EMBO/SourceData huggingface dataset:
Figure 3(D, E) Still frames of a movie showing endogenously tagged mVenus::βH-spectrin in the cross section of a gastrulating wild type (D) and twist mutant (E) embryo imaged using two-photon microscopy. The top frame corresponds to the time point where an initial bending of the apical surface was observed, the middle frame was taken 2 min and the lower frame 3 min afterwards. Scale bars, 30 µm.(F, G) Confocal images showing apical βH-spectrin (endogenously tagged mVenus::βH-spectrin) in a wild type (F) and twist mutant (G) embryo. Scale bars, 20 μm. Medio-apical βH-spectrin fibers observed in wild type embryos were absent in twist mutant embryos.(H-Q) Confocal or two-photon microscopy images of the apical surface of an embryo mounted with the dorsal side facing the objective and co-expressing endogenously tagged mVenus::βH-spectrin (green, H, K, M, P), the myosin-II marker Sqh::mCherry (magenta, I, N), and the optogenetic modules as explained in (C). The apical surface is shown before photoactivation (H-L) and 1 min after photoactivation (M-Q). Panels (J, O) show a merge of βH-spectrin and myosin-II and the dashed rectangle indicates regions shown in (L and Q) at higher magnification. Panels (K, L) and (P, Q) are zoom-ins into the region indicated in (J) an (O), respectively. Scale bars, 20 μm (H-J, M-O). 2.5 μm (K, L, P, Q).(R) Quantification of apical βH-spectrin upon optogenetic stimulation of Rho signaling. The graph shows the mean intensity of βH-spectrin (green) at the apical surface over time as the fold change relative to the initial time point (before photo-activation). The semi-transparent region indicates the standard deviation and dots indicate individual data points (n = 5 embryos).(S-T) Two-photon microscopy images of apical βH-spectrin (green) 1.5 min after optogenetic stimulation of Rho signaling in embryos that were previously injected with water (S) or the ROCK-inhibitor Y-27634 (T). Dashed boxes indicate photo-activated cells. Y-27634-injected embryos did not undergo apical constriction and did not accumulate βH-spectrin upon photo-activation, while water-injected embryos constricted and showed increased βH-spectrin levels. Scale bars: 10 µm.
Figure 1(B, C) Kinetics of the binding between the scFv of FD20 (scFD20) and RBD from SARS-CoV-2 (B) or SARS-CoV (C). Biolayer interferometry (BLI) assay was performed with RBD immobilized and scFD20 as analyte at indicated concentrations (nM). Raw data are shown in color and fitted lines are shown in grey. KD values were calculated using 1:1 global fitting. Note, although the fitting of the binding curves in D gave a KD of 4.8 nM, the accuracy of the kinetic parameters may be compromised owing to the mismatch between the raw data and the fitted curves at the initial dissociation phase. The reason for the abnormal BIL profile is unknown but it may be caused by possible denaturation of the commercial SARS-CoV RBD during storage. It was also noted that the BLI signal in D was ~5 times less than that with SARS-CoV-2 RBD under similar conditions (C).(D) Neutralizing assay of scFD20 against SARS-CoV-2 (black) and SARS-CoV (red) pseudoviruses.(E) Neutralization assays using FD20 against pseudoviruses harboring the wildtype SARS-CoV-2 S (blue), S D614G mutant (red), or SARS-CoV S (black).
Figure 2(A) Neutralization assay of FD20 using SARS-CoV-2 pp harboring spike derived from four Variants of Concern as indicated. Mean ± s.d. are plotted (n = 3 biological replicates). Numbers in brackets indicate IC50 values in nM.
Figure 3(A) Body weights of hamsters treated with antibodies were measured at indicated days after inoculation with SARS-CoV-2. The mean % of starting weight ± s.e.m. (n = 5 biological replicates) are plotted.(B-E) SARS-CoV-2 viral RNA (B and D) or infectious virus (C and E) was detected in the lung (B and C) and nasal turbinates (D and E). The mean copy number or the mean infectious titer is shown, error bars represent s.e.m. (n = 5 biological replicates).
Figure 4(D) Modest perturbation of scFD20 for RBD-ACE2 interaction. A sensor with RBD immobilized was soaked in 200 nM of scFD20 before being further soaked in scFD20-containing buffer with (red) or without (blue) 25 nM of ACE2 for BLI signal recording. As a control, the ACE2-RBD interaction was monitored in the absence of scFD20 (black).
Figure 5(A) Inhibition of cell-cell fusion by mAbs. Cell fusion was quantified by measuring the luciferase expression induced by full cell membrane fusion and mixing of cytoplasm, and the rate of inhibition by indicated antibodies compared to the condition without antibody is reported. Data depict mean ± s.d. (n = 4 biological replicates).(C) FD20 pre-incubation with SARS-CoV-2 pp in the absence of cells strongly impairs entry. To test direct action on particles, SARS-CoV-2 pp and FD20 were co-incubated for 1 h, then the premixes were used to infect VeroE6-hACE2 cells for 6 h at 37 °C. Mean ± s.d. are plotted (n = 3 biological replicates). Numbers in brackets indicate the gained neutralization (%) by pre-incubation. As control, cells were incubated at each step with an equal volume of PBS or Y112R. Percentages of primary infection were calculated according to viral titers of PBS control conditions. p < 0.0001 (****) unless specified otherwise (2-tailed, unpaired student's t-test). ns, not significant.(D) Inhibition of cell surface binding of SARS-CoV-2 VLP by FD20. VLP-GFP were preincubated, or not, with FD20 and RBM-targeting mAbs (CB6, REGN10933, REGN10987) for 30 min at 37 °C. Then the VLP premixes were incubated with VeroE6-hACE2 for 1 h at 37 °C and the binding detected by flow cytometry. Control binding assays were performed using soluble ACE2 as a competitor. Data are representatives from three experiments.
Figure 6(B) FD20 acts irreversibly on particles in the presence or absence of other mAbs. As described in (A), 5 different conditions were used to evaluate the effect of SARS-CoV-2 pp pre-incubation with mAbs. In some experiments, FD20 was mixed with other mAbs and synergic effects were observed. Inhibition rate was calculated relatively to infection without antibodies at the similar SARS-CoV-2 pp input (diluted 20 times or not, Group 2/1 in A). Mean ± s.d. are plotted (n = 3 biological replicates). p < 0.0001 (****) unless specified otherwise (one-way ANOVA with Tukey's test); ns, not significant. Source data is available online.(C-F) The destruction of SARS-CoV-2 S by FD20. (C) Negative-stain of FD20. (D-F) Negative-stain image (i) and 2D class averages (ii) of S alone (D), and S incubated with FD20 (E) or 5E1 (F) which is an unrelated mAb targeting a protein called Hedgehog (Maun et al, 2010). Bar = 100 nm. In D and F, red circles indicate typical side views of S. Results are representative of two independent experiments with two different S purification batches.
Figure 1Graph represents brain levels of TKIs over a 24 hr period after IP injection with Imatinib, Nilotinib, Bosutinib and DMSO (N = 7). [Correction added after publication on 4 July 2013: The y axis of the graph shown in Fig. 1A has been corrected from "concentration (mM)" to "concentration (nM)"]WB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) show total Abl (top blot) T412 Abl (2nd blot), soluble parkin level (3rd blot), and LC3 (4th blot) relative to MAP-2 (N = 9).Quantitative ELISA showing soluble (STEN extract) and insoluble (4 M urea extract) mouseparkin in Tg-APP (N = 9). Parkin−/−brains were used as a specificity control.(D) Blots represent immunoprecipitatedBeclin-1 in mice probed with parkin antibody(D) Blots represent immunoprecipitatedBeclin-1 in mice probed with parkin antibody, and (E) control IgG in parallel with immunoprecipitates.(F) immunoprecipitatedBeclin-1 probed with parkin and the reverse experiment in post-mortem human AD cortex analyzed on 4-12% SDS-NuPAGE gel.In situ proximity ligation assay (PLA) shows endogenous parkin-Beclin-1 complexes in (G) WT C57BL/6mice (N = 5) and (H) parkin−/− as control.PLA in Tg-APPmice IP injected once daily for 3 weeks with (I) DMSO (J) 5 mg/kg Bosutinib and (K) 10 mg/kg Nilotinib (N = 5).PLA in human post-mortem brains in the (L) cortex of a normal subject and (M) cortex of an AD patient; the hippocampus of (N) a normal subject and (O) an AD patient; the caudate of (P) a normal subject and (Q) an AD patient.Graph represents human Aβ1-42ELISA in rat B35 neuroblastoma cells transfected with human cDNA Aβ1-42 (or LacZ) or Beclin-1 shRNA for 24 hr, and then treated with 1 μM Bosutinib for an additional 24 hr (N = 12). *Significantly different to control or as indicated, Mean ± SEM, ANOVA with NeumannKeuls multiple comparison.
