EMBO/SourceData huggingface dataset:
Figure 6A) Turbidity of N is increased in the presence of torula yeast RNA and homopolymeric RNAs. Error bars represent standard deviation of three replicates. B) DIC micrographs of MBP-N in the absence or presence of TEV (to cleave MBP from N to initiate with indicated RNA. In the absence of TEV, MBP-N in the presence of torula yeast RNA, polyC, or polyU RNA does not undergo LLPS. However, after cleavage of MBP, N phase separates. polyA RNA increases LLPS of N. Both in the absence and presence of TEV, polyG RNA induces aggregation of MBP-N. Sample conditions:50 µM MBP-N, 0.5 mg/mL RNA/polyX, 70 mM NaCl, 25˚C, 50 mM Tris pH 7.4. Scale bar represents 100 µm.
Figure 7A) N partitions into hnRNPA2 LC droplets even with the MBP tag attached.B) N partitions into TDP-43 CTD droplets even with the MBP tag attached.C) N partitions into FUS LC droplets only after cleavage of the MBP tag.D-F) N partitions into hnRNPA2 FL (D), TDP-43 FL (E), FUS FL (F) droplets.
Figure 1(A-B) Representative MA currents recorded in whole cell patch clamp experiments at -60 mV in large DRG neurons in response to repetitive mechanical indentations with a blunt glass probe; experiments were performed as described in the methods section. The applications of 25 μM baclofen (B) or vehicle (A) are indicated by the red horizontal lines. The downwards spikes represent individual MA currents induced by repetitive mechanical stimuli every 30 s for 200 ms stimulus duration.(C) Quantification of normalized MA current amplitudes from DRG neurons, vehicle (white, n=7), neurons responding to baclofen (R, red) (n=6, **P<0.01, Paired t-test and non-responding DRG neurons (NR, blue, n=6). Striped column shows the average normalized current amplitudes 3-10 minutes after removal of baclofen in responding neurons (Washout) (n=6). Data are shown as mean ± SEM and scatter plots.(D-E) Representative MA currents recorded at -60 mV in large DRG neurons in response to repetitive mechanical stimulation with a blunt glass probe displaced 2-6 µm in 0.4 µm increments every 15 s (step protocol) before (black) and after (red) exposure to 25 µM Baclofen. Not every trace is shown for clarity. Inset above the current traces show the mechanical step protocol indicating the displacement of the mechanical probe.(F) Quantification of normalized MA currents of responding large DRG neurons [n=13, *P<0.05, **P<0.01, ***P<0.001, before (black) and after (red) baclofen treatment. Dashed grey line shows average MA currents 3-15 minutes after washout of baclofen. Repeated measures ANOVA with student's t-test no corrections was used to compare baseline and baclofen treatment. Data are shown as mean ± SEM.(G) Quantification of normalized MA currents of non-responding DRG neurons (n=9). Data are shown as mean ± SEM.(H-I) Quantification of MA current inactivation rate (Tau) measured for responding (R) large DRG neurons (n=13, **P<0.01, Paired t-test) and non-responding large (NR) DRG neurons (n=9, NS, not significant) before (gray) and after (red) baclofen application. Data are shown as mean ± SEM and scatter plots.
Figure 2(A-B) Representative MA currents recorded in whole cell patch clamp experiments at -60 mV in HEK293 cells transiently transfected with Piezo2 +GABAB1+ GABAB2 + GFP (for visualization) in response to repetitive mechanical stimulation with a blunt glass probe displaced 2-5.6 µm in 0.4 μm increments every 15 s before (black) and after (red) exposure to 25 µM baclofen. Not every trace is shown for clarity.(C) Quantification of normalized MA currents of HEK293 cells transfected with Piezo2+GABAB1+ GABAB2 + GFP [C, n=13, *P<0.05, **P<0.01, before (black) and after (red) baclofen treatment, Repeated measures ANOVA with student's t-test no corrections]. Data are shown as mean ± SEM.(D) Quantification of MA current inactivation rate (Tau) in HEK293 cells transfected with Piezo2+GABAB1+ GABAB2 + GFP before (gray) and after (red) baclofen application (n=13, NS, not significant; Paired t-test). Data are shown as mean ± SEM and scatter plots.Quantification of normalized MA currents of HEK293 cells transfected Piezo2 + GFP (E, n=7) before (black) and after (red) application of 25 µM baclofen. Data are shown as mean ± SEM.Quantification of normalized MA currents of HEK293 cells transfected Piezo2+GABAB1+ GABAB2 + βARKct-mCherry (F, n=8) before (black) and after (red) application of 25 µM baclofen. Data are shown as mean ± SEM.
Figure 3Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+D2RS-YFP (A, n=13) before (black) and after (red) application of quinpirole 200 nM. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.Quantification of normalized MA Piezo2 currents in HEK293 cells transfected with Piezo2 and GFP (B, n=5) before (black) and after (red) application of quinpirole 200 nM. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.(C) Quantification of Piezo2 current inactivation rate (Tau) in HEK cells transfected with Piezo2+D2RS-YFP. Data are shown as mean ± SEM and scatter plots (n=13)(D,E) Quantification of normalized MA currents in responding large DRG neurons (D, n=5) versus non-responding DRG neurons (E, n=3) before (black) and after (red) application of sumatriptan 10 µM to activate the Gi-coupled Htr1d receptors. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.(F) Quantification of MA current inactivation rate (Tau) measured for responding (R) large DRG neurons (n=5, Paired t-test). Data are shown as mean ± SEM and scatter plots.(G-H) Quantification of normalized MA currents recorded from large DRG neurons dialyzed with 50 ng/ml Gβγ (G, n=7) or with 0.0001% Lubrol (vehicle for Gβγ) (H, n=6). *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.(I,J) Quantification of MA current inactivation rate (Tau) measured for large DRG neurons dialyzed with Gβγ (n=7, **P<0.01, Paired t-test) and vehicle (Paired t-test). Data are shown as mean ± SEM and scatter plots.
Figure 4Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+ GABAB1+ GABAB2 + GFP (A, n=12)Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+ GABAB1+ GABAB2 + GFP (A, n=12), treated with the PI3K inhibitor Wortmannin 50 nM (B, n=10) Cells were treated with wortmannin 30 min before the experiments, and were present throughout the measurements.Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+ GABAB1+ GABAB2 + GFP (A, n=12), treated with the MAPK inhibitor U0126 10 µM (C, n=7). Cells were treated with U0126 for 30 min before the experiments, and were present throughout the measurements.
Figure 2A. HZZ-OTOFop, carrying a C-terminal glycosylation site (opsin tag) was purified alone or in complex with wild-type or an ATPase-deficient mutant version of TRC40 and incubated in the absence or presence of ER-derived rough microsomes (RM). Membrane integration (glycosylation) was monitored by SDS-PAGE and immunoblot using an anti-opsin antibody. Where indicated, EndoH was used to remove N-linked oligosaccharides.B. HZZ-OTOFop in complex with wild-type or mutant TRC40 was incubated with RM in the presence or absence of ATP and membrane insertion was monitored by opsin-specific immunoblot.C. HZZ-OTOFop in complex with wild-type TRC40 was incubated in the presence of RM, trypsin-treated rough microsomes (TRM) or in the presence of WRBcc or CAMLcyt. Membrane insertion was monitored by opsin-specific immunoblot.D. Quantification of relative protein glycosylation shown in B (n = 3). Data are represented as mean ± SEM. TRC40gr, mutated version of TRC40; **p<0.01; ***p<0.001 (Student's two-sample t-test).E. Quantification of relative protein glycosylation of the data shown in C (n = 3). Data are represented as mean ± SEM. ***p<0.001 (Student's two-sample t-test)
Figure 3A. Grand averages of auditory brainstem responses (ABR) from Wrbfl/fl:CreA (red traces; SEM pink) and Wrb+/+:CreA (black traces; SEM grey) mice. ABRs were recorded for three separate age groups (A-A'', as indicated in the graph) using click stimulation at 80 dB (peak equivalent) and 20 Hz stimulation rate. There was an age- progressive reduction of ABR amplitude in Wrbfl/fl:CreA mice.B. ABRs from Wrbfl/fl:CreA mice showed a progressive threshold increase, Wrb+/+:CreA mice and Wrbfl/fl mice lacking Cre recombinase had normal thresholds. Statistical comparison of the threshold at 12 kHz was done by Kruskal-Wallis test, p values are from post-hoc Dunn's multiple comparison test. Measurements in which no ABR was observed at the maximal available tone burst level (90 dB) or click level (120 dB) scored as 100 dB threshold. Data are represented as mean ± SEM.
Figure 4A-D. Representative maximum projections of confocal sections of (A-B) P14 and (C-D) P20 Wrb+/+:CreA and Wrbfl/fl:CreA apical turn organs of Corti following immunolabeling against otoferlin (green) and Vglut3 (magenta) processed and imaged under identical conditions. A'-D' show individual otoferlin stainings with an intensity-coded lookup table. A''-D'' present single xz projections at a central point through a representative IHC to illustrate otoferlin subcellular distribution. Scale bars in A-D' represent 10 μm, in A''-D'' 2 µm. Note the dramatic reduction in otoferlin fluorescence and the alteration in its distribution as well as a change in cell shape and position of nuclei (for detailed analysis of cell shape and nuclei position, please refer to Figure EV5).E-G. Semiquantitative analysis of otoferlin immunofluorescence in the apical parts of IHCs from Wrbfl/fl:CreA mice using averaged regions of interests in cell's maximal projections (outlined with a dotted line in G for a single representative IHC) normalized to the maximum values observed in the respective control IHCs. Averaged supra nuclear coronal line profiles as illustrated with a dashed line in G are shown for (F) P14-16 and (F') P18-21 IHCs. ***p<0.001 versus Wrb+/+:CreAH-J. Semiquantitative analysis of otoferlin immunofluorescence in the basal parts of IHCs from Wrbfl/fl:CreA mice using averaged regions of interests in cell's maximal projections (outlined with a dotted line in J for a single representative IHC) normalized to the maximum values observed in the respective control IHCs. Averaged supranuclear coronal line profiles as illustrated with a dashed line in G are shown for (I) P14-16 and (I') P18-21 IHCs. ***p<0.001 versus Wrb+/+:CreA
Figure 5A. Representative electron micrograph of a Wrb+/+:CreA IHC ribbon synapse. Scale bar: 120 nm.B-B'. In Wrbfl/fl:CreA (depicted are two consecutive ultrathin sections) IHCs, accumulations of large partially amorphous vesicles were observed close to ribbon synapses (red arrows), but also further away in the cytoplasm (cyan arrows). Moreover, cisternal structures resembling (r)ER are found unusually close to the ribbon (orange arrow in B'). Scale bar: 120 nm.E. Quantification in random ultrathin sections (Wrb+/+:CreA n = 28, from two animals; Wrbfl/fl:CreA n = 44, from three animals) of the numbers of membrane-proximal "MP" vesicles (in close proximity to membrane and ribbon); vesicles within a 100 nm range along the active zone membrane; vesicles at the lower "proximal" and the upper "distal" half of the ribbon as well as vesicle >70 nm ("large vesicles") in a radius of 800 nm around the ribbon. The vesicle number was significantly reduced in Wrbfl/fl:CreA IHCs at both ribbon parts but not at the presynaptic membrane.F. Average vesicle diameters of the membrane-proximal vesicles and the vesicles at both halves of the ribbon. The mean vesicle diameter increases significantly at both parts of the ribbon in Wrbfl/fl:CreA IHCs. Data are represented as mean ± SEM. Student's two-sample t-test ***p<0.001
Figure 6A. Ca2+ current-voltage relationship of P14-P17 IHCs of Wrbfl/fl:CreA (n = 17 IHCs) and Wrb+/+:CreA (n = 15 IHCs) mice in 2 mM extracellular [Ca2+] showed no change in amplitude or voltage-dependence of the Ca2+ current. Data are represented as mean ± SEM.B. Representative Ca2+ current (top) and Cm changes (bottom) in Wrbfl/fl:CreA and Wrb+/+:CreA IHCs in response to a 20-ms step depolarizations to -14 mV (the potential eliciting the maximum Ca2+ current).C. Representative Ca2+ current (top) and Cm changes (bottom; finite impulse response filtered) in Wrbfl/fl:CreA and Wrb+/+:CreA IHCs in response to a 100 ms step depolarizations to -14 mV.D. Exocytic ΔCm (top) and corresponding Ca2+ current integrals, QