EMBO/SourceData huggingface dataset:
Figure 4HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the IFN-β promoter , plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for RIG-I 2CARD, RIG-I, MDA-5, MAVS or stimulated with poly (I:C), for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the IFN-β promoter plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for TBK1 TRIF , for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the the NF-κB promoter plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for TRAF 6, TRAF 2, NEMO for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the NF-κB promoter plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for TBK1 TRIF , for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).Interaction between MARCH2 and NEMO in response to viral infection. HEK293T cells (E) were infected with NDV-GFP in a time-dependent manner in the presence of MG132 (proteasome inhibitor, 10 μM). Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.Interaction between MARCH2 and NEMO in response to viral infection. Raw264.7 cells (F) were infected with NDV-GFP in a time-dependent manner in the presence of MG132 (proteasome inhibitor, 10 μM). Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.G Confocal microscopy was conducted to examine time-dependent colocalization of MARCH2 and NEMO in HeLa cells upon NDV infection (MOI = 1) in the presence of MG132 (10 μM). Arrow indicates the co-localized NEMO and MARCH2 protein.Interaction between MARCH2 and NEMO in response to bacterial infection. Raw264.7 cells were infected with L. monocytogenes (H) or S. typhimurium (I) in a time-dependent manner in the presence of MG132. Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.J Confocal microscopy was conducted to examine time-dependent colocalization of MARCH2 and NEMO in HeLa cells expressing TLR2 upon zymosan treatment (100 μg/ml) in the presence of MG132 (10 μM). Arrow indicates the co-localized NEMO and MARCH2 protein.
Figure 6(B) HEK293T cells were transfected with Strep-tagged MARCH2 domain constructs together with GST-tagged NEMO. Whole cell lysates were subjected to a Strep-PD assay, followed by immunoblotting with an anti-GST antibody.(D) HEK293T cells were transfected with GST-tagged NEMO domain constructs together with Strep-tagged MARCH2-TM. Whole cell lysates were subjected to a Strep-PD assay, followed by immunoblotting with an anti-GST antibody.(F) HEK293T cells were transfected with HA-tagged NEMO domain constructs together with GST-tagged MARCH2. Whole cell lysates were subjected to a GST-PD assay, followed by immunoblotting with an anti-HA antibody.G HEK293T cells transfected with His-tagged ubiquitin and HA-tagged NEMO WT or NEMO lacking the CC1 domain together with Strep-tagged empty vector or MARCH2 were treated with MG132 (10 μM) for 6 h before harvest. Lysates were subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with an anti-His antibody. WCL were subjected to immunoblotting with anti-HA, anti-Strep, or anti-β-actin antibody.(I) HEK293T cells transfected with GST-tagged NEMO mutants together with Strep-tagged empty vector or MARCH2 were subjected to immunoblotting with anti-GST, anti-Strep, or anti-β-actin antibody.J HEK293T cells transfected with HA-tagged ubiquitin and GST-tagged NEMO mutants together with Strep-tagged empty vector or MARCH2 were subjected to pulldown with GST beads, followed by immunoblotting with anti-Lys48 (K48)-linkage specific polyubiquitin (K48-ub), anti-Lys63 (K63)-linkage specific polyubiquitin (K63-ub), or anti-Strep antibody. Whole cell lysates were determined by immunoblotting with the indicated antibodies.
Figure 7A HEK293T cells were transfected with GST-tagged NEMO-WT and its point mutants with or without Strep-tagged MARCH2. Whole cell lysates were subjected to immunoblotting with the indicated antibodies.B HEK293T cells were transfected with GST-tagged NEMO-WT and its point mutants together with Strep-tagged MARCH2 in the presence of MG132. Whole cell lysates were subjected to a GST-PD assay, followed by immunoblotting with an anti-K48 linkage specific polyubiquitin antibody.C In vitro ubiquitination assay for NEMO-K326R. HEK293T cells were transfected with GST-tagged NEMO, GST-tagged NEMO-K326R or Strep-tagged MARCH2 individually. Immunoprecipitates were incubated with a reaction mixture containing ubiquitin, E1, and UbcH6 (E2), followed by immunoblotting with an anti-K48-linkage specific polyubiquitin antibody.HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. (D-F) Viral replication was determined by fluorescence microscopy (D)HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. , fluorescence absorbance (E)HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. plaque assay (F).HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. (G) IFN-β and IL-6 in the cell supernatants were measured in an ELISA.H, I IFN-β (H) and NF-κB (I) luciferase reporter assay.
