EMBO/SourceData huggingface dataset:
Figure 1C Calculated BRET ratios for the indicated protein pairs. The positive control protein PA-mCit-NL and the interacting fusion proteins NL-BCL2L1 and PA-mCit-BAD show high BRET ratios.D Calculated LuC ratios for the indicated protein pairs. The positive control proteins PA-NL and PA-mCit-NL and the interacting proteins NL-BCL2L1 and PA-mCit-BAD show high LuC ratios.
Figure 2Reproducibility of LuTHy PPI mapping experiments with positive and negative reference sets. The scatter plots show the mean cBRET (A) ratios from two independent experiments (Exp 1 and Exp 2), the calculated two-tailed Pearson correlation coefficient is indicated; ***p<0.001.Reproducibility of LuTHy PPI mapping experiments with positive and negative reference sets. The scatter plots show the mean cLuC (B) ratios from two independent experiments (Exp 1 and Exp 2), the calculated two-tailed Pearson correlation coefficient is indicated; ***p<0.001.C Receiver operating characteristic (ROC) analysis of cBRET (AUC=0.80±0.04) and cLuC (AUC=0.72±0.04) data determines the thresholds to define true positive binary interactions.D Performance of BRET and LuC assays in benchmarking studies with hPRS and hRRS.
Figure 3C Recovery rates of PPIs from affinity-based sub-groups with BRET and LuC assays. Significance was calculated by two-sided Fisher's exact test; *p<0.05. D Success of PPI detection from AIRS sub-groups considering single and double LuTHy readouts.E Scatter plot depicting the relationships between published binding affinities (KD) and in-cell BRET50 measurements for 25 interactions in AIRS; BRET50 values are the mean from two independent experiments. Significance was calculated by two-tailed Spearman correlation; ***p<0.001.
Figure 4A Live-cell bioluminescence imaging (BLI) of HEK293 cells co-producing the indicated fusion proteins. mCitrine was excited at 500 nm, emitted fluorescence was detected at 535 nm. After substrate addition, short (460) and long (535) band-pass (BP) filters in a dual-view adapter were used to detect the emitted luminescence simultaneously at the respective wavelengths. BRET images were calculated by dividing the 535 BP by the 460 BP images using ImageJ. Scale bar = 20 μm.B Quantification of the rapamycin-induced interaction between NL-FKBP12 and PA-mCit-FRB. Plasmids encoding the fusion proteins were co-transfected in HEK293 cells and 24 h later treated with the indicated concentrations of rapamycin. After an additional incubation for 4 h BRET was quantified upon substrate addition. After cell lysis in buffer with indicated rapamycin concentrations, LuC ratios were determined. EC50 values were obtained with non-linear curve fitting.Plasmids encoding the fusion proteins were co-transfected in HEK293 cells and After an additional incubation for 4 h BRET was quantified upon substrate addition. LuC ratios were determined. EC50 values were obtained with non-linear curve fitting. C Effects of Nutlin-3 on the interaction between NL-MDM2 and PA-mCit-p53. for 6 h treatment with Nutlin-3.D Time resolved HSF1 oligomerization by Hsp90 inhibition. HEK293 cells co-producing NL-HSF1 and PA-mCit-HSF1 for 24 h were transferred to 384-well plates containing Ganetespib or Geldanamycin to reach a final concentration of 1 µM. Luminescence was measured at the indicated time points and the calculated BRET ratios normalized to the respective untreated control. Data are the mean of two biological replicates. All values are mean ± sem. Significance was calculated by two-way ANOVA followed by Dunnett's multiple comparison's post-hoc test.E Schematic depiction of a heat shock experiment. HEK293 cells co-producing NL-HSF1/PA-mCit-HSF1 for 48h were subjected to a heat shock at 42°C for 6 h, after which the cells were recovered at 37°C for 24h. Luminescence was measured at the indicated time points: immediately before the heat shock, during the heat shock at 90, 180 and 360 min and after a recovery phase of 24 h. Calculated cBRET ratios were normalized to a control plate that was not heat treated. All values are mean ± sem from three independent experiments. Significance was calculated by one-way ANOVA followed by Dunnett's multiple comparisons post-hoc test; *p<0.05.
