EMBO/SourceData huggingface dataset:
Figure 3(B) Representative confocal pictures showing these changes in live cells. Cells were transfected with the indicated constructs and imaged before and after addition of rapamycin (100 nM) for 10 min. Note that the Rab7 recruiter itself is dissociating from the membranes after PI(4,5)P2 generation. Left panels show some cells enlarged. Scale bars: 20 µm.(C) Quantification of the changes in BRET experiments. Here the BRET sensor contained the PLCδ1PH-fused to Sluc and the Venus targeted with Rab7 expressed from a single vector and it was co-transfected with the Rab7-targeted FRB (tagged with iRFP) and the FKBP12-fused PIP5Kγ in which the CFP was mutated to eliminate its fluorescence (CFP*). Recruitment of the PIP5Kγ but not its kinase-dead version caused an increase in the BRET signal indicating the increased PI(4,5)P2 in this compartment (means ± S.E.M., from three separate experiments performed in triplicates).(D) PI(4,5)P2 increases were larger when the Venus part of the BRET sensor was targeted with the GTP-locked form of Rab7 (Q67L) and was negligible with the GDP-locked form of Rab7 (N125I) in the BRET construct. Also note that the Rab7 wild-type-based BRET signal slowly returned toward the green trace consistent with Rab7 falling off from the membrane both in the BRET and the recruiting constructs (means ± S.E.M., from three separate experiments performed in triplicates).(E) The response of PI4K2A K/O cells was significantly reduced Means ± S.E.M. are shown from three experiments performed in triplicates.(F) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments (means ± S.E.M., n=3). One-way ANOVA with Dunnett's multiple comparisons was used for statistical analysis (* P = 0.0478 and 0.0287 for #20 and #26 clones, respectively).
Figure 4(A) Representative confocal images of HEK293-AT1 cells, which show that treatment with OSW1 (20 nM), a drug that inhibits the cholesterol-PI4P transport protein, OSBP, causes accumulation of PI4P in the Golgi and in the endosomes (upper panels). Note that the accumulation is much reduced in the endosome but less so in the Golgi in the PI4K2A K/O cells. Scale bars: 20 µm(B) Quantification of these changes by BRET analysis. The PI4P-Rab7 BRET sensor construct was transfected into the respective cell lines and the cells treated with OSW1 (20 nM) for the indicated times. The BRET ratios were expressed relative to those of DMSO treated cells. Data information: Means ± S.E.M. are shown from three experiments performed in triplicates.(C) Detection of endosomal PI(4,5)P2 in cells pre-treated with OSW1 for 1 h in serum-free medium using confocal microscopy and the PLCδ1PH-GFP PI(4,5)P2 reporter. Angiotensin II (AngII, 100 nM) (a Gq and PLC-activating agonist) was used to liberate the PLCδ1PH-GFP PI(4,5)P2 reporter from the PM, which was necessary to detect the endosomal PI(4,5)P2. The signal is transient as the AngII receptors show desensitization. Note the reduced signal in the two PI4K2A K/O clones. Scale bars: 20 µm.(D) Quantification of the same changes by BRET analysis in wild-type cells using the PI(4,5)P2 sensor with Rab7-Q67L-targeting. Note the transient signal increase after AngII stimulation only in cells treated with OSW1. (E) This increase is barely detectable in the PI4K2A K/O cells. D Data information: Means ± S.E.M. are shown from three experiments performed in triplicates. The values in (D) and (E) were measured in the same 96-well plate at the same time.
Figure 5(A) BRET experiment in parental HEK293-AT1 cells in OSW1-pretreated cells after RNAi mediated knock-down of PIP5Kβ or PIP5Kγ. (means ± S.E.M. from three experiments, each performed in triplicates). Note the reduced response in the PIP5Kγ knock-down cells and the slight reduction in the case of PIP5Kβ knock-down. (B) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments shown in panel A (means ± S.E.M., n=3). One-way ANOVA with Dunnett's multiple comparisons was used for statistical analysis (* P=0.0241; **P=0.0088). ( (C) These reductions are proportional to the reduction in PIP5Kγ levels as assessed by Western blot analysis of cell lysates obtained from cells treated with siRNAs at the same time.(D) Increased appearance of GFP-LC3 positive vesicles in PI4K2A K/O cells and reduced acidification of the LC3-positive vesicles using the GFP-mCherry-LC3 reporter. Cells were transfected with the indicated constructs for one day and observed by confocal microscopy. Scale bars: 20 µm. (E) Quantification of the LC3 positive vesicles. (n = 67, 34, 26, 13, 30, 27 for the various groups, respectively from left to right). *** designates P < 0.0001 using unpaired t-test. The same data set was also analyzed with the non-parametric Mann-Whitney test showing a P = 0.0004 and 0.0012 for #20 and #26 clones relative to control, respectively. (F) Co-localization analysis using the Pearson coefficient (n = 31, 21 and 14 cells analyzed for the different groups from left to right). *** designates P < 0.0001 using unpaired t-test. The same data set was also analyzed with the non-parametric Mann-Whitney test showing a P value < 0.0001 for both groups.
Figure 7(A) HEK293-AT1 cells were transfected with PLEKHM1-GFP-Rab7 along with CFP-FKBP-PIP5Kγ and iRFP-FRB-Rab7 constructs for one day and examined live by confocal microscopy. Representative cells show reduced PLEKHM1 localization when PIP5Kγ was recruited to the Rab7 endosomes (lower row images). (B) This response is reduced in PI4K2A K/O cells. ( Data information: Scale bars: 20 µm.(C) Quantification of these changes by BRET analysis where the full-length PLEKHM1 was fused to Sluc and Venus was fused to Rab7 in the BRET construct. For PIP5Kγ recruitment a mutant dark CFP(W66A)-FKBP-PIP5Kγ was used with an iRFP-FRB-Rab7. BRET values were normalized to those of DMSO treated cells. Comparisons were made for parental (blue) and PI4K2A K/O (green and orange) cells. (D) Similar BRET analysis where the PLEKHM1 was replaced by either RILP or Vps35 in the BRET construct. D Data information Means ± S.E.M. are shown from three experiments performed in triplicates.
Fig. 1.lines.Analysis of 2019-nCoV-S expression (A) by Western blot. Representative blots from three experiments are shown. ß-Actin (cell lysates)ines.Analysis of 2019-nCoV-S pseudotype incorporation (B) by Western blot. Representative blots from three experiments are shown. VSV-M (particles) served as loading controls.(C) Cell lines of human and animal origin were inoculated with pseudotyped VSV harboring VSV-G, SARS-S or 2019-nCoV-S. At 16 h postinoculation, pseudotype entry was analyzed. Shown are the combined data of three experiments. Error bars indicate SEM.
Fig. 2.(C) 293T cells transiently expressing ACE2 of human (dark blue) or bat (light blue) origin, human APN (purple) or hDPP4 (green) were inoculated with pseudotyped VSV harboring VSV-G, SARS-S, 2019-nCoV-S, MERS-S or 229E-S. At 16 h postinoculation, pseudotype entry was analyzed. The average of three independent experiments is shown. Error bars indicate SEM.
Fig. 3.priming.Ammonium chloride (A), were added to the indicated target cells before transduction with pseudotypes bearing the indicated glycoproteins.E64d (CatB/L inhibitor) (B) and/or camostat (TMPRSS2 inhibitor) (B) were added to the indicated target cells before transduction with pseudotypes bearing the indicated glycoproteins.(C) 293T cells transiently expressing ACE2 alone or in combination with TMPRSS2 were incubated with CatB/L inhibitor E64d or PBS as control and inoculated with pseudotypes bearing the indicated viral surface proteins. The average of three independent experiments is shown in panels A-C. Error bars indicate SEM. Statistical significance was tested by two-way ANOVA with Dunnett posttest.
