EMBO/soda-nel huggingface dataset:
Chimeric Pneumoviridae fusion proteins as immunogens to induce cross-neutralizing antibody responses (2017)
Figure 6

Figure 6. Antibody responses of mice inoculated twice with either wild-type or chimeric proteins based on prefusion hRSV F. Groups of BALB/c female mice (n = 5) were inoculated twice with 10 µg of the proteins indicated at the top of each panel, folded in either the prefusion conformation for hRSV F, F-410 and F-411 or in the postfusion conformation for hMPV F. One week after the last dose, mice were sacrificed and their blood collected. A) Each mouse serum was tested in ELISA for antibody binding to the F proteins indicated at the bottom of each panel. *P = 0.00046 and **P = 0.02459. B) Mouse sera were also tested in microneutralization assays with hRSV and hMPV, indicated at the bottom. *P = 0.04138, **P = 0.04831 and ***P = 0.00339. The results in A and B are presented as in Fig. 3.
Genome-scale, single cell-type resolution of microRNA activities within a whole plant organ (2019)
Figure 2

Figure 2. Cell-type specific impact of miRNA action on the Arabidopsis root translatome.

(A) Schematics of layer-specific polysome profiling comparing, here, the WT versus hyl1 columella. Profiling was conducted with RPL18 expressed under each cell-type-specific promoter, or the ubiquitous 35S promoter, providing a control for the total roots' signal.

(B) Representative confocal images of cell-type-specific promoter::GFP transcriptional fusions in hyl1 vs WT roots.

(C) Hierarchical clustering of validated miRNA targets between different cell layers in either WT or hyl1 roots.

(D) Total number of up-(pink) or down-(blue)-regulated transcripts in hyl1 versus WT background, compared to the total number of validated miRNA targets (grey) isolated via total, or cell-type-specific, polysome profiling.

(E) qRT-PCR validation of three distinct classes of direct miRNA target transcripts from total root-, total root polysome- or epidermis-specific polysome-associated RNA. The AT2G37050 kinase is positively regulated by LAC17.

(F) miRoot snapshots of whole-root (left) and layer-specific (right) LAC17 mRNA accumulation in WT versus hyl1, as assessed by polysome profiling.

Data information: Scale bars: 50μM. All qPCR values were normalized to the internal control ACTIN2. Error bars represent +/-SD of three biological replicates. * p<0.05 (1-tailed T-test).
Genome-scale, single cell-type resolution of microRNA activities within a whole plant organ (2019)
Figure 3

Figure 3. Integration of miRNAome and targetome data into the miRoot browser.

(A) miRoot snapshot of cell-type specific miRNAs showing, in this case, a sum group of the miR156 family. The right panel shows the gradient of expression revealed upon removal of the stele (blue box).

(B) Snapshot of the SPL6 miR156 translatome in WT and hyl1 backgrounds. Removal of the stele (blue box) reveals subtleties of miR156-mediated gene regulation in the other layers, resulting in a near-mirror image of (A).
Genome-scale, single cell-type resolution of microRNA activities within a whole plant organ (2019)
Figure 4

Figure 4. Cell-type specific analysis reveals hitherto unknown miRNA-mediated gene regulatory programs.

(A) Confocal image of pmiR396a::H2B:GFP.

(B) Normalized read counts from AGO1-IP-Seq and independent northern analysis of AGO1-loaded miR396.

(C) Representative confocal image of the pGRF2::H2B:GFP translational fusion.

(D) Focused miRoot analysis of the GRF2 translatome signal in WT versus hyl1 columella. Graph: GRF2 normalized read counts.

(E) Representative confocal images of the pGRF2::GRF2:GFP translational fusion in WT or hyl1 backgrounds.

(F) Focused miRoot analysis of the PLT1 translatome signal in WT versus hyl1 columella.

(G) Schematic demonstrating multiple principles of miR396-mediated transcript regulation.