Figure 2Staining of 20 μm brain sections shows plaque formation with 6E10 antibody and DAB in the brain in different (A,B) Tg-APP + DMSO and (C) thioflavin-S staining in Tg-APP treated with DMSO (N = 7). (D,E) Tg-APP treated with 5 mg/kg Bosutinib for 3-weeks and (F) thioflavin-s staining.Staining of 20 μm thick brain sections shows (G) parkin, (H) Aβ1-42 and (I) merged figure in cortex of Tg-APPmice after 3 weeks of DMSO treatment, and (J) parkin, (K) Aβ1-42 and (L) merged figure in cortex of Tg-APPmice after 3 weeks of 5 mg/kg Bosutinib treatment. (M) Parkin, (N) Aβ1-42 and (O) merged figure in hippocampus of Tg-APPmice after 3 weeks of DMSO treatment, and (P) parkin, (Q) Aβ1-42 and (R) merged figure in cortex of Tg-APPmice after 3 weeks of Bosutinib treatment (N = 7).(S) Graphs represent quantification of amyloidplaques in Tg-APP with and without Bosutinib.WB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) showing (T) levels of BACE1 (1st blot), ADAM-10 (2nd blot) and presinilin-1 (3rd blot) relative to actinWB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) showing (U) total APP (top blot), CTFs (2nd blot) and phospho-tyrosine (3rd blot) relative to actin (N = 9).
Figure 3Graphs represent ELISA of human Aβ1-42 in (A) brain levels and (B) bloodAβ1-42 levels in 8-month old Tg-APPmice injected I.P. every other day for 6 weeks with Bosutinib or Nilotinib (N = 10).ELISA of human soluble and insoluble brain (C) Aβ1-42 and (D) Aβ1-40 levels in 8-month old Tg-APPmice injected IP daily for 3 weeks with 5 mg/kg Bosutinib (N = 9).ELISA of (E) mouse p-Tau levels in 8-month old Tg-APP mice injected IP daily for 3 weeks with 5 mg/kg Bosutinib (N = 9).WB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) show total Tau (1st blot), serine 396 p-Tau (2nd blot), threonine 231 (AT180, 3rd blot) and serine 199 p-Tau (AT8, 4th blot) relative to actin (N = 7).
Figure 4Staining of 20μm brain sections shows intracellular Aβ1-42 within the (A) hippocampus of lentiviralAβ1-42 injected WT mice, and (B) Bosutinib clearance of intracellular Aβ1-42. Staining of 20μm brain sections shows intracellular Aβ1-42 within the (C) cortex of WT mice with the lentiviralAβ1-42, and (D) Bosutinib clearance of intracellular Aβ1-42 (N = 7).ELISA of human soluble and insoluble Aβ1-42 at 6 weeks post-injection of lentiviralAβ1-42 and daily treatment with 5 mg/kg Bosutinib (N = 9) for 3 weeks. * Significantly different to control or as indicated, Mean ± SEM, ANOVA with Neumann Keuls multiple comparison.WB on 4-12% NuPAGE SDS gel of total brain lysates in 1 year old wild type and parkin−/−mice treated with 5 mg/kg Bosutinib for 3 weeks on 4-12% NuPAGE SDS gel (Invitrogen) showing parkin (1st blot)total Abl (2nd blot), T412 Abl (3rd blot), Beclin-1 (4th blot) and LC3 (5th blot) relative to actin (N = 7).Staining of 20μm brain sections shows plaque formation with 6E10 antibody and DAB 6 weeks post-injection with (G) lentiviralAβ1-42 + 3 weeks DMSO treatment and (H) IP injection with 5 mg/kg Bosutinib 3 weeks post-lentiviral expression (3 weeks treatment) clears plaques in WT mice (N = 7).(I) Lentiviral Aβ1-42 + DMSO and (J) IP injection with 5 mg/kg Bosutinib 3 weeks post-lentiviral expression (3 weeks treatment) in parkin−/−mice (N = 7).
Figure 5Graphs represent ELISA (N = 5) in autophagic vacuoles (AVs) in the brain of 4 and 8 months old Tg-APPmice treated with 10 mg/kg Nilotinib or 5 mg/kg Bosutinib (N = 5) for 3 weeks showing (A)humanAβ1-42 (Insert is WB analysis of AVs showing LC3-B in AV10 and AV20 (1st blot) and LAMP2a in Lys (2nd blot) fraction) and (B)Aβ1-40,(C) p-Tau and (D) parkin.ELISA in autophagic vacuoles of 1 year old WT and parkin−/−mice (N = 5) injected with lentiviralAβ1-42 for 3 weeks and treated with 10 mg/kg Nilotinib and 5 mg/kg Bosutinib for 3 additional weeks showing (E) humanAβ1-42 and (F) p-Tau levels.
Figure 6(A) Represents results of Morris water maze test in lentiviralAβ1-42-injected ± Bosutinib WT (N = 12) and parkin−/− (N = 10) mice, and (B) heat maps for each group.Graphs represent total number of entry into platform area and distance travelled.Represents results of Morris water maze test in Tg-APP ± Bosutinib (N = 12) mice.Graphs represent total number of entry into platform area and distance travelled.
Fig 1.Histopathological (A-D) and virological (E) examinations of the lung tissues from an ill Malayan pangolin naturally infected with Pangolin-CoV (A and B, 200× and 400×, respectively) and an uninfected Malayan pangolin (C and D, 200× and 400×, respectively). Interstitial pneumonia with extensive infiltration of inflammatory cells is seen in the lung tissue from the infected pangolin. Viral particles are seen in double-membrane vesicles in the transmission electron microscopy image (small scale = 50 nm) taken from the Verno E6 cell culture inoculated with supernatant of homogenized lung (E), with morphology indicative of coronavirus (inserts at the upper right corner of E).
Fig 2. Similarity plot of the near full-length genome sequence (A) and S gene sequence (B) of Pangolin-CoV to sequences of 2019-nCoV_WIV02, Bat-CoV RaTG13, Bat-CoV ZC45 and Bat-CoV ZXC21. While Pangolin-CoV has a high sequence identity to 2019-nCoV and Bat-CoV-RaTG13 in most regions of the S gene, it is more similar to Bat SARSr-CoV ZXC21 and Bat SARSr-CoV ZC45 at the 5′ end (B). More importantly, Pangolin-CoV is highly similar to 2019-nCoV in the functional RBD region of the S protein, with only one amino difference (insert in B). Because of the presence of genetic recombination, there is a discrepancy in cluster formation among the outcomes of phylogenetic analyses of different regions of the S gene (C).
Fig 4.Free energy (kcal/mol) for the binding between S proteins of four SARSr-CoV viruses and ACE2 of four likely hosts.
Figure 1Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. A mRNA expression of CD4+ cytokines Il2, IL4, IL8 and IL22 relative to endogenous controls, n=13/11/10/8 biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. D mRNA expression of CD8+ cytokines IFNγ, PRF1, TNFα, GZMB and CTLA4 in stimulated human T cells relative to internal controls, n=15/14/10/14/9 biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. F Foxp3 mRNA relative to internal control in human PBMCs and quantification of CD4+CD25+Foxp3+ regulatory T cells (Treg) following 5 days of Treg differentiation with a representative Foxp3 histogram plot (right side), n=7/11 biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. G IL10 and TGFβ1 mRNA and protein expression of human Treg cells, n=8/9 (IL10), 5/9 (TGFβ1) biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.
Figure 2Human PBMCs were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan T cells, CD4+ and CD8+ T cells were isolated through magnetic cell separation. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. (A-C) OCR, maximum respiration, spare respiratory capacity and basal respiration were measured in A pan T cells, B CD4+ T cells and C CD8+ T cells, n=9 (pan T), n=3/4 (CD4 unstimulated), n=7/8/9 (CD4 stimulated), n=5 (CD8 unstimulated) and n=8/7/7 (CD8 stimulated) individual experiments. Data depicted as mean ± SEM (OCR) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.