Fig. 4.entry.Pseudotypes harboring the indicated viral surface proteins were incubated with different dilutions of serum from a convalescent SARS patient and subsequently inoculated onto 293T cells that transiently express ACE2 in order to evaluate cross-neutralization. The results from a representative experiment with triplicate samples are shown and were confirmed in a separate experiment. Error bars indicate SD. Statistical significance was tested by two-way ANOVA with Dunnett posttest.
Figure 1(B) Structure of FRH from the large crystallographic cell with an accordingly colored domain map below.
Figure 2(A) FRH large cell (cyan), Mtr4 (pdb 2XGJ, green, RNA backbone in orange), and FRH small alternate cell (blue) differ primarily in the orientation of the arch domain. The FRH RNA-bound structure also crystallizes in the small-cell configuration. The two different FRH structures have an overall RMSD of 3.9 Å and the RMSD between Mtr4 and FRH is 2.9 Å (over 734 residues, for large cell FRH).(B) SAXS data indicates that FRH-Δ114 is monomeric and flexible. The crystal structure of the large cell structure predicts the solution scattering curve (left, χ2 = 0.92) and fits well to the calculated molecular envelope (right).
Figure 3(A) Comparison of the FRH N-terminus (large cell structure, magenta) to that of Mtr4 (green) aligned on their RecA domains. Only the additional elements of FRH are shown (cyan). The FRH N-terminal extension has little ordered secondary structure, although regions 143-147 and 129-135 form small turns that interact with the helicase core. (B) The very N-terminus of FRH extends away from the RecA-2 domain to interact with a symmetry related molecule in the crystal (below), whereas the Mtr4 N-terminus turns back to form an outer β−strand. Center panel shows the crystal contacts, with close-ups for each region highlighted as insets on the left and right.
Figure 4(A) The two RecA domains are rotated relative to one another in FRH (cyan, large cell) compared to Mtr4 (green, RNA backbone in orange). The change in positioning of RecA-2 with respect to RecA-1 results in an overall displacement from the Mtr4 domain juxtaposition that ranges from 1.1 - 4.0 Å throughout RecA-2 with the largest differences at the external helices.(B) Comparison of arch domains conformations from Mtr4 (green) and FRH (large cell in cyan, small cell in marine) after superposition of the most N-terminal helical region. The isolated KOW modules of FRH, Mtr4 (with FRH superimposed), and Ski2 have considerably different loop configurations.
Figure 5(A) 3.8 Å resolution mFo-DFc omit map density (green, calculated with sharpened B-factors and contoured at 2.0) reveals the presence of ADP and a short segment of RNA bound to FRH.(B) 3.8 Å resolution 2mFo-DFc calculated without ADP or RNA contributing to the model. Green electron density shown for ADP and RNA (0.6orange density shown for surrounding protein (0.6.(C) Comparison of Mtr4 and FRHADP binding (FRH in cyan with ADP, RNA, and F179 in orange bonds; Mtr4 in green with ADP, RNA, and F148 in yellow bonds). (c) Unwound DNA from Hel308 (pdb 2PR6; magenta, DNA in orange bonds) overlaid in FRH with RNA and ADP (FRH in cyan with yellow RNA and cyan ADP in stick) after superposition of the respective RecA domains.(d) Hel308 DNA helicase DNA (pdb 2PR6; backbone in orange, bases in gray bonds) after aligning the RecA domains of Hel308 with those of FRH (cyan, RNA in yellow); see also Appendix Fig S4. The superposition indicates that the KOW module is positioned to interact with double stranded RNA that is being unwound by the RecA and DHSCT domains.
Figure 6(A) Key residues for FRH function shown on the large-cell structure: K208 and S324 compose the ATP binding site, E294 lies between the ADP and RNA pockets and participates in ATP hydrolysis; P871 resides in the elbow region of the arch domain. K811, R806, R712 and K766 contribute to a positively charged surface on the KOW domain. R806H disrupts the clock. Q131, G132 and V142 in the N-terminus provide interactions to the helicase RecA domains (FRH domains are colored according to Fig1, ADP and RNA are in orange bonds). (B) Same as (A) after rotation about a horizontal axis.(C) Close-up of the ADP-binding pocket and N-terminal interaction for the large-cell structure (cyan ribbons) and the small-cell structure (blue ribbons).(D) Electrostatic potential surface of FRH (blue positive, red negative) shown with the same orientation as in (B).(E) Close-up of the KOW domain showing the large number of basic (aqua sticks) and acidic side chains (yellow sticks) on the surface that also includes the mutational sites R806, R712, K811 and K766 (grey sticks)
Figure 7(A) Race tube assays with FRH mutants. Every circadian cycle N. crassa lays down aerial hyphae that appear as fluffy yellow/orange conidial bands. The distance between the bands reflects the clock period. The strains were grown in constant darkness and growth fronts were labeled every 24 h (black vertical lines), rhythmicity on right (τ= period in hours, σ = standard deviation, n = number of race tubes, ARR, arrhythmic). The race tubes shown here are representative samples from three replicate tubes.(B) Pull-down assays evaluating interactions among FRH, FRQ, WC-1 and WC-2. Ratio of FRQ, WC-1 and WC-2 binding to the V5 tagged FRH mutants as compared to the inter-strain variations in FRH levels, error bars represent standard deviation for the assay run in triplicate. *P < 0.05 (unpaired two-tailed t-test with Gaussian distribution assumption). Representative immunoblots are shown in Appendix Fig S5.
Figure 3(a) Relative quantification (RQ) of CG7630 transcript after RNAi with a 120-bp inverted-repeat sequence (act5c-gal4>UAS-CG7630RNAi) compared to control (act5c-gal4>+).(b) Relative percentage of egg to adult viability of CG7630 RNAi cross (act5c-gal4/CyO.GFP x UAS-CG7630RNAi) and control cross (act5c-gal4/CyO.GFP x w1118), calculated at three developmental stages (eggs, pupae and adults), (n > 250, Chi-square test 9.920 df(2), **p = 0.007).(d) Kinetic enzyme activity of individual MRC complexes in CG7630 RNAi and control individuals normalized by citrate synthase (CS) activity.(e) In gel-activity assays for MRC complexes II and IV in DDM-solubilized mitochondria from RNAi (act5c-gal4>UAS-CG7630RNAi) and control larvae (act5c-gal4>+).(f) BN-PAGE, Western blot immunodetection of MRC complexes from CG7630 RNAi (act5c-gal4>UAS-CG7630RNAi) and control (act5c-gal4>+) larvae using antibodies against specific subunits: anti-UQCRC2 (complex III), anti-NDUFS3 (complex I), anti-COX4 (complex IV) and anti-SDHA (complex II).(g) Western blot analysis of steady state levels (SDS-PAGE) of MRC subunits COX4 (complex IV), SDHA (complex II) and UQCR-C2 (complex III) in whole lysates from CG7630 RNAi (act5c-gal4>UAS-CG7630RNAi) and control (act5c-gal4>+) larvae.(h) Western blot analysis of anti-HA affinity purified samples from S2R+ D. melanogaster cells. EV = samples from cells carrying the empty expression vector. CG7630-HA = samples from cells expressing HA-tagged CG7630. Tot = total mitochondrial-enriched fractions; unb = unbound material; IP:HA = immunoprecipitated material. Samples were probed with antibodies against MRC subunits of complexes I (NDUFS3), II (SDHA), III (UQCR-C2), IV (COX4) and V (ATP5A).
Figure 6(a) Relative quantification (RQ) of CG7630 transcript in wild-type D. melanogaster S2R+ cells treated with a dsRNA targeting CG7630 (CG7630 KD) and a mock control dsRNA (mock).(b) Relative quantification (RQ) of CG7630 and COX7B transcripts in stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). Cells were treated with a dsRNA targeting CG7630 (striped bars, CG7630 KD) and a mock control dsRNA (solid bars, mock). N/D = not detected.(c) Immunoblot analysis of stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). The CG7630 transcript was knocked-down (KD) in cells by a dsRNA targeting CG7630 and compared with cells treated with a mock control dsRNA (mock). Samples were probed with an antibody anti HA tag and with an antibody against Hsp70 as an endogenous control.(d) Kinetic enzyme activity of COX normalized by citrate synthase activity (CS) in stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). Cells were treated with a dsRNA targeting CG7630 (striped bars, CG7630 KD) and a mock control dsRNA (solid bars, mock).(e) Steady-state levels of MRC subunits in samples from rescue experiment performed as in (d).
Figure 1A-B Representative image of a proliferative cell in the DG labeled with an anti-PH3 antibody (green) (A) and quantification of the number of PH3+ cells in the DG of WT and Tau-/- mice (B) (mean ± SEM; n= 10 mice WT, n= 8 mice Tau-/-; Student´s t-test).C-D Representative image of an apoptotic cell in the DG labeled with an anti-fractin antibody (red) (C) and quantification of the number of fractin+ cells in the DG of WT and Tau-/- mice (D) (mean ± SEM; n= 10 mice WT, n= 8 mice Tau-/-; Student´s t-test).E-F Schematic diagram of the cell survival experimental design using thymidine analogs (F) and quantification of the percentage of surviving cells in Tau-/- mice as compared to WT mice at each cell age (1-, 2-, 4-, 6- and 8-week-old cells) (F) (mean ± SEM (normalized data); Student´s t-test).H Quantification of the percentage of 1-, 4- and 8-week-old newborn neurons that express the DCX neuroblast marker and the NeuN mature neuron marker in WT and Tau-/- mice (χ2 test).I-J Representative images of progenitor cells in the DG labeled with anti-Sox2 (red) and anti-BLBP (green) antibodies (I), and neuroblasts and immature neurons labeled with anti-DCX (red) and anti-calretinin (green) antibodies, respectively (J).K-N Quantification of the number of Sox2+ (K), BLBP+ (L), DCX+ (M) and calretinin+ (N) cells in the DG of WT and Tau-/- mice (mean ± SEM; n= 10 mice WT, n= 8 mice Tau-/-; Student´s t-test).
Figure 2A Representative images of 4-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus. A schematic representation of Sholl´s analysis is shown in the WT image.B-C Quantification of total dendritic length (B) and Sholl´s analysis (C) of 4-week-old newborn granule neurons in WT and Tau-/- mice.D Representative images of 8-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus.E-F Quantification of total dendritic length (E) and Sholl´s analysis (F) of 8-week-old newborn granule neurons in WT and Tau-/- mice.G Representative images of 4-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus and their corresponding high-power magnifications showing PSDs (green).H-IQuantification of the number of PSDs/µm (H) and PSD area (I) in each dendritic branching order of 4-week-old newborn granule neurons in WT and Tau-/- mice.J Representative images of 8-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus and their corresponding high-power magnifications showing PSDs (green).K-L Quantification of the number of PSDs/µm (K) and PSD area (L) in each dendritic branching order of 8-week-old newborn granule neurons in WT and Tau-/- mice.
Figure 3A Representative traces of mEPSCs recorded at a holding potential of −65 mV from WT (black traces) and Tau-/- (red traces) mice.B Exemplary traces of mEPSCs from one cell from WT (black trace) and Tau-/- (red trace) mice.C Resting membrane potential of WT and Tau-/- mice (mean ± SEM; n= 13 cells WT, n= 10 cells Tau-/-; *0.05 > p ≥ 0.01, Student´s t-test).D-E mEPSC amplitude (D) and mEPSC frequency (E) of WT and Tau-/- animals (mean ± SEM; n= 13 cells WT, n= 10 cells Tau-/-; Student´s t test or Mann-Whitney´s U test).F Cumulative probability plot of the total mEPSC amplitudes, small mEPSCs (i) and big mEPSCs (ii) from both WT and Tau-/- mice (black and red traces, respectively) (***p < 0.001; Kolmogorov-Smirnov Z test).