Figure 1(E) (left) Western blot of NANOG, OCT4, and SOX2. (right) Relative NANOG levels as estimated by densitometry (n=8). NANOG was nearly 7-fold more in PD, which is twice that of 2iL/SL2i. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns = not significant (paired two-tailed student's t-test).(F) Western blot of pERK and ERK at 0, 1, 4, 8, 12, 16, and 24 hrs after media change in indicated treatments (n=3).(G) Western blot of pERK and ERK in SLPD, SLCHIR, 2iL, and SL2i after 8, 12, 16, and 24 hrs of culture relative to SL, where the cells in SL were harvested 24 hrs after the media change (n>3).(H) (left) Western blot of pERK and ERK in 1μM PD and increasing concentrations of CHIR in serum-free N2B27 media. (right) Relative pERK levels (n=3). (I) (left) Western blot of pERK and ERK in 3μM CHIR and increasing concentrations of PD in serum-free N2B27 media. (right) Relative pERK levels (n=6). Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in H, and I. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns = not significant (paired two-tailed student's t-test).
Figure 2(G) (left) Western blot of pERK in TNERT, TNERTFgfr1-/-, TNERTFgfr2-/-, TNERTFgf4-/-, and TNERTFgfbp1-/- with or no OHT. (Right) relative pERK levels estimated by densitometry (n=4). Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in G, *P < 0.05, **P < 0.01, ***P < 0.001, and ns = not significant (paired two-tailed student's t-test).
Figure 3(E) Western blot of FGFR1, FGFR2, and pERK in TNERT at one-hour intervals at indicated time points after OHT treatment (n>3).
Figure 5(C) Western blot of pERK and ERK in TNERT and TNERTNono-/- cells treated with or no OHT (n=3).(D) Immunoprecipitation analysis showing interactions between ERK and NONO in the presence or absence of Nanog induction by Doxycycline in TDiN cells.(E) (left) Western blot of pERK and ERK in E14Tg2a cells treated with PD or FGF4. (Middle and right) Relative levels of pERK and NONO in E14Tg2a cells treated with PD or FGF4 (n>4). Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in E *P < 0.05, **P < 0.01, ****P < 0.0001 and ns = not significant (paired two-tailed student's t-test).(L) Cycloheximide chase assay of NANOG in SL, SLPD, and SLFGF4 in E14Tg2a cells.
Figure 6(A) Immunofluorescence of pERK (red) and NANOG (green) in the indicated ESCs. Scale bars 25 μM. (B)The normalized fluorescence intensity of pERK was plotted against the normalized fluorescent intensity of NANOG.(C) (left) Immunofluorescence of pERK and NANOG in 10% NANOG-high NiRFP2A cells at indicated time cultured in the same media collected from cells before FACS. (Right) The normalized fluorescence intensity of pERK was plotted against the normalized fluorescent intensity of NANOG at different time points. Scale bars 25 μM.(D) (left) Western blot analysis of pERK, and NANOG in 10% NANOG-high NiRFP2A cells at indicated time cultured in the same media collected from cells before FAC sorting. (Right) Expression levels of NANOG and pERK in 10% NANOG-high NiRFP2A cells relative to 0 hrs during the time course of culture. The NANOG and pERK expression oscillate between high and low levels in the cells during culture. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in D. *P < 0.05, **P < 0.01 and ns = not significant (paired two-tailed student's t-test).
Figure 1Analysis of DNA content by flow cytometry.Analysis of BrdU incorporation by BrdU-IP-chip 45 minutes after release into S phase in cells treated with 0.1% MMS as described in (A). A representative region on chromosome VII is shown. The position of replication origins is indicated above the map. Asterisks: late origins firing in mrc1∆ cells.Quantification of the BrdU incorporation (signal log ratio) at 384 early and late replication origins in wild type, rad9∆ and mrc1∆ strains exposed to 0.1 or 0.033% MMS. Red lines indicate median values. Median replication times (Trep) are indicated for bins of 96 origins.
Figure 2Wild type, mrc1∆ and rad9∆ cells were released into S phase in the presence of BrdU and 0.033% MMS, as described in Fig. 1A. DNA combing analysis was performed for samples collected 45 and 60 min after release into S phase. Representative DNA fibers are shown. Red: DNA, green: BrdU. Bar is 20 kb.Distribution of center-to-center distances in cells released for 45 minutes into S phase.Distribution of BrdU track lengths in cells released for 45 or 60 minutes into S phase, respectively.Wild type, mrc1∆ and rad9∆ cells were synchronized in G1 with α-factor prior to release with pronase in the presence of 0.033% MMS. Cells were collected at the indicated times and Rad53 phosphorylation was monitored by western blot The asterisk indicates a non-specific band detected by the anti-Rad53 antibody.Wild type, mrc1∆ and rad9∆ cells were synchronized in G1 with α-factor prior to release with pronase in the presence of 0.033% MMS. Cells were collected at the indicated times and Rad53 phosphorylation was monitored with an in situ phosphorylation assay - ISA (F).
Figure 3Analysis of Rad53 phosphorylation by Western blot.Analysis of DNA content by flow cytometry.Analysis of DNA replication by DNA combing in cells released for 45 and 60 minutes into S phase in the presence of 100 μg/ml Zeocin. IdU tracks (red) correspond to initiation events occurring during the HU treatment. Elongation events after HU removal appear as CldU tracks (yellow) flanking IdU tracks. New initiation events as detected as isolated CldU tracks (asterisks). Bar is 20 kb.Number of new initiation events per DNA fibers detected at 60 minutes in the experiment shown in (D). For each sample, at least 65 individual DNA fiber larger than 200 kb were analyzed, for a total of >17 Mb.2D gel analysis of the initiation of DNA replication at the early origin ARS305 and the late origin ARS1212. The experiment was performed as in (A) for the indicated times. Arrows indicate late origin firing.Length distribution of CldU tracks 45 minutes after release into S phase in the DNA combing experiment shown in (D). Median values are indicated in red. For each sample, at least 250 tracks were analyzed in two biological replicates.
Figure 42D gel analysis of initiation and elongation in wild type and rad9∆ cells at the early origin ARS719 (probe 1) and at a locus 7 kb away of the origin (probe 2). Time in Zeocin after the HU wash is indicated. Arrows point to Y arcs (fork signals) with the strongest intensity.2D gel analysis of fork progression in wild type and rad9∆ cells through a 70-kb region of chromosome VI for which all active origins downstream of ARS607 have been deleted. The position of probes and the time after release from HU are indicated. Arrows point to Y arcs (fork signals) with the strongest intensity.
Figure 5Analysis of DNA content by flow cytometry.Analysis of Rad53 phosphorylation in G1 prior to Zeocin addition (G1) and at indicated times. The asterisk indicates a non-specific band detected by the anti-Rad53 antibody.DNA combing analysis of replication profiles 60 minutes after release into S phase in the presence of Zeocin. Representative DNA fibers are shown. Red: DNA, green: BrdU. The green channel is also shown separately. Bar: 20 kb.Distribution of BrdU tracks length in wild type, mrc1∆ and mrc1AQ cells. DNA fibers are fully labeled in the case of rad9∆ cells. Median values are indicated in red and correspond to the analysis of at least 150 tracks from two biological replicates. ns: non-significant Mann-Whitney rank sum test.
Figure 6Cells from the indicated strains were released synchronously into S phase in the presence of Zeocin as described in Fig. 5A and DNA content was analyzed by flow cytometry.Analysis of chromosome mobility by Pulse Field Gel Electrophoresis (PFGE) after staining of DNA with ethidium bromide. In G1, the chromosomes are linear and are sorted according to their length. Upon entry into S phase (30 min), they cannot enter the gel due to the presence of replication intermediates. Quantitation of chromosomes re-entering the gel shows completion of DNA replication in 40% of rad9∆, rad53-11 and rad9∆ rad53-11 mutants, but not in wild type cells and mrc1 mutants as determined for a representative experiment. Error bars indicate SD between the signal intensity determined for the three larger chromosomes (arrowheads).DNA combing analysis of replication profiles 30 minutes after release into S phase in the presence of Zeocin. Representative images are shown. Red: DNA, green: BrdU. Bar is 20 kb.Length distribution of BrdU tracks measured at that time for wild type, rad9∆, rad53-11 and rad53-11 rad9∆ cells. Median lengths are indicated in red. For each sample, at least 200 tracks were measured from two biological replicates.
Figure 1C-E. Full length GFP:Mud shows the wild type pattern of localization in mud3/mud4 mitotic follicle cells (C) and rescues spindle orientation (D E). GFP:Mud lacking the Pins binding domain localizes only at spindle poles (C') in mud3/mud4 mitotic follicle cells and fails to rescue spindle orientation (D E). Statistical significance in spindle orientation was determined using the Mann-Whitney test.
Figure 2C, D. Pins:Tom and GFP:Mud have overlapping but not identical localization patterns in mitotic follicle cells. Representative pictures (C) and quantification from 9 cells (D) are shown. Scale bars = 5 microns.
Figure 3A-C. Pins-myr-GFP and myr-RFP show a similar localization pattern to Pins:Tom during mitosis. Representative pictures are shown (A, B); a heatmap lookup table (A', B') emphasizes the relative enrichments of Pins-myr-GFP and myr-RFP along the cortex. Quantification (Pins-myr: 4 cells, myr-RFP: 2 cells) (C).D-F. Random spindle orientation in pinsp62/pinsp62 null mutant follicle cells is rescued by the expression of full-length Pins or Pins-myr-GFP. Representative pictures (D, E) and quantification (F) are shown. Scale bars = 5 microns. Statistical significance in spindle orientation was determined using the Mann-Whitney test. **** p<0.0001.
Figure 4A. Spindle orientation is randomized in Dlg1P20/Dlg1P20 and Dlg1P20/Dlg2 mutant follicle cells. B. Random spindle orientation in pinsp62/pinsp62 null mutant follicle cells is rescued by the expression of full length Pins or phosphomimetic (S436D) Pins. Unphosphorylatable (S436A) Pins fails to rescue. C. Quantification of spindle orientation phenotypes. Statistical significance in spindle orientation was determined using the Mann-Whitney test. **** p<0.0001, *** p<0.001. D.H. Quantification of spindle orientation phenotypes in the Stage 9/10 embryonic ectoderm. n = 17 cells from 2 embryos in w1118 and n = 18 cells from 2 embryos in Dlg1P20/Dlg2 embryos. Statistical significance in spindle orientation was determined using the Mann-Whitney test. ** p=0.0015.
Figure 5A-C. Multiple genetic approaches were used to test a role for either Khc73 or GUKHolder in metaphase spindle orientation. Representative pictures (A-B) and quantification (C) are shown. Scale bars = 5 microns. Spindle angle randomization in pinsp62/pinsp62 null mutant follicle cells is included as a positive control for comparison.
Figure 6A-D. Two distinct patterns of Inscuteable localization are observed in the follicular epithelium when UAS-Inscuteable is driven by Traffic jam-GAL4. (A, C) In the first pattern (InscA), Inscuteable is highly enriched at the apical cell surface. (B, D) In the second pattern (InscB), Inscuteable is observed at both the lateral and apical cortex. Mud and Pins localizations are also shown. Representative pictures (A, B) and quantifications (InscA: average of 5 cells, InscB: average of 7 cells) (C, D) are shown.E. aPKC localization in InscA and InscB follicle cells. F. Quantification of spindle orientation phenotypes shows that mitotic spindles are reoriented in InscA cells, but not InscB cells. Reorientation relies on Mud but not on the interaction between Pins and Dlg. G
Figure 1Domain architecture and structure of Miro. (A) Bar diagram of Miro domain structure. Numbering corresponds to dMiro isoform D, domain names are described in the text (B) Ribbon diagram of the MiroS structure in the 'side view'. Note three distinct domains: ELM1 (blue), ELM2 (green), cGTPase (grey/yellow), joined by two linkers: Lnk1 (orange), and Lnk2 (red). The structure shown is MgGDP‐MiroS, with the Ca2+ ion from the Ca‐MiroS structure overlaid for orientation ( Ca2+/Mg2+/GDP as yellow/purple/multicoloured spheres). cGTPase, C‐terminal GTPase; dMiro, Drosophila Miro; ELM, EF hand pair with ligand mimic.
Figure 2(A) Top, MiroS shown in the 'top view'. ELM1 and ELM2 are related by pseudo twofold symmetry, represented by the dyad between them. Bottom, overlay of the two ELM domains (rmsd of 1.8 Å over 84 Cα positions). ELM1 (blue), ELM2 (green) and identical or similar conserved residues in identical positions are shown as sticks.(B) Comparison of the ELM domain hydrophobic cores. The ELM1 core contains the UNL, modelled here as homoserine shown in spheres, occupying a ∼442 Å3 cavity highlighted with mesh. The ELM2 core includes three tryptophans in close proximity (W367, W391, W395 in orange). Hydrophobic residues in the LM/EF hand pair interfaces are also shown in sticks. LM, ligand mimic; UNL, unidentified ligand.
Figure 4(A) The ELM2/cGTPase interface can be divided into three regions (insets 1-3). Select residues are shown as sticks, polar contacts as dashed lines, and Mg2+ and MgGDP as spheres. Region 1: αC2 and the hEF2 loop form both backbone and side‐chain contacts with αG1 and Switch I in the cGTPase, including the Switch I threonine T481. Region 2: αD2 in hEF2 packs against the twisting cGTPase β‐strands Gβ1‐3. Region 3: the candidate Pink1 phosphorylation site residue S324 in Lnk1 hydrogen bonds with R444 in Lnk2, which also forms a salt bridge with D521, at the end of the Switch II loop.(B) cGTPase rearrangements in MgGDP binding. Displayed is the MiroS‐MgGDP structure, with select residues from the aligned apo‐MiroS structure shown in orange. GDP is shown as sticks (2Fo‐Fc map, 1.2σ), and Mg2+ as a purple sphere. In the apo state, D505 in the Switch II 503DIDV506 motif is far from the nucleotide‐binding pocket and is hydrogen bonded to R507, which is also stabilized by interactions with P512. In the MgGDP‐bound state, the D505 side‐chain rotates ∼7 Å to interact with the β‐phosphate (arrow), locking the Switch II loop in a β‐strand conformation and extending Gβ3. V506 rotates away from the nucleotide‐binding pocket, and both R507 and Y537 become solvent exposed. cGTPase, C‐terminal GTPase; Sw I/II, Switch I/II; P loop, phosphate‐binding loop.
Figure 1.(A-D) Cells expressing Rpl5-GFP (A and B) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (A and C, left) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts. (right) The ratio between cleaved GFP and full-length protein was quantified for every time point in four independent experiments. a.u., arbitrary unit.(A-D) Cells expressing Rpl5-GFP (A and B) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (B and D) Cells before starvation or starved for 24 h (starvation) were examined both by fluorescence microscopy and differential interferential contrast (DIC). Note that 100% of wild-type (WT) and mutant cells expressed GFP-tagged proteins in the cytoplasm before starvation. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole.(A-D) Cells expressing Rpl25-GFP (C and D) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (A and C, left) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts. (right) The ratio between cleaved GFP and full-length protein was quantified for every time point in four independent experiments. a.u., arbitrary unit.(A-D) Cells expressing Rpl25-GFP (C and D) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (B and D) Cells before starvation or starved for 24 h (starvation) were examined both by fluorescence microscopy and differential interferential contrast (DIC). Note that 100% of wild-type (WT) and mutant cells expressed GFP-tagged proteins in the cytoplasm before starvation. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole.
Figure 2.(A) GFP or Rpl25-GFP were expressed in cells transformed (+) or not transformed (−) with a plasmid encoding 6His-ubiquitin. (top) Expression levels of GFP and Rpl25-GFP in whole cell lysates were verified with anti-GFP blotting. (middle) Purified 6His-ubiquitin conjugates were examined by anti-GFP blotting. (bottom) 6His-ubiquitin expression and efficiency of purification was controlled using an anti-6His antibody. White lines indicate that intervening lanes have been spliced out.(B) Analysis of ubiquitylated forms of Rpl25-GFP in wild-type and the indicated mutant cells as in A.(C) Expression of Rpl25-GFP and its modified form were analyzed by anti-GFP blot of extracts from wild-type and mutant cells starved for the indicated periods. Ub, ubiquitin; WT, wild type.