated for 5h at the non-permissive temperature (37°C).

(E) Epifluorescence and phase contrast microscopy of living tul1∆ and tul1∆ sec13-4 mutants expressing GFP-Orm2 (green) and dsRed-HDEL (red) incubated at 26°C or shifted for 90 minutes to the non-permissive temperature (37°C).

Data information: Scale bars 5µm.
Endosome and Golgi-associated degradation (EGAD) of membrane proteins regulates sphingolipid metabolism (2019)
Figure 5

Figure 5: Orm2 is retro-translocated by the Dsc complex and Cdc48 for proteasomal degradation.

(A) WT (pdr5∆) cells expressing FLAG-Orm2 and Sec61-GFP (integral ER membrane protein) treated with MG-132 were fractionated into insoluble membrane (P100) and soluble cytoplasmic (S100) fractions and analyzed by SDS-PAGE and Western blot with the indicated antibodies.

(B, C) WT cells, tul1∆ and cdc48-3 mutants (all in a pdr5∆ strain background) were left untreated or treated with the proteasome inhibitor (MG-132) as indicated and subsequently subjected to subcellular fractionation as in (A). S100 and P100 fractions were subjected to denaturing FLAG-Orm2 immunoprecipitations (IP). Input and eluted fractions (with FLAG peptide) were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.

(D) P100 fractions from MG-132-treated WT (pdr5∆) cells expressing FLAG-Orm2 and Sec61-GFP were extracted with Na2CO3 solution at the indicated pH and subsequently fractionated by 100.000 x g centrifugation into soluble (S) and insoluble membrane (M) fractions, subjected to denaturing FLAG-immunoprecipitations and analyzed by Western blotting with the indicated antibodies. The asterisk indicates reactivity of the secondary antibody with IgG heavy chains.

(E) Schematic representation of the degradation of Orm2 by the EGAD pathway.
Endosome and Golgi-associated degradation (EGAD) of membrane proteins regulates sphingolipid metabolism (2019)
Figure 6

Figure 6: EGAD-degradation of Orm2 contributes to sphingolipid homeostasis.

See also Figure EV5.

(A) The levels of two major ceramide (Cer) species in lipid extracts from WT (black), tul1Δ (dark gray) or Orm2-K25,33R mutants (light grey) were measured using LC-MS and quantified using spiked non-yeast ceramides as internal standard. Data are normalized to Cer44:0;4 levels of WT cells (set to 1) and presented as mean ±standard deviation from three independent experiments. Pairwise statistical significance was assessed by Student's t-test. p values are given above the respective bars.

(B) Autoradiogram of sphingolipid extracts from [3H]serine radiolabelled WT cells and tul1∆ mutants were separated by thin layer chromatography. IPC, Inositolphosphorylceramide; MIPC, mannosylinositolphosphorylceramide; MIP2C, mannosyldiinositolphosphorylceramide. X indicates an unknown lipid species that is insensitive to myriocin treatment.

(C) SDS-PAGE and Western blot analysis with the indicated antibodies of input and elution (with FLAG peptide) from native anti-FLAG co-immunoprecipitations from cells expressing FLAG-Orm2-WT or FLAG-Orm2-K25,33R. Control cells expressed untagged Orm2 WT.

(D-E) SDS-PAGE or Phos-tag-PAGE and Western blot analysis with the indicated antibodies of total cell lysates (D) from WT cells expressing FLAG-Orm2 or FLAG-Orm2-K25,33R;

(E) from WT cells and tul1∆ mutants expressing FLAG-Orm2, FLAG-Orm2-3A or FLAG-Orm2-3D with or without Myriocin treatment (1.5 µM) for one hour.

(C,D) The asterisks label unspecific cross-reactions of the respective antibodies.

(F) Schematic representation of the regulation of SPT activity via TORC2-Ypk1/2-dependent phosphorylation of Orm proteins and EGAD-dependent degradation of Orm2.
Endosome and Golgi-associated degradation (EGAD) of membrane proteins regulates sphingolipid metabolism (2019)
Figure 7

Figure 7: ER exit of Orm2 is regulated by TORC2-Ypk1 phosphorylation and essential for Orm2 degradation.

See also Figure EV5.

(A) Left panel: In tor1-1 avo3∆1274-1430 cells rapamycin specifically inhibits TORC2 but not TORC1 (Gaubitz et al., 2015). tor1-1 avo3∆1274-1430 cells were treated with cycloheximide (CHX) and rapamycin or vehicle (methanol) for the indicated times. Right panel: WT or Orm2 mutant cells were treated with CHX for the indicated times. The protein levels of FLAG-Orm2 or of the indicated FLAG-Orm2 mutants from total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.

(B) Graphs display the FLAG-Orm2 protein levels determined by densitometric quantification of Western blots in cell lysates of WT cells and the indicated mutants 0, 90 and 180 min after the addition of CHX. Each experiment was repeated at least three times. FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each timepoint was related to t=0 min (set to 1). Data are presented as mean ±standard deviation.

(C) Denaturing immunoprecipitation (IP) of FLAG-Orm2, FLAG-Orm2-3A and FLAG-Orm2-3D. The control strain expresses untagged Orm2. Input and elutions (with FLAG peptides) were analyzed by SDS-PAGE and Western blot with the indicated antibodies.

(D) Epifluorescence and phase contrast microscopy of living tul1∆ mutants expressing GFP-Orm2, GFP-Orm2-3A or GFP-Orm2-3D (green) with dsRed-HDEL (red). Arrowheads point to GFP-Orm2 accumulations outside the endoplasmic reticulum (ER). Scale bars = 5 µm.

(E) SDS-PAGE and Western blot analysis with the indicated antibodies of input and elution (with FLAG peptide) from native anti-FLAG co-immunoprecipitations from cells expressing FLAG-Orm2-K25,33R, FLAG-Orm2-K25,33R-3A or FLAG-Orm2-K25,33R-3D. The control strain expresses untagged Orm2.

(F) The levels of the long chain bases (LCB) C18-dihydrosphingosine (C18-DHS) and C18-phytosphingosine (C18-PHS) and of two major ceramide (Cer) species in lipid extracts from WT, tul1∆, Orm2-K25,33R, Orm2-3A and Orm2-3D mutants were measured using LC-MS and quantified using spiked non-yeast LCBs/ceramides as internal standard. Data are normalized to WT levels of the most abundant LCB/ceramide species (set to 1) and presented as mean ±standard deviation from three independent experiments. Pairwise statistical significance was assessed by Student's t-test. p values are given above the respective bars.

(G) Model for the dual function of the Dsc complex in EGAD-dependent proteasomal as well as ESCRT-dependent lysosomal membrane protein degradation.

Expanded view Figures legends
PABPN1L Mediates Cytoplasmic mRNA Decay as a Placeholder during the Maternal-to-Zygotic Transition (2020)
Figure 1

Figure 1. Expression and function of PABPN1L during MZT.

A Quantitative RT-PCR (RT-qPCR) results showing relative expression levels of mouse Pabpn1l in somatic tissues, oocytes, and preimplantation embryos. n = 3 biological replicates. Error bars indicate S.E.M.

B Western blot results of PABPN1L, BTG4, and phosphorylated ERK1/2 (pERK1/2) in oocytes and embryos. Total protein from 400 oocytes or embryos is loaded in each lane. DNA damage-binding protein 1 (DDB1) is blotted as a loading control.

C Western blot results of PABPN1L in MII oocytes of wild type (WT) and Pabpn1l−/− females. DDB1 is blotted as a loading control.

D Cumulative pup numbers of WT and Pabpn1l-/- female mice. n = 5 for each genotype. Error bars, S.E.M. ***P < 0.001 by two-tailed student's t-test.

E Quantification of preimplantation embryos derived from WT and Pabpn1l-/- females when WT embryos reached the corresponding stages. The number of analyzed embryos is indicated (n). Error bars, S.E.M. ***P < 0.001 by two-tailed Student's t-test.

F Representative images of the embryos collected from the oviducts of mated WT and Pabpn1l-/- females at the indicated time points after hCG injection. Scale bar = 100 μm.

Data information: For all western blot results, at least three independent experiments were done with consistent results.
PABPN1L Mediates Cytoplasmic mRNA Decay as a Placeholder during the Maternal-to-Zygotic Transition (2020)
Figure 2

Figure 2. Transcriptome analyses in Pabpn1l-deleted oocyte and embryos during the MZT.

A Scatter plot comparing the transcripts of GV oocytes and zygotes from WT and Pabpn1l-/- females. Transcripts that increased or decreased by more than 3-fold in Pabpn1l-deleted GV oocytes or zygotes are highlighted in red or blue, respectively.

B Venn diagrams showing the overlap in transcripts that are significantly degraded during the MZT (FPKM[GV/zygote] > 3) of WT oocytes and transcripts that are accumulated in this process after Pabpn1l knockout (FPKM[Pabpn1l♀−/♂+/WT] in zygote) > 3).

C Venn diagrams showing the overlap in transcripts that are stabilized during the MZT in Pabpn1l♀−/♂+ and Btg4♀−/♂+ zygotes (FPKM[GV/zygote] > 3 in WT, but < 3 in KO).

D RT-qPCR results for relative expression levels of the indicated maternal transcripts in oocytes and zygotes from WT and Pabpn1l −/− females. n = 3 biological replicates. Error bars, SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed Student's t-test.

E Poly(A) tail assay results showing poly(A)-tail length of indicated transcripts in WT and Pabpn1l-deleted oocytes and embryos. The poly(A) tails of Actin are used as an internal control. Each sample was prepared from 100 oocytes or embryos. Plots show the averaged relative signal intensity (y-axis) and length of the PCR products based on mobility (x-axis).
PABPN1L Mediates Cytoplasmic mRNA Decay as a Placeholder during the Maternal-to-Zygotic Transition (2020)
Figure 3

Figure 3. Maternal PABPN1L is essential for ZGA.

A Scatter plot comparing the transcripts of 2-cell embryos from WT and Pabpn1l-/- females. Transcripts that increased or decreased by more than 3-fold in Pabpn1l-deleted oocytes or embryos are highlighted in red or blue, respectively.

B Venn diagram and schematic showing the overlap in transcripts that are activated during zygote-to-2-cell transition in WT (FPKM[2-cell/zygote] > 3) and the transcripts that are decreased in Pabpn1l♀−/♂+ compared to WT at the 2-cell stage (FPKM[WT/Pabpn1l♀−/♂+] > 3 in 2-cell embryos).

C 5-Ethynyl uridine (EU) fluorescence (green) showing RNA transcription in WT and Pabpn1l♀−/♂+ 2-cell embryos. Some WT embryos were treated with α-amanitin as early as the zygote stage and cultured to the 2-cell stage. The phosphorylated RNA polymerase II CTD repeat YSPTSPS (pS2) (red) is co-stained to label the RNA polymerase II activity. Nuclei are labeled by DAPI (blue). Scale bar = 20 μm.

D Quantification of EU and pS2 signals in (A). The numbers of analyzed embryos are indicated (n). Error bars, S.E.M. ***P < 0.001 by two-tailed Student's t-test.

E Relative intensity of tdTomato signals in (F). The numbers of analyzed embryos are indicated (n). Error bars, S.E.M. ***P < 0.001 by two-tailed Student's t-test.

F Representative images of MuERV-L::tdTomato relative to GFP signal in the same embryo when WT embryos reached the corresponding stages. Zygotes were injected with the MuERV-L::tdTomato reporter plasmid and polyadenylated Gfp mRNA (as a positive control of microinjection), then allowed to develop in vitro. Cultured embryos were imaged at 24 h and 40 h after hCG injection. DIC, differential interference contrast. Scale bar = 100 μm.