Data information: Scale bars: 50μM.
Genome-scale, single cell-type resolution of microRNA activities within a whole plant organ (2019)
Figure 5

Figure 5. Spatial deconvolution of miRNA-mediated gene regulation.

(A) Layer-specific normalized read counts of miR169 paralogs (separated into 4 different groups based on sequence identity: a, b-c, d-g, h-n) from AGO1-IP-deep-Seq and independent northern analysis, as in Fig.1F.

(B) Cell-type specific AGO1-IP signals for each miR169 sequence-group with (upper) or without (lower panel) the miRoot scale normalization option.

(C) Predicted base-pairing of indicated miR169 sequence-group to four NF-YA target transcripts. Pairing with expectation value greater than 3 was not considered. The color gradient (pale: low, dark: strong) qualitatively approximates the pairing contribution of each miR169 sequence-group according to the upper panel of (B).

(D) miRoot snapshots of layer-specific translatome signals of four out-of-seven miR169 NF-YA targets in WT versus hyl1 backgrounds.
Genome-scale, single cell-type resolution of microRNA activities within a whole plant organ (2019)
Figure 6

Figure 6. Non-cell autonomous miRNA action.

(A) Browser representation and independent northern analysis and AGO1-loaded miR165/166.

(B) miRoot snapshot of PHB translatome signals in WT versus hyl1 backgrounds.

(C) Representative confocal images of pmiR395(a/c/e)::H2B:GFP transcriptional fusions under SO4--deficient or SO4--proficient growth conditions.

(D) Northern analysis of AGO1-loaded miR395 under the conditions in (C).

(E) Representative confocal images of the pAPS4::APS4:GFP translational fusion under the conditions in (C).

(F) The principles of non-cell-autonomous miRNA-mediated gene regulation within a whole organ.

Data information: Scale bars: 50μM.
Genome-scale, single cell-type resolution of microRNA activities within a whole plant organ (2019)
Figure 7

Figure 7. Mechanisms and examples of miRNA movement in different tissue types

(A,B) (A) Representative confocal image of the pSHR::GFP:P19 translational fusion and (B) principle of P19-based miRNA duplex sequestration.

(C) Binding of miR395 by SHR::HA:P19 under SO4--sufficient (+) and -deficient (-) growth conditions.

(D) qRT-PCR analysis of polysome-associated APS4 in the cortex or epidermis in the presence (+) or absence (-) of SHR::HA:P19. Signals are relative to SHR::HA:P19 (-) lines given a value of 1.

(E) Representative confocal image of the pmiR397b::H2B:GFP transcriptional fusion (left) and the browser AGO1-loaded signal for miR397b (right).

(F) Cross section of Arabidopsis leaf showing cell-types isolated via Meselect.

(G) miR160 northern analysis in whole-leaf, vasculature and epidermis. U6: loading control.

(H) Western analysis of the promoter::H2B:GFP translational fusions shown in (5C) in the cell types isolated in (G).

(I) qRT-PCR analysis of ARF17 in whole-leaves or in separated cell types in WT versus hyl1 backgrounds.

Data information: Scale bars: 50μM. CB: Coomassie blue. Error bars are +/-SD of 3 biological replicates. * p<0.05 (1-tailed T-test).

END OF MAIN MANUSCRIPT
EMC is required for biogenesis of Xport-A, an essential chaperone of Rhodopsin-1 and the TRP channel (2021)
Figure 1

Figure 1. EMC is required for the biogenesis of a subset of tail-anchored proteins.

(A) The Drosophila proteome was screened to identify TA proteins using the PolyPhobius (Käll et al, 2005) algorithm within the TOPCONS (Tsirigos et al, 2015) web server. Proteins with a single TMD within 35 AAs or less upstream of the C terminus and lacking a signal peptide or mitochondrial targeting peptide were selected for further interrogation. The DeepLoc (Almagro Armenteros et al, 2017) predictor of protein sub-cellular localization was used to eliminate soluble proteins from the list. These procedures yielded 254 proteins, which were scored for their tail charge and hydrophobicity using the "transmembrane tendency" score of Zhao & London (Zhao & London, 2006).

(B) A subset of the predicted TA proteins was screened by overexpression in Drosophila larval eye imaginal discs containing clones of a previously isolated EMC3 mutant allele, EMC3Δ4 (Satoh et al, 2015). TA protein predicted membrane localization is shown to the left: G (Golgi apparatus), ER (endoplasmic reticulum), C (cytoplasmic membrane), N (nucleus), M (mitochondria) and P (peroxisome). The ratio of fluorescence intensity for the tested TA proteins in EMC3 homozygous mutant cells over WT cells (EMC3Δ4/WT) was measured and plotted. Proteins were classified into four groups according to the ratio measured: strongly reduced (ratio<0.5; bars in red), reduced (ratio 0.5-0.8; bar in pink), normal (ratio 0.8-1.2; bars in blue) and increased (ratio>1.2; bars in dark blue). For quantification, at least 3 mutant patches were quantified per eye imaginal disc, and at least three eye imaginal discs derived from distinct flies were used (N=3). Error bars correspond to standard deviation (stdev).

(C-J) All panels show Drosophila 3rd instar larval eye imaginal discs with eyeless-Flippase-induced clones of cells homozygous for EMC3Δ4 (Satoh et al, 2015), labelled by the absence of ubiGFP (green). The red channel shows ELAV, which labels the nuclei of photoreceptor cells. All UAS constructs were expressed under the control of GMR-GAL4. (C) Expression of UAS-Rh1 (4C5, in blue) and (D) Na+K+ATPase (A5-C, in blue) is strongly reduced in EMC3Δ4 homozygous mutant cells. (E) Expression of UAS-Xport-A-HA (anti-HA, in blue) and (F) UAS-fan-HA (anti-HA, in blue) is strongly reduced in EMC3Δ4 homozygous mutant cells. (G) Expression of UAS-rtv-HA (anti-HA, in blue) and (H) hid (anti-hid, in blue) is increased in EMC3Δ4 homozygous mutant cells. (I-J) Normal expression of two TA proteins in EMC3Δ4 homozygous mutant cells. (I) UAS-CG8814-HA (anti-HA, in blue) is a TA protein containing a TMD of low hydrophobicity (J) UAS-Syx13-HA is a TA protein with high hydrophobicity TMD. Scale bars represent 10 μm.
EMC is required for biogenesis of Xport-A, an essential chaperone of Rhodopsin-1 and the TRP channel (2021)
Figure 2

Figure 2. EMC3 is required for the expression of Xport-A, but not Xport-A4L.

(A) The TMD of Xport-A was identified using TOPCONS (Tsirigos et al, 2015) and is in bold/underlined, together with the immediate flanking residues. Red residues indicate mutations made in the Xport-A TMD to increase its hydrophobicity, creating the Xport-A4L mutant. To the right of each sequence is the "transmembrane tendency" score, calculated according to Zhao & London (Zhao & London, 2006).

(B-D) - Immunostaining of 3rd instar larval eye imaginal discs with eyeless-Flippase-induced clones of cells homozygous for EMC3Δ4, labelled by the absence of ubiGFP (green) shows reduced expression of (B) UAS-HA-Xport-A (anti-HA, in blue) but normal expression of (C) UAS-HA-Xport-A4L (anti-HA, in blue) or (D) UAS-Xport-A4L-HA (anti-HA, in blue) in EMC3 mutant homozygous cells. ELAV (red in B and C) marks photoreceptor cells and Na+K+ATPase (red in D) acts as a positive control for the presence of EMC3Δ4 homozygous mutant clones. The UAS constructs were expressed under the control of GMR-GAL4. Scale bars represent 10 μm.

(E-F) Immunostaining of mosaic adult retina with eyeless-Flippase-induced clones of cells homozygous for EMC3e02662, labelled by the absence of RFP (red), show loss of (E) UAS-HA-Xport-A (anti-HA, in blue) in EMC3 mutant homozygous cells. (F) Expression of UAS-HA-Xport-A4L (anti-HA, in blue) is normal in EMC3e02662 homozygous mutant clones. The rhabdomeres are stained for actin (Phalloidin, in green). Scale bars represent 10 μm.

(G) The ratio of fluorescence intensity in EMC3 homozygous mutant cells over that of WT cells (EMC3mut/WT) was measured and plotted. Proteins were classified into two groups according to the ratio measured: strongly reduced (ratio<0.5; bars in red) and normal (ratio 0.8-1.2; bars in blue). For quantification, at least 3 mutant patches were quantified per eye imaginal disc, and at least 2 mutant patches were quantified per adult retina. At least three eye imaginal discs or adult retina derived from distinct flies were used (N=3). Error bars correspond to standard deviation (stdev). Significance was determined by Welch's t-test: ****p ≤ 0.0001.
EMC is required for biogenesis of Xport-A, an essential chaperone of Rhodopsin-1 and the TRP channel (2021)
Figure 3

Figure 3. EMC is required for the biogenesis of Xport-A in mammalian cells.

(A) Schematic representation of the reporter constructs used for analysis of protein biogenesis by flow cytometry. All constructs contain an N-terminal GFP and a C-terminal RFP, separated by a viral 2A peptide that mediates peptide bond skipping. Changes in the stability of SQS, Xport-A and Xport-A4L fused to RFP change the RFP:GFP fluorescence ratio.

(B-D) Histograms of flow cytometry data monitoring the fluorescence protein ratio in the indicated U2OS cell lines for each construct. "EMC5KO+EV" indicates a knockout of EMC5 stably harbouring empty vector, while ''EMC5KO+EMC5" indicates EMC5KO cells rescued by inducible re-expression of a stably integrated EMC5.

(E-G) Histograms of flow cytometry data monitoring the fluorescence protein ratio in the indicated U2OS cell lines for each construct. "EMC6KO+EV" indicates a knockout of EMC6 stably harbouring empty vector, while ''EMC6KO+EMC6" indicates EMC6KO cells rescued by re-expression of a stably integrated EMC6.
EMC is required for biogenesis of Xport-A, an essential chaperone of Rhodopsin-1 and the TRP channel (2021)
Figure 4

Figure 4. EMC6 is required for membrane insertion of Xport-A TMD.

(A) Schematic representation of the constructs used to monitor insertion efficiency in the ER membrane. All constructs contain a HA-tagged SQS cytosolic domain fused to a varying TMD sequence, which is C-terminally fused to an opsin tag (sequence indicated). Glycosylation of the C-terminal opsin tag acts as an insertion readout.

(B) Sequences of the TMD regions used for cloning and the immediate flanking residues are represented. The regions defined as TMDs were determined using TOPCONS (Tsirigos et al, 2015) and are in bold/underlined. Red residues indicate mutations made in the Xport-A TMD to increase its hydrophobicity. To the right of each sequence is the "transmembrane tendency" score of Zhao & London (Zhao & London, 2006).

(C-G) (C-D, F) Western-Blot of WT and EMC6KO (6KO) U2OS cell lines transiently transfected with the opsin-tagged constructs probed for HA. When indicated, denatured samples were treated with PNGase to confirm glycosylation. Glycosylated proteins shift upwards in the gel and are indicated by arrows (). (E, G) Western-Blot of WT and EMC6KO (6KO) U2OS cell lines transiently transfected with the opsin-tagged constructs probed for Actin and EMC6.
EMC is required for biogenesis of Xport-A, an essential chaperone of Rhodopsin-1 and the TRP channel (2021)
Figure 5

Figure 5. Expression of Xport-A4L can rescue Rhodopsin-1 and TRP biogenesis defects in EMC3 mutant clones.