Figure 3Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. A Quantification of cellular reactive oxygen species (ROS) using CellROX dye, indicated by mean fluorescence intensity (MFI) FITC in human pan/CD4+/CD8+ T cells, n=8/6/6 biological replicates. B Mitochondrial superoxide production quantified through MitoSOX staining, depicted as MFI Phycoerythrin (PE) in human pan/CD4+/CD8+ T cells, n=8/14/14 biological replicates. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. C Confocal microscopy images of human CD4+/CD8+ T cells, stained with MitoTracker green, representative of two individual experiments.Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Flow cytometry analyses were performed using the indicated dyes and antibodies identifying T cells and CD4+/CD8+ T cell subsets. K Flow cytometric quantification of memory T cells: CD4+/CD8+ T cells were stained for CCR7 (PE+/-) and subsequently defined memory phenotype for CD45RO+ (Pacific Blue+) and CD45RA- (PerCP-) staining. Histogram plots depicting the exemplary change of CD45RO and CD45RA distribution for CD8+ T cells following memory cell differentiation (left side; NC=black, BHB=red). Fractions of CCR7+CD45RA-CD45RO+ central memory (CM) and CCR7-CD45RA-CD45RO+ effector memory (EM) CD4+/8+ T cells (right side), n=12/11/8/7 biological replicates. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.
Figure 3Tau phospho-variants generated by in vitro phosphorylation of non-phosphorylated Tau from E. coli with different kinases (PKA, GSK3β, or Cdk5) show differences in phosphorylation at specific phospho-site (pT205, pS262/pS356, pS324 and pS404) tested by Western blot. Non-phosphorylated Tau and p-Tau from insect cells are loaded for comparison. Position of tested p-sites is indicated in the Tau sequence cartoon. Western blot for total Tau (Tau13 antibody) confirms equal loading (0.2 μg Tau per lane) of the Tau preparations.Phos-tag gel shows degree and heterogeneity of phosphorylation in the different Tau phospho-variants. Intensity along the lanes are plotted as colored lines next to gel image.
Figure 4Tubulin co-condensation with Tau. Addition of tubulin-Alexa647 (5 μM, 10:1 unlabeled:labeled tubulin) to a Tau-Dy488 solution (25 μM) induces Tau condensation. Addition of tubulin to Tau:PEG, Tau:RNA (polyA-Cy3), and Tau:PEG:RNA condensates leads to partitioning of tubulin into existing condensates and the occurrence of additional small Tau:tubulin condensates. Condensates with high tubulin-HiLyte647 intensity have low polyA-Cy3 intensity, and vice versa. In Tau:PEG:polyA condensates, individual condensates host RNA- and tubulin-rich domains (arrow heads) at homogeneous Tau-Dy488 intensity. Scale bars=20 μm. Average intensity of RNA (top graph) normalized to Tau intensity in individual condensates. Addition of tubulin reduces the average amount of RNA in condensates. Intensity of tubulin (middle graph) normalized to Tau intensity in individual condensates. The presence of RNA increases the amount of tubulin in the condensates. Data shown as mean±SD. Data +/- tubulin and +/- polyA were compared by two-tailed Student's t-tests. Tubulin and RNA intensity correlate negatively on an individual condensate level (bottom graph). Line represent linear regression to all data. N=3 experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Condensate size (are on surface) relative to tubulin (yellow data points) and RNA (pink data points) intensity. Smaller (new) condensates are high in tubulin, larger (old) condensates have more RNA. Lines represent linear regressions to tubulin (yellow) and RNA (pink) data.MT polymerization out of Tau:PEG and Tau:PEG:RNA condensates. Addition of tubulin-Alexa647 (5 μM, 10:1 unlabeled:labeled tubulin) and GTP to Tau-Dy488 (25 μM) diluted in BRB80 polymerization buffer induces MT polymerization. Images taken 30 min after addition of tubulin show thin partially curved MT bundles in Tau:PEG system, and thick bundles attached to persisting Tau:RNA condensates in the Tau:PEG:RNA system. Scale bar=5 μm. Cross-sectional intensity plots (line plots) of MT bundles differ between Tau:PEG (yellow/red line plots) and Tau:PEG:RNA (blue/green line plots) systems. Four line plots at random positions are shown per condition.Nocodazole treatment (120 μM for 30 min) of MTs formed from Tau:PEG:RNA and Tau:PEG condensates. MT formed in absence of Tau shown as control. Graphs show surface coverage (%) of fibrillar MT structures before and after nocodazole treatment. N=3 experiments, 3 images analyzed per experiment and condition. Data shown as mean±SD. Two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bar=20 μm.