EMBO/soda-nel huggingface dataset:
A Zeb1/MtCK1 Metabolic Axis Controls Osteoclast Activation and Skeletal Remodeling (2023)
Figure 7

Figure 7. The Zeb1/MtCK1 axis controls sealing zone formation and osteoclast activity.

A, B Human monocyte-derived macrophages (hMDMs) were transfected with either shCTRL or siRNA targeting ZEB1, differentiated into osteoclasts, and relative mRNA expression of CKMT1 (A) and mitochondrial creatine kinase activity determined (B; n = 3).

C PCr/Cr ratio of osteoclasts differentiated from non-targeting siCTRL- or siZEB1-transfected hMDMs cultured atop plastic substrata as determined by LC/MS analysis (n = 3).

D, E ATP levels (D) and RhoA activity (E) of osteoclasts differentiated from non-targeting siCTRL- or siZEB1-transfected hMDMs.

F Mitochondrial function parameters analyzed by XF Cell Mito Stress Assay in non-targeting siCTRL- or siZEB1-transfected osteoclasts after sequential treatment of compounds modulating mitochondrial function (n = 4).

G, H siCTRL- or siZEB1-transfected human osteoclasts were cultured atop bone slices for 6 days and stained with phalloidin (red). Osteoclasts were removed and resorption pits visualized by WGA-DAB staining (G). Scale bar, upper 20 μm, lower 100 μm. The actin ring area per cell, resorption pit area, and the number of TRAP+ MNCs were quantified (H; n = 6).

I-L Calvaria isolated from wild-type and Zeb1ΔM/ΔM mice were cultured in the presence or absence of 200 μM cyclocreatine, and stained for phalloidin (red) and Vpp3 (green) (I). Scale bar, 10 μm. The TRAP activity of whole cell lysates, actin ring area per cell, and supernatant CTX-I were quantified (J-L; n = 6).

Data information: Bars and error bars represent mean ± SEM. Data are representative of at least 3 independent experiments with biological replicates. Data were analyzed using one-way ANOVA with Bonferroni correction (A-F, H, J-L). ns, not significant; *P < 0.05, **P < 0.01.

EV Figure Legends
SHP-1 dephosphorylates histone H2B to facilitate its ubiquitination during transcription (2022)
Figure 1

Figure 1: Nuclear localized SHP-1 associates with Paf complex and Histone H2B.

(A) Localization of endogenous SHP-1 in hematopoietic and non-hematopoietic cells was determined by confocal imaging after staining with SHP-1 antibody. DAPI was used for nuclear staining, Scale bar:5µm. Fluorescence intensities along the line drawn in a representative cell were obtained using Zen software and the co-localization plots were made using GraphPad (Red: SHP-1, Blue: DAPI).

(B) Network of nuclear proteins associated with SHP-1 is shown. SFB-GFP purification was used as control to filter out non-specific interactions. The list of SFB-SHP-1 and SFB-GFP associated proteins was derived from our earlier study (Kumar et al., 2017). Paf complex components and Histone H2B were highlighted in blue in the network.

(C) SFB tagged PAF1, SFB-LEO1, SFB-CTR9, SFB-CDC73, SFB-RTF1, SFB-WDR61 along with Myc SHP-1 or Myc PTPN7 were transfected in 293T cells and interaction of SHP-1 with PAF complex components was detected by immunoblotting with Myc antibody after S protein beads pulldown in presence of TurboNuclease.

(D) SFB empty vector, SFB SHP-1 or SFB PTPN7 was co-transfected with Myc tagged H2B in 293T cells. The interaction of H2B with SHP-1 was detected through immunoblotting using Myc antibody after pull down with streptavidin sepharose beads.

(E) Endogenous association of SHP-1 with Paf1 complex subunits (CDC73, RTF1, WDR61, LEO1, Paf1, CTR9) was detected by immunoblotting after performing immunoprecipitation with either IgG or SHP-1 antibody.

(F) Immunoprecipitation (IP) with control IgG or anti-SHP-1 antibody was performed with extracts prepared from MCF7 cells and endogenous association of H2B with SHP-1 was detected by immunoblotting.

(G) Glutathione Sepharose beads immobilized with bacterially expressed recombinant GST or GST-H2B proteins were incubated with bacterially purified recombinant MBP-SHP-1. The association of SHP-1 with H2B was detected by immunoblotting with MBP antibody. Expression of GST, GST-H2B and MBP SHP-1 was shown by Coomassie staining.
SHP-1 dephosphorylates histone H2B to facilitate its ubiquitination during transcription (2022)
Figure 2

Figure 2: SHP-1 is required for efficient H2B ubiquitination.

(A) Nuclear extracts of WT and SHP-1 knockout cells (derived from two independent guide RNAs) were made and the levels of H2BUb were detected.

(B) Wild type (WT) 293T cells along with SHP-1 KO cells transfected with either Myc vector, Myc SHP-1 ∆SH2 or Myc SHP-1 NLS mutant were lysed. H2BUb levels were determined using specific antibody.

(C) WT and SHP-1 KO 293T cells transfected with Myc vector, Myc SHP-1 or Myc SHP-1 C/S (C453S) mutant were lysed. H2BUb levels were determined using specific antibody.

(D) H2B WT along with the indicated Y/F and Y/D mutants were transfected into 293T cells. Histone extract was prepared from cells transfected with each construct and subjected to western blotting with anti-H2B and anti-H2BUb antibodies.

(E) In vitro ubiquitination assay was performed using bacterially purified GST, GST tagged H2B and its mutants as substrates. Recombinant E1, E2(UBE2A) and ubiquitin were included in each reaction. Ubiquitination was detected by H2BUb antibody.

(F) In vitro ubiquitination was performed using biotin tagged non-phospho and phospho (pY121) peptide of H2B as substrates in the presence of E1 and E2. H2BUb was detected by immunoblotting with specific antibody.
SHP-1 dephosphorylates histone H2B to facilitate its ubiquitination during transcription (2022)
Figure 3

Figure 3: Histone H2B is a new substrate for SHP-1.

(A) In vitro phosphorylated GST H2B by Syk kinase was incubated with bacterially purified recombinant wild type SHP-1 or elution buffer and the amount of released phosphate was assayed colorimetrically using the malachite green reagent (A620 nm). Data represent mean absorbance from four independent experiments. Error bar indicates SD; *p<0.05, by Student's t test.

(B) Bacterially purified GST H2B was subjected to in vitro kinase assay using HEK293T cell lysate, and then phosphorylated proteins were incubated with either bacterially purified MBP tagged constitutively active (SHP-1 ΔSH2), or catalytically inactive SHP-1 C/S mutant. The phosphorylation status of recombinant H2B was detected by immunoblotting with phospho-tyrosine antibody.

(C) Phospho (pH2B) Y121 and non-phospho peptide of H2B was incubated with constitutively active (SHP-1 ΔSH2) or catalytically inactive mutant (C/S) of SHP-1 and the released phosphate was assayed colorimetrically using the malachite green reagent (A620nm). n=3 independent experiments, Error bars indicate SD; ***p<0.001 (one-way ANOVA, Tukey's multiple comparison test).

(D) 293T cells were transfected with vector, Myc tagged SHP-1 WT or C/S mutant. The phosphorylation status of endogenous H2B was detected by immunoblotting with pY121-H2B antibody.

(E) Nuclear extracts of 293T WT and SHP-1 KO cells were made and the levels of H2B phosphorylation were detected using pY121-H2B antibody.

(F) Wild type (WT) along with SHP-1 KO 293T cells transfected with either Myc vector, Myc SHP-1 or Myc SHP-1 C/S mutant were lysed. Levels of pY121-H2B and H2BUb were determined using specific antibodies.

(G) 293T Cells were treated with SHP-1 inhibitor TPI (50nm) for 1hr and levels of pY121-H2B was determined using pY121H2B antibody.

(H-J) MCF7 cells were transduced with (H) CTR9 shRNA, (I) Paf1 shRNA, or (J) RTF1 shRNA along with control shRNA. Post 72 h, cells were collected and lysed to isolate soluble and nuclear fractions. Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies.
SHP-1 dephosphorylates histone H2B to facilitate its ubiquitination during transcription (2022)
Figure 4

Figure 4: H2B Y121 dephosphorylation by SHP-1 is necessary for E2 binding.

(A) Cell lysates derived from 293T cells transfected with empty vector, SFB-tagged H2B, H2B Y121F or H2B Y121D constructs were pulled down with S-protein agarose beads. RNF20 interaction with H2B was detected by immunoblotting with specific antibody.

(B) HEK293T cells were co-transfected with the SFB-tagged H2B, H2B Y121F or H2B Y121D constructs along with Myc-tagged UBE2A. Cell lysates were pulled down with S-protein beads, and interaction was detected by immunoblotting with Myc antibody.

(C) Glutathione Sepharose beads bound with bacterially expressed recombinant GST-H2B, GST H2B Y121F and GST-H2B Y121D proteins were incubated with bacterially purified recombinant His UBE2A, and interaction of UBE2A with H2B was detected by immunoblotting with His antibody. Recombinant protein expression and pulldown was shown by Coomassie staining.

(D) Bacterially purified GST, GST H2B and GST H2B-p (phosphorylated using Syk) was incubated with recombinant His UBE2A, and interaction of UBE2A with H2B was detected by immunoblotting with His antibody. Recombinant protein expression and pulldown was shown by Coomassie staining.

(E) WT 293T cells along with SHP-1 KO were co-transfected with SFB vector or SFB H2B along with Myc UbE2A. Cell lysates were pulldown using S-protein beads, and interaction of UBE2A with H2B was determined by Western blotting with the indicated antibodies.

(F) HEK293T cells were transfected with SFB vector or SFB H2B along with Myc UBE2A.Cells were treated with SHP-1 inhibitor TPI (50nm) for 1hr after 24hrs of transfection. Cells lysates were subjected to pull-down with S-protein beads, and western blotting was performed using specific antibodies.
SHP-1 dephosphorylates histone H2B to facilitate its ubiquitination during transcription (2022)
Figure 5

Figure 5: SHP-1 participates in Pol II mediated transcription

(A) Representative super-resolution images of MCF7 cells immunostained with SHP-1 in combination with CDK9, total RNA Polymerse II, RNA polymerase II Ser2P, and RNA Polymerase II Ser5P. Scale bar: 5µm. Fluorescence intensities along the line drawn in a representative section of cell were obtained using Zen software and the co-localization plots were made using GraphPad.

(B) Co-localization of SHP-1 with CDK9, total RNA Polymerase II, RNA Polymerase II S2P, and RNA Polymerase II S5P was measured by Pearson correlation coefficient using ImageJ. Values presented as the mean ± SD, n=10 (from three independent experiments). ***p value < 0.001. (One-way ANOVA).

(C) Endogenous interaction of SHP-1 with RNA Polymerase II, RNA polymerase II Ser2P, and RNA Polymerase II Ser5P and CDK9 was detected by immunoblotting after performing immunoprecipitation with either IgG or SHP-1 antibody using TurboNuclease treated cell lysate.

(D) Nuclear extracts of MCF-7 WT and SHP-1 knockout cells were made and the levels of RNA Polymerase II, RNA Polymerase II Ser2P and RNA Polymerase II Ser5P were detected by immunoblotting using specific antibodies.

(E) Nuclear extracts of cells treated with either DMSO or SHP-1 inhibitor TPI (50nm) were used to detect total RNA Polymerase II and its modifications by immunoblotting using specific antibodies.

(F) Nuclear extracts of MCF-7 WT and SHP-1 knockout cells transfected with Myc vector, H2B Y121F or H2B Y121D constructs were made and the levels of RNA Polymerase II, RNA Polymerase II Ser2P, RNA Polymerase II Ser5P and Lamin B1 were detected by immunoblotting using specific antibodies.

(G) Immunofluorescence of WT or SHP-1 knockout MCF7 cells labelled with the 5-Ethynyl Uridine (EU) for 60 minutes to capture nascent RNA synthesis. Scale bars 10μm.

(H) The EU incorporation in MCF-7 WT and SHP-1 KO cells was quantified in three independent experiments. Values presented as the mean ± SD, ***indicates p<0.001 (One-way ANOVA).

(I) Representative images of immunofluorescence of DMSO or TPI treated (50nm) MCF7 cells labelled with EU for 60 minutes was shown. Scale bar: 40µm.

(J) The EU incorporation in MCF7 cells treated with DMSO or TPI was quantified in three independent experiments. Values presented as the mean ± SD, ***indicates p<0.001 (One-way ANOVA).
SHP-1 dephosphorylates histone H2B to facilitate its ubiquitination during transcription (2022)
Figure 7

Figure 7: SHP-1 facilitates H2Bub and Pol II occupancy at transcriptional sites via H2B dephosphorylation

(A) ChIP-qPCR using either anti-IgG or anti-SHP-1 antibody was performed in MCF-7 cells and the binding of SHP-1 at TSS region adjacent to the promoter region of the ATG2B, ATG13 and ULK1 genes was determined. The Y-axis represents the percentage input signal. The data shown is derived from three independent experiments. Error bars indicate the mean ± SD; **P<0.01; *P<0.05, Student's t-test.

(B) Expression of indicated genes in WT and SHP-1 knockout MCF7 cells was measured by qRT-PCR from three independent experiments. Values presented as the mean ± SD. *P<0.05 and ***p value < 0.001, Student's t-test.

(C, D) MCF-7 WT cells along with SHP-1 KO cells transfected with either vector, H2B Y121F or H2B Y121D mutant were used for ChIP q-PCR analysis of the H2BUb at indicated genes. The data shown is derived from two independent experiments. Error bars indicate the mean ± SD.

(E) MCF-7 WT cells along with SHP-1 KO cells transfected with either Myc vector, H2B Y121F or H2B Y121D mutant were used for ChIP-qPCR analysis of the RNA Pol II at indicated genes. The data is derived from two independent experiments. Error bars indicate the mean ± SD.

(F) Expression of indicated genes in WT and SHP-1 knockout MCF7 cells transfected with Myc vector, Myc H2B Y121F or Myc H2B Y121D mutant was measured by qRT-PCR from three independent experiments. Values presented as the mean ± SD. *P<0.05, **P<0.01, one-way ANOVA.
SHP-1 dephosphorylates histone H2B to facilitate its ubiquitination during transcription (2022)
Figure 8

Figure 8: SHP-1 is required for maintenance of basal autophagic flux

(A) Lysates of WT and SHP-1 knockout MCF7 cells were made and the levels of LC3-I and II were detected by immunoblotting.

(B) Levels of LC3 II were normalized against total LC3 (LC3 I+LC3 II). The data shown is derived from three independent experiments. Error bars indicate the mean ± SD. *p value < 0.05, Student's t-test.

(C) Representative super- resolution images of WT and SHP-1 KO MCF7 cells immunostained with LC3 antibody, (Scale bar: 5µm).

(D) Quantification of LC3 puncta accumulation per cell done by using ImageJ was shown. Bars represents mean ± SD.(N=60) ***p value < 0.001, Student's t-test.

(E) WT along with SHP-1 KO MCF7 cells transfected with either Myc vector, Myc SHP-1 or Myc SHP-1 NLS mutant were lysed. Levels of LC3 and actin were determined using specific antibodies.

(F) WT and SHP-1 knockout MCF7 cells transfected with the Myc vector or Myc-tagged H2B, H2B Y121F or H2B Y121D constructs were immunostained with endogenous p62 and Myc antibody. Image acquisition was done using LSM 700, (Scale bar: 20µm).

(G) Accumulation of p62 was quantified by counting p62 positive puncta per cell. (N= 25); values presented as the mean ± SD. ***p value < 0.001 (One-way ANOVA).

(H) Representative super-resolution images of LC3 and LAMP2 immunostaining in WT and SHP-1 KO MCF7 cells. (Scale bar: 20 µm for merge & 5 µm for Zoom).

(I) LC3 puncta co-localizing with LAMP2 per cell were quantified using ImageJ. (N=45); values presented as the mean ± SD. ***p value < 0.001, Student's t-test.

(J) A proposed model to depict the role of nuclear SHP-1 during active transcription. SHP-1 dephosphorylates H2B at Y121 residue, thereby allowing ubiquitination of K120 site and productive transcription. Loss of SHP-1 leads to impaired transcription as H2B stays in a constitutively phosphorylated state at Y121 thus precluding interaction of E2 enzyme and failure to ubiquitinate H2B.

Expanded view figures
Intellectual disability associated gene ftsj1 is responsible for 2'-O-methylation of specific tRNAs (2020)
Figure 1

Figure 1. Subcellular localization and protein-protein interactions of FTSJ1.

(A) Immunofluorescence labeling of FTSJ1-Flag (red) in HEK293T cells. The nucleus was stained by DAPI (blue). Scale bar, 20 μm.

(B) Subcellular localization of FTSJ1. Cytoplasmic (C) and nuclear (N) fractions were separated from HEK293T cells expressing FTSJ1-Flag. α-Tubulin and Lamin A/C were used as indicators of the cytoplasmic and nuclear fractions, respectively.

(C) Co-IP assays of the interaction between FTSJ1 and WDR6 in co-transfected HEK293T cells. Anti-HA IP (top) and anti-Flag IP (middle) were immunoblotted by anti-HA and anti-Flag antibodies, respectively. Bottom: cell lysates immunoblotted by anti-HA or anti-Flag antibodies, with GAPDH serving as a loading control.

(D) Co-IP assays of the interaction between FTSJ1 and WDR6 with RNase A addition.
Intellectual disability associated gene ftsj1 is responsible for 2'-O-methylation of specific tRNAs (2020)
Figure 2

Figure 2. FTSJ1 directly binds to WDR6.

(A) Schema showing a series of truncated FTSJ1 mutants used to map FTSJ1 domain(s) responsible for WDR6 binding.

(B, C) Co-IP assays of the interaction between FTSJ1-Flag or truncated FTSJ1 mutants and WDR6-HA in co-transfected HEK293T cells. Anti-Flag IP (top) were respectively immunoblotted by anti-HA and anti-Flag antibodies. Bottom: cell lysates immunoblotted by anti-HA or anti-Flag antibodies, with GAPDH serving as a loading control.

(D) The structure of DIZPK and scheme showing the capture of FTSJ1 interacting proteins using DIZPK.

(E) IP assays of FTSJ1-Asn221DIZPK. The empty vector was transfected as a negative control.

(F) A list of top FTSJ1-Asn221DIZPK putative interacting proteins identified by IP-MS in HEK293T cells.
Intellectual disability associated gene ftsj1 is responsible for 2'-O-methylation of specific tRNAs (2020)
Figure 3

Figure 3. Knockout of ftsj1 and/or wdr6 affect Cm, Gm, m1G and o2yW levels of tRNAPhe(GAA).

(A, B) Schematic depiction of ftsj1 and wdr6, and target sites of mutations introduced by the CRISPR-Cas9 system in HEK293T cells. Shaded and open boxes indicate coding regions and untranslated regions of exons, respectively. Lines indicate introns. For ftsj1, two sgRNA sequences for targeting exons 2 and 3 are noted. For wdr6, two sgRNA sequences for targeting exons 1 and 2 are noted. Sequences of both alleles of ftsj1 or wdr6 in KO1 and KO2 cell lines are aligned. Deleted nucleotides are indicated as dashed lines.

(C) Secondary structures of human tRNAPhe(GAA) with modified nucleosides.

(D-H) Mass chromatograms of the nucleosides, Cm (Q1/Q3=258.1/112.1), Gm (Q1/Q3=298.1/152.1), m1G (Q1/Q3=298.1/166.1), m7G (Q1/Q3=298.1/166.1) and m2G (Q1/Q3=298.1/166.1), o2yW (Q1/Q3=541.2/409.0), m5C (Q1/Q3=258.1/126.1) and A (Q1/Q3=268.1/136.2) of tRNAPhe(GAA) isolated from WT, ftsj1 KO and wdr6 KO cells. Target peaks are indicated by black triangles. m2G, 2-methylguanosine; D, dihydrouridine; m22G, N2,N2-dimethylguanosine; Cm, 2'-O-methylcytidine; Gm, 2'-O-methylguanosine; m5C, 5-methylcytidine; m7G, 7-methylguanosine; m5U, 5-methyluridine; m1A: 1-methyladenosine; ψ: pseudouridine; o2yW, peroxywybutosine. Q1/Q3: the mass of the precursor ion and the mass of the product ion. Combined the retention time of standard product, we marked the o2yW in Figure 3G. Considering the change of this peak area was consistent with that of o2yW, we speculated the other peak was generated by the intermediate product of o2yW with nature isotope labelled.
Intellectual disability associated gene ftsj1 is responsible for 2'-O-methylation of specific tRNAs (2020)
Figure 4

Figure 4. FTSJ1-WDR6 catalyzes Gm34 formation on tRNAPhe(GAA) with m1G37 in vitro.

(A) Secondary structures of the anticodon loop of three types of tRNAPhe(GAA)s: tRNAPhe(GAA), △ftsj1_tRNAPhe(GAA), and m1G37_tRNAPhe(GAA).

(B, C) Quantification of the Cm/A and Gm/A of tRNAPhe(GAA), △ftsj1_tRNAPhe(GAA), and m1G37_tRNAPhe(GAA) after incubation with FTSJ1, WDR6, and FTSJ1-WDR6 by UPLC-MS/MS analysis, respectively. Error bars represent the standard deviation of three independent experiments. n.d., not detected.

(D) UPLC-MS/MS analysis of 2'-O-methylation of m1G37_tRNAPhe(GAA), -G34A, -G34C and G34U after incubation with FTSJ1-WDR6. 2 μL of Gm (1 ng/mL), Am (1 ng/mL), Cm (1 ng/mL) and Um (5 ng/mL) standards were injected to UPLC-MS/MS as control. cps, counts per second.
Intellectual disability associated gene ftsj1 is responsible for 2'-O-methylation of specific tRNAs (2020)
Figure 5

Figure 5. The substrate binding affinity of FTSJ1.

(A) The SAM binding affinity of FTSJ1 as measured by ITC.

(B, C) The binding affinity of FTSJ1 alone or FTSJ1-WDR6 for tRNA analyzed by the gel mobility shift assay. For the reaction in figure C, 0.2 μΜ GST, 0.2 μΜ GST-WDR6, 1 μM FTSJ1, the mixture of 1 μM FTSJ1 with 0.2 μΜ GST-WDR6 or 0.2 μΜ GST, 0.5 mM SAM and 250 nM tRNAPhe(GAA) transcript were incubated.
Intellectual disability associated gene ftsj1 is responsible for 2'-O-methylation of specific tRNAs (2020)
Figure 6

Figure 6. The Nm32/A% and Nm34/A% ratios of some tRNAs.

The representative graphs for quantification of the expected Nm32/A% and Nm34/A% ratios of G37-tRNAPhe(GAA), tRNALeu(UAA), tRNATrp(CCA), tRNAArg(ACG), tRNAArg(UCG), and A37-tRNAMet(CAU), which were isolated from WT, ftsj1 KO, and wdr6 KO cells by UPLC-MS/MS analysis. Error bars represent the standard deviation of three independent experiments. p values were determined using two-tailed Student's t-test for paired samples. ***p < 0.001. n.s., no significance. n.d., not detected.
Intellectual disability associated gene ftsj1 is responsible for 2'-O-methylation of specific tRNAs (2020)
Figure 7

Figure 7. FTSJ1 affects cell proliferation and translation efficiency of the UUU codon.

(A, B) The growth curve of WT and two ftsj1 KO HEK293T cell lines under normal culture or in the culture condition with 0.2 mg/mL paromomycin assayed by cell counting kit-8 proliferation analysis.

(C, D) The growth curve of WT with or without overexpressing tRNAPhe(GAA) under normal culture or with 0.2 mg/mL paromomycin.

(E, F) The growth curve of ftsj1 KO cells with or without overexpressing tRNAPhe(GAA) under normal culture or with 0.2 mg/mL paromomycin.

(G) The RNA reporter vector encodes F-luc as the primary reporter and R-luc on the same plasmid as the internal transfection control. The effect of tRNAPhe(GAA) in protein translation was revealed by a reporter assay. 6×TTC (Phe) or 6×TTT (Phe) were inserted after the PGK promoter region of F-luc as the positive reporter.

(H, I) The translation efficiency of F-luc with the 6×UUC codons or 6×UUU codons.

(J, K) The translation efficiency of F-luc with all UUC codons or all UUU codons.

(L) The frequency usage analysis of UUU and UUC codons showed that in 488 genes at least one transcript was found to use the UUU codon for more than 80% of Phe codons. Among the 488 genes, 189 genes are involved in the nervous system and brain functions.

Data information: Error bars represent the standard deviation of three independent experiments. p values were determined using two-tailed Student's t-test for paired samples. *p < 0.05, **p < 0.01. n.s., no significance; OD450, optical density at 450 nm.
The fitness cost and benefit of phase separated protein deposits (2019)
Figure 1

Figure 1. Model proteins and design of experimental system

A. Protein phase separation can lead to the formation of protein assemblies with varying dynamics. Soluble proteins may phase separate into liquid-like droplets or insoluble deposits that according to their viscoelastic properties and their ability to exchange components with the cytoplasm vary from being dynamic to static.

B. Design of the modular system to measure the effect of protein phase separation on cell fitness.

C. Growth media composition and the essential/non-essential/toxic roles of Ura3p.

D. The model proteins consist of a fusion between Ura3p and GFP (URA3sol, left) and the amyloid-β-peptide of 42 residues (Aβ) (URA3agg, right). After 18 hours of expression Ura3psol remains homogeneously distributed whereas Ura3pagg is accumulated into intracellular foci.