Figure 4A Representative image of a mouse subjected to forced swimming in the Porsolt test.C Representative images of 1-week-old newborn granule neurons labeled with CldU (green) belonging to WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice. Cell nuclei were labeled with DAPI (blue). Scale bar 50 µm.D-E Quantification of the number of 1-week-old CldU+ cells (D) and apoptotic fractin+ cells (E) in each experimental group.
Figure 5B Representative images of 8-week-old newborn granule neurons of WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice infected with a PSD95-GFP-expressing retrovirus. In the WT CNP image a schematic representation of Sholl´s analysis is shown.C-D Quantification of total dendritic length (C) and Sholl´s analysis (D) of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).E-F Representative images of an 8-week-old newborn granule neuron, in which proximal dendrites are highlighted (E), and of PSDs (green) in proximal dendrites belonging to WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice (F).G-H Quantification of the number of PSDs/µm (G) and PSD area (H) in each dendritic branching order of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).I-J Representative Western Blot (I) and quantification (J) of the levels of GluR1 subunit of AMPA receptors in hippocampi of WT and Tau-/- mice subjected or not to the Porsolt test (n= 6 mice per experimental condition).K Representative image showing 8-week-old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field (indicated by a white square).L-M Representative images (L) and quantification of the area (M) of MFTs of 8-week-old newborn neurons in the CA3 region of WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice (n= 3 mice per experimental condition). Arrows indicate MFTs.
Figure 6A Representative images of control housing (CH) and environmental enrichment (EE) cages.B Schematic diagram of the experimental design.C-D Representative images of DCX+ cells in the DG of CH and EE animals (C), and quantification of the number of DCX+ cells of WT and Tau-/- animals in CH or EE conditions (D).E Representative images of 8-week-old newborn granule neurons labeled with IdU (red) belonging to WT CH, WT EE, Tau-/- CH and Tau-/- EE mice.F-G Quantification of the number of 8-week-old IdU+ cells (F) and apoptotic fractin+ cells (G).
Figure 7A Representative images of the whole hippocampus showing GABAergic innervation labeled with GAD-65 antibody (red), and high-power magnifications of the DG molecular layer showing GAD-65+ terminals in WT CH, Tau-/- CH, WT EE and Tau-/- EE mice. Cell nuclei were labeled with DAPI (blue). White scale bar 300 µm. Red scale bar 50 µm. Green scale bar 5 µm.B-C Quantification of the density (B) and area (C) of GAD-65+ terminals in the molecular layer of mice in each experimental condition (n= 10 mice WT CH, n= 8 miceTau-/- CH, n= 6 mice WT EE, n= 10 miceTau-/- EE).
Figure 8B Representative images of 8-week-old newborn granule neurons of WT CH, WT EE, Tau-/- CH, and Tau-/- EE mice infected with a PSD95-GFP-expressing retrovirus. In the WT CH image a schematic representation of Sholl´s analysis is shown.C-D Quantification of total dendritic length (C) and Sholl´s analysis (D) of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).E-F Representative images of an 8-week-old newborn granule neuron in which proximal and distal dendrites are highlighted (E) and of PSDs (green) in distal and proximal dendrites of WT and Tau-/- mice housed under either CH or EE conditions (F).G-H Quantification of the number of PSDs/µm (G) and PSD area (H) in each dendritic branching order of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).I Representative image showing 8-week-old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field (indicated by a white square).J-K Representative images (J) and quantification of the area (K) of MFTs of 8-week-old newborn neurons in the CA3 region of WT CH, WT EE, Tau-/- CH, and Tau-/- EE mice (n= 3 mice per experimental condition). Arrows indicate MFTs.