(A-B) Immunostaining of 3rd instar larval eye imaginal discs with eyeless-Flippase-induced clones of cells homozygous for EMC3Δ4, labelled by the absence of ubiGFP (green) show loss of (A) UAS-Xport-A-HA (anti-HA, in blue) and UAS-Rh1 (4C5, in red). (B) Expression of UAS-Xport-A4L (anti-HA, in blue) and UAS-Rh1 (4C5, in red) is observed in EMC3Δ4 homozygous cells. The UAS constructs were expressed under the control of GMR-GAL4. Scale bars represent 10 μm.

(C-D) Immunostaining of mosaic adult retinas with eyeless-Flippase-induced clones of cells homozygous for EMC3e02662, labelled by the absence of RFP (red), show loss of (C) TRP (Mab83F6, in blue) when UAS-HA-Xport-A is expressed. (D) Expression of TRP (Mab83F6, in blue) is observed in EMC3e02662 homozygous cells, when HA-Xport-A4L is expressed. The rhabdomeres are stained for actin (Phalloidin, in green). The UAS constructs were expressed under the control of Rh1-GAL4. Scale bars represent 10 μm.

(E) The ratio of fluorescence intensity in EMC3 homozygous mutant cells over that of WT cells (EMC3mut/WT) was measured and plotted. Proteins were classified into three groups according to the ratio measured: strongly reduced (ratio<0.5; bars in red), rescue (ratio 0.5-0.8, bars in light blue) and normal (ratio 0.8-1.2; bar in dark blue). For quantification, at least 3 mutant patches were quantified per eye imaginal disc, and at least 2 mutant patches were quantified per adult retina. At least three eye imaginal discs or adult retina derived from distinct flies were used (N=3). Error bars correspond to standard deviation (stdev). Significance was determined by Welch's t-test: ***p≤ 0.001, ****p ≤ 0.0001.
EMC is required for biogenesis of Xport-A, an essential chaperone of Rhodopsin-1 and the TRP channel (2021)
Figure 6

Figure 6. Glycosylated Rhodopsin-1 TMD1-5 accumulates in Xport-A homozygous mutants.

(A) Schematic representation of endogenous Rh1. Predicted glycosylation sites (N20 and N196) are shown in yellow.

(B-D) Schematic representation of the Rh1 TMD1, TMD1-3 and TMD1-5 truncation constructs used to monitor glycosylation efficiency in Xport-A1 mutants. Predicted glycosylation sites are shown in yellow.

(E-G) Western blot of fly heads labelled for V5, TRP, Rh1, Xport-A (Xp-A) and tubulin.

(E) Immunoblot of fly heads expressing Rh1Gal4 (driver) (left) or Rh1 TMD1-V5 (right) in Xport-A heterozygous (Xport-A1/TM6B) or homozygous mutant flies (Xport-A1/Xport-A1). Extracts were untreated (-) or treated (+) with PNGase F enzyme (lanes 2, 4, 6 and 8). For Rh1Gal4, 4 fly heads were loaded per well, while for Rh1 TMD1-V5, 8 fly heads were loaded per well. (F) Immunoblot of fly heads expressing Rh1 TMD1-3-V5 in Xport-A heterozygous (Xport-A1/TM6B) or homozygous mutant flies (Xport-A1/Xport-A1). Extracts were untreated (-) or treated (+) with PNGase F enzyme (lanes 2 and 4). Eight fly heads were loaded per lane. (G) Immunoblot of fly heads expressing Rh1 TMD1-5-V5 in Xport-A heterozygote (Xport-A1/TM6B) or homozygous mutant flies (Xport-A1/Xport-A1). Extracts were untreated (-) or treated (+) with either Endo-H (lanes 2 and 5) or PNGase F enzyme (lanes 3 and 6). Each lane contains approximately 2,5 fly heads.

Expanded View Figure Legends
The UPR sensor IRE1α promotes dendritic cell responses to control Toxoplasma gondii infection. (2020)
Figure 1

Figure 1. The IRE1α/ XBP1s pathway is activated in BMDCs infected with T. gondii.

(A, B) BMDCs were infected with Pru parasites (at a MOI of 4 parasites/BMDC) or treated with tunicamycin (TN) for 16h and the expression of UPR target genes were analyzed by RT-qPCR. The results are expressed as (Log2) Fold change (A) or relative mRNA level (B) compared to non-infected (NI) conditions. IRE1α/XBP1s pathway: Ern1 (IRE1α), Xbp1s, Dnajb9 (ERdj4), Sec24D, Bloc1s1 and Sec61a1; PERK/ATF4 pathway: Atf4, Ddit3 (CHOP) and Ppp1r15a (GADD34); ATF6 pathway: Atf6 and Herpud1; classical ER chaperons: Hspa5 (BIP) and Hsp90b1 (GRP94). Results are normalized to the housekeeping gene Gapdh. Unpaired Student T-test, ns: p>0.05; **: p<0.01; ***: p<0.001; mean ± S.E.M (n=2-5 independent experiments depending of the studied gene, 3 mice / independent experiment).

(C, D) Immunoblot analysis of IRE1α, PERK, Phospho-PERK (detected by the presence of a second upper band after migration on a 6% acrylamide gel), Phospho-eIF2α, CHOP and XBP1s protein expression in non-infected BMDCs, BMDCs treated with TN and Pru infected BMDCs (C) and in non-infected and Pru infected Flt3/Notch-DCs (D) for the indicated times. GAPDH and tubulin were used as loading controls. The same blots are shown in Figure EV3B-C.
The UPR sensor IRE1α promotes dendritic cell responses to control Toxoplasma gondii infection. (2020)
Figure 2

Figure 2. UPR and cytokine responses are under the control of MyD88 protein in BMDCs infected with T. gondii.

(A) Mean percentage of Pru parasite infection of WT and MyD88KO BMDCs; mean ± S.E.M (n=3 mice, 1 representative experiment from 2 independent experiments is shown). Mann-Whitney U test, ns: p>0,05.

(B) Mean percentage of parasitophorous vacuoles containing 1, 2, 4 or 8 parasites/vacuole (p/v) in WT and MyD88 KO BMDCs after 16h of infection with Pru parasites. Mean values ± S.E.M. n= 3 independent experiments (biological replicates). Unpaired Student T-test, ***p<0,001.

(C) IL-6, IL-12 and IL-23 cytokine levels from WT and MyD88KO BMDCs non-infected (NI) and infected with Pru parasites for 16h. Two-way ANOVA Tukey's multiple comparisons, ****: p<0.0001; mean ± S.E.M (n=4-6 mice pooled from 2 independent experiments).

(D) mRNA relative expression level of Xbp1s, Dnajb9 (ERdj4), Ddit3 (CHOP) and Ppp1r15a (GADD34) from WT and MyD88KO BMDCs infected with Pru parasites for 16h compared to non-infected (NI) conditions. Results are normalized to the housekeeping gene Gapdh. Two-way ANOVA Tukey's multiple comparisons, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001, ****: p<0.0001; mean ± S.E.M (n=4-5 mice pooled from 2 independent experiments).
The UPR sensor IRE1α promotes dendritic cell responses to control Toxoplasma gondii infection. (2020)
Figure 3

Figure 3. Replicating parasites modulate the UPR.

(A) BMDCs (CD11c+) were incubated with Pru-Tomato parasites for 16h. Infected cells (red gating) and non-infected bystander cells (green gating) were sorted-purified and further analyzed.

(B) The expression of UPR target genes were analyzed by RT-qPCR in infected and non-infected bystander cells compared to non-infected BMDCs (NI). The results are expressed as relative mRNA level compared to non-infected conditions. Results are normalized to the housekeeping gene Gapdh. Unpaired Student T-test, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; mean ± S.E.M (n=6 mice pooled from 2 independent experiments).