Figure 5B,C,D VCP and the UBX domain-containing proteins were co-expressed as N- and C-terminal NL and PA-mCit fusion proteins in eight different orientations for which cBRET (B) and cLuC (C) ratios were generated. cBRET and cLuC values over the threshold of ≥0.01 and ≥0.03, respectively, are colored in cyan. D The cBRET and cLuC ratios were normalized to the highest of the obtained eight values to easily identify the orientation among the eight tested interactions with the highest cBRET and cLuC value.
Figure 6Tukey box plots of the mean cBRET (B) ratios obtained from PPI experiments with wild-type CSPα-mCit-PA and its mutant variants.Tukey box plots of the mean cLuC ratios (C) obtained from PPI experiments with wild-type CSPα-mCit-PA and its mutant variants.D Number of screened presynaptic proteins manually annotated according to their function and localization in the presynaptic terminal. Number of proteins identified with wild-type (wt) CSPα-mCit-PA or its mutant variants (ΔL116 and L115R) are indicated.E Influence of mutations in CSPα (ΔL116, L115R) on the interactions with ZDHHC17 in primary hippocampal neurons. BRET and LuC ratios were systematically quantified for interactions with wild-type and mutant CSPα fusion proteins.F Immunoblot analysis of SHEP cells transfected with plasmids encoding wild-type CSPα-mCit-PA and mutant variants. Blot was developed with an anti-CSP antibody; loading control: anti-Histone-H3 antibody. Arrowhead and bracket indicate monomeric CSPα-mCit-PA and SDS-insoluble protein aggregates, respectively.G Immunofluorescence image of SHEP cells co-transfected with NL-ZDHHC17 and CSPα-mCit-wt, -ΔL116 or -L115R constructs lacking the PA-tag. NL-ZDHHC17 was stained with an anti NanoLuc antibody. Scale bar: 20 µm.
Figure 1A Flow cytometric quantification of lymphocytes, monocytes, B-cells, natural killer (NK) cells, dendritic cells (DCs) and granulocytes in peripheral venous blood samples from non-diabetic (ND) and type-2 diabetic (T2D) patients with COVID-19.B Quantification of monocyte subpopulation phenotypes as classical (CD14Hi CD16-), intermediate (CD14Hi CD16+) or non-classical (CD14Lo CD16+).C Principle component analysis of lymphocyte and monocyte population frequencies in ND and T2D COVID-19 patients and in T2D patients without COVID-19.
Figure 2A Quantification of CD14 expression and size (FSC) in monocyte populations from non diabetic (ND) and type-2 diabetic (T2D) COVID-19 patients.B Frequency of conventional (FSCLo) and large (FSCHi) monocytes in ND and T2D COVID-19 patients.C Expression of CD16, CD14 and HLA-DR in FSCLo and FSCHi monocytes from COVID-19 patients.D Proportions of classical, intermediate and non-classical monocytes within FSCLo and FSCHi monocytes from COVID-19 patients.E Correlative analyses of IL8 expression in peripheral blood mononuclear cells (PBMCs) to monocyte FSC.F IL8, IL6 and CCL2 mRNA expression in PBMCs from ND and T2D COVID-19 patients.G Monocyte size quantified in ND and T2D COVID-19 patients treated in general wards (Gen) or in intensive care unit (ICU).
Figure 3A Quantification of IRF5 and IFNB1 mRNA expression in peripheral blood mononuclear cells of non-diabetic (ND) and type 2 diabetic (T2D) COVID-19 patients (A).B Histograms of IRF5 expression on different populations analysed by flow cytometry on venous blood samples.C IRF5 median fluorescence intensity (MFI) in monocytes of ND and T2D patients with COVID-19.D IRF5 expression overlaid onto monocyte phenotypic gating.E IRF5 expression (MFI) in monocyte subtypes from ND and T2D COVID-19 patients.F HLA-DR and IRF5 expression in monocytes from COVID-19 patients.G IRF5 expression in monocytes of ND or T2D COVID-19 patients admitted to intensive care unit (ICU) or treated exclusively in general wards (Gen) .H IRF5 expression in monocytes of ND or T2D COVID-19 patients admitted ICU or treated exclusively in Gen.
Figure 4B Proportions of lymphocytes and monocytes in peripheral venous blood from T2D patients with COVID-19 either treated in Gen or requiring ICU admission.C Phenotypic analysis of lymphocyte and monocyte subpopulations in peripheral venous blood from T2D COVID-19 patients treated in Gen or requiring ICU treatment.
f1a) QF-FRL of unilamellar liposomes containing 95 mol% of phosphatidylcholine and 5 mol% of a phosphoinositide, labelled with GST-PX. Only liposomes containing PtdIns(3)P were labelled intensely. GST-PXR58A, a PX-domain mutant that does not bind PtdIns(3)P, yielded virtually no labelling in PtdIns(3)P-containing liposomes. Scale bar, 0.2 μm.(b) QF-FRL of unilamellar liposomes containing 0-5 mol % of PtdIns(3)P. Scale bar, 0.2 μm. The labelling density increased roughly in proportion to the PtdIns(3)P content of the liposomes. The experiment was repeated twice and the number of colloidal gold particles per μm2 was measured in more than 75 randomly selected liposomes. Values for all concentrations are statistically different from those in the adjacent conditions (for example, 1 versus 2 mol%; mean±s.e.m.; Student's t-test; P0.05).
f2(b) QF-FRL in wild-type yeast in log phase. PtdIns(3)P was labelled with GFP-PX and marked with colloidal gold (10 nm). The vacuolar membrane PF was labelled intensely, whereas other organellar membranes did not exhibit significant labelling. Scale bar, 0.5 μm.c) Quantification of the labelling intensity of PtdIns(3)P (mean±s.e.m.). The experiment was repeated three times and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images.(d,e) Negative controls. Both wild-type yeast incubated with GST-PXR58A (d) and vps34Δ yeast incubated with GST-PX (e) were virtually unlabelled. Scale bar, 0.5 μm.
f3(a) Wild-type yeast cultured in S(-NC) for 4 h. PtdIns(3)P was labelled intensely in the EF of autophagosomal membranes. A lower but still significant amount of labelling was also seen in the PF of autophagosome membranes as well as in the EF of the vacuolar membrane. Scale bar, 0.5 μm.(b) Autophagosomes in wild-type yeast cultured in S(-NC) for 4 h. The autophagosome was identified as a smooth-surfaced double-membrane structure, and seen in two different patterns, as shown in the diagrams in c. Upper panel: convex structures displaying the outer membrane EF and the inner membrane PF. Lower panel: concave structures displaying the outer membrane PF and the inner membrane EF. In both cases, PtdIns(3)P was labelled more intensely in the EF than in the PF. Scale bars, 0.2 μm.
f4(a) Wild-type yeast cultured in S(-NC) for 4 h. PtdIns(3)P was labelled intensely in the EF of autophagic body membranes in the vacuolar lumen. A lower amount of labelling was also seen in the PF of autophagic body membranes. Scale bar, 0.5 μm. ((c) Labelling intensity of PtdIns(3)P in wild-type yeast cultured in S(-NC) for 4 h (average±s.e.m.). PtdIns(3)P was found in both leaflets of the autophagosome and autophagic body membrane, and the labelling was much stronger in the EF than in the PF. In the vacuolar membrane, significant labelling was found in the EF, albeit at much lower levels than in the PF. The experiment was repeated three times, and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images.
f5(a) Huh7 cells expressing GFP-LC3 treated with 0.25 μM Torin1 and 0.4 μM bafilomycin A1 for 1 h. The autophagosome was identified as the IMP-poor double-membrane structure labelled with anti-GFP antibody (large gold, 10 nm). Both PtdIns(3)P (small gold, 6 nm) and GFP-LC3 were seen exclusively in the PF in both convex and concave structures. The small and large gold particles are shown as green and red dots, respectively, in the figures at right. Scale bars, 0.1 μm. Figures displaying wider areas, as well as additional examples, are presented in Supplementary Fig. 4c. ((b) NIH3T3 cells expressing Atg4C78A were incubated in Earle's balanced salt solution for 1 h. The isolation membrane was observed as parallel membranes with few IMPs. PtdIns(3)P was labelled only in the PF. Scale bar, 0.1 μm. Other examples are presented in Supplementary Fig. 5.(c) Labelling density of PtdIns(3)P in mammalian autophagosomal membrane (mean±s.e.m.). The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images. Some labelling was observed in the EF but the majority of gold particles in the EF occurred near the EF-PF boundary (white circles; see Supplementary Fig. 6c for more examples), suggesting that many of them actually represent PtdIns(3)P in the PF.
f6(a) ymr1Δsjl3Δ cells cultured in S(-NC) for 4 h. PtdIns(3)P was labelled in comparable densities in PF and EF of autophagosomal membranes. Scale bar, 0.2 μm.(b) The PF/EF ratio of the PtdIns(3)P labelling density in autophagosomal membrane (mean±s.e.m.). The ratio was small (that is, PF) in WT, but close to 1 (that is, PF≅EF) in ymr1Δsjl3Δ. The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images.
Figure 1(A-B) Live third instar discs incubated in DiBAC. DiBAC fluorescence is observed in the wing imaginal disc in both apical (A-A'), and basal (B-B') optical sections. Increased fluorescence is observed in the center of the pouch (white arrow, B'), and at the dorsoventral (D-V) compartment boundary in the anterior compartment (yellow arrow, B', and inset, D).(C-E) Comparison of DiBAC with the voltage-insensitive dye FM4-64. Incubation of live discs in FM4-64 (E) shows more uniform fluorescence when compared to DiBAC (D). Quantitative comparison of fluorescence in the two white boxes in each panel is shown in (C). N=7 discs for each treatment, data compared using an unpaired t test, ** indicates p<0.001, error bars are standard deviations. The region boxed in yellow in (D) is shown at higher magnification to show DiBAC fluorescence at the D-V compartment boundary.(F, G) Incubation in ouabain results in brighter and more uniform DiBAC fluorescence.(H-H'') Live wing discs expressing UAS-RFP anterior to the compartment boundary, under the control of ptc-Gal4, were incubated in DiBAC, showing that the stripe of increased DiBAC fluorescence coincides with the posterior edge of ptc expression. White arrowhead in (H) indicates the stripe in the wing pouch; yellow arrowheads indicate the stripe in the dorsal and ventral hinge.(I, I') Early L3 discs incubated in DiBAC. Patterned depolarization is evident throughout the third larval instar, with the stripe of increased fluorescence becoming narrower in more mature discs with developmental time (Compare I with A, bracket in I indicates width of increased DiBAC fluorescence).
Figure 2Expression of Rpk is increased anterior to the A-P compartment boundary, and in two rows of cells flanking the D-V compartment boundary in the A compartment.(B-B'') Blockade of DEG/ENaC channels by incubation in amiloride abolishes the stripe of increased DiBAC fluorescence along the A-P compartment boundary (white arrowhead). White boxes indicate regions used to calculate the average DiBAC fluorescence intensity ratio across the compartment boundary = 0.98, standard deviation = 0.05, n = 5 discs. Increased fluorescence at the D-V boundary in the A compartment is still observable (yellow arrowhead).(C, C') Amiloride incubation does not diminish Rpk expression. White arrowhead indicates approximate position of the A-P compartment boundary, yellow arrowhead indicates approximate position of the D-V compartment boundary.(D, E) Expression of rpk RNAi using dpp-Gal4 results in diminished DiBAC fluorescence along the A-P compartment boundary (white arrowhead), while increased fluorescence at the D-V boundary in the A compartment is still observable (yellow arrowhead).
Figure 3(A-A''') Immunostaining of discs expressing ptc>RFP with antibodies to Rpk and ATPα showing elevated levels of both proteins anterior to the compartment boundary. White arrowheads indicate approximate position of the A-P compartment boundary.(B-C') L3 discs that are temperature-sensitive for hh following shift to the restrictive temperature for 12 h show loss of increased expression of Rpk (B, B') and ATPα (C, C') anterior to the compartment boundary. Controls shown in Figure EV2. White arrowheads indicate approximate position of the A-P compartment boundary.(D-E''') Clones of cells expressing a constitutively activated form of the transcription factor Ci show elevated Rpk and ATPα expression in the anterior compartment (white box) as well as the posterior compartment (yellow arrowhead). (E-E''') A single clone in the A compartment is shown at higher magnification.(F-F') A clone of cells in the anterior compartment expressing ci3M following incubation in DiBAC.(G-G') A clone of cells in the anterior compartment expressing ptc RNAi following incubation in DiBAC. The clones also express RFP.
Figure 4(A-A''') Immunostaining of Smo protein (red) and full-length Ci (light blue) in discs expressing en-Venus. Smo is expressed at higher levels in the P compartment and in a stripe 5-10 cells wide just anterior to the A-P compartment boundary. White arrowhead indicates cells just anterior to the A-P compartment boundary.(B-C) Effect of expressing an RNAi against the ENaC channel rpk in the dorsal compartment of the disc using ap-Gal4. Knockdown of Rpk in the dorsal compartment results in decreased accumulation of the Hh signal transducer Smo (B, B'), and the activator form of Ci (B, B''). The expression of ptc, visualized using an anti-Ptc antibody (C), a downstream target gene of Hh signaling, is also diminished. The Rpk RNAi line BL:39053 is used in all panels shown, but the phenotype was validated with a second RNAi line (BL:25847).
Figure 5(A-C) Localization of Smo protein (blue) following expression of 71B-Gal4 (A), 71B-Gal4 and UAS-NaChBac (B) or 71B-Gal4 and UAS-Kir2.1 (C). Expression of the sodium channel NaChBac increases membrane localization of Smo while expression of the potassium channel Kir2.1 reduces membrane localization, and increases cytosolic Smo (B, C).(D) Quantification of the ratio of membrane Smo fluorescence to cytosol fluorescence. N=7 salivary glands per genotype. Data were compared using ANOVA followed by Tukey test for significance (** indicates p<0.01, *** indicates p<0.001), error bars are standard deviations.(E, F) Expression of the Hh target gene ptc is visualized using anti-Ptc immunostaining. Compared to 71B-Gal4 (E), Ptc expression is increased in glands expressing 71B-Gal4 and UAS-NaChBac (F).(G-I) Effect of expression and activation of the depolarizing channelrhodopsin ChR2 on Smo protein membrane abundance. After dissection, glands were kept in either dark conditions (G) or exposed to 15 minutes of blue light (H). (I) Quantification of membrane fluorescence; N=13 glands, data compared using an unpaired t test (** indicates p<0.01), error bars are standard deviations.(J-L) Effect of expression and activation of the Cl--selective channelrhodopsin ChloC. An overall reduction in Smo immunostaining was observed following channel activation (K), as compared to control glands kept in the dark (J). (L) Quantification of membrane fluorescence; N=10 glands, data were compared using an unpaired t test, (*** indicates p<0.001), error bars are standard deviations.(M-Q) Time course of change in Smo localization. Salivary glands expressing the channelrhodopsin ChR2 were subjected to increasing intervals of activating light, then fixed and stained for Smo protein (M-P''). (Q) Quantification of membrane-associated fluorescence; N=7 glands/timepoint, data were compared using ANOVA followed by Tukey test for significance (** indicates p<0.01), error bars are standard deviations.
Figure 1All ORFs for the sense non-AUG-dependent translation of the (G4C2)n~188, DPR coding for (GA), (GP), (GR) can be detected and quantified using a filter-trap binding assay probing for the c-terminal HA tag of the C9 DPR reporter construct. The (GA) ORF is the most abundantly detected DPR and GR the lowest in HEK293T cell 24 h post transient transfection of the C9 DPR reporter construct. n = 5.The C9orf72 NRE non-AUG-dependent reporter constructs recapitulate localization patterns and pathological features identified in C9orf72 NRE models. The Dendra2 (green) c-terminal fusion was used to monitor cellular localization and fluorescence intensity for each DPR translated from the G4C2 strand in HEK293T cells transiently cotransfected with an NES-mIFP-mIFP-NES (NES-mIFP) fusion protein cytoplasmic marker.C9 DPR reporter constructs show cell-type variability in the number of cells that are DPR positive and the overall DPR levels. Transient cotransfections of HEK293T cells robustly undergo G4C2 -Dendra2 non-AUG-dependent translation compared to NSC34 or rat primary cortical neurons. DPR positive cells and DPR fluorescent intensity were analyzed and calculated 24 h post-transfection only in cells expressing the co-transfected NES-mIFP construct. DPR fluorescent intensity was normalized to NES-mIFP fluorescent intensity to adjust for cell specific AUG-dependent translation levels; represented as fold-change relative to HEK293T. HEK293T: n = 5 with m > 500 cells analyzed per n. NSC34: n = 5 with m > 500 cells per n. Rat primary cortical neuron: n = 5 with m > 40 cells per n. (***P < 0.001, **P < 0.01, *P <0.05) See also Figure EV1-2.
Figure 2Quantitative fluorescent microscopy imaging shows a dose-dependent increase in the number of C9 DPR reporter positive cells with increasing concentrations of an apoptotic stimulator, staurosporine (SSP). The C9 DPR reporter in the GP frame was utilized for these experiments. n = 4.Filter trap binding assays using DPR-specific antibodies show all DPR levels are increased for the sense-coding C9 DPR reporter when NSC34 cells are challenged with stress-inducing compounds. These stress-inducing compounds broadly cover distinct or overlapping cellular pathways (see Table EV2) Antibodies specific for the C9orf72 NRE DPRs, poly-GA, -GP, or -GR, were used to quantify DPR levels normalized to GAPDH 24 h post transient transfection of the C9 DPR reporter constructs. n = 4.Quantitative fluorescent microscopy imaging shows that NSC34, rat primary cortical neurons have increased relative DPR fluorescent intensity levels following compound induced cellular stress. In NES-mIFP cotransfection positive NSC34 and rat primary cortical neurons there is a significant increase in C9 DPR reporter fluorescent intensity levels 24 post treatment with most stress-inducing compounds. Fluorescent intensity was not normalized to NES-mIFP fluorescent levels. NSC34: n = 10 with m > 500 cells analyzed per n. Cortical neuron: n = 6 with m > 40 cells per n. Endogenously expression of DPRs in iPS spinal motor neurons (iPS sMN) derived from C9orf72 NRE patients increases following induction of cellular stress. DPR fluorescent intensity in iPS sMN was measured using the DPR-specific antibodies described above. IPS sMN: n = 4 with m > 20 cells per n.
Figure 4Microscopy images show increased DPR fluorescent intensity levels from medium and high frequency stimulation of ChR2 in primary cortical neurons transfected with the C9 DPR reporter.Relative fluorescent intensity quantification shows a significant increase in DPR levels with increases in neuronal activity through medium and high stimulation in primary cortical neurons. No stimulation and low stimulation frequencies did not increase levels of DPR. The C9 DPR reporter, monitoring the GP DPR (ORF2), was cotransfected with ChR2. Cortical neuron: n = 4 with m > 10 cells per n. (****P < 0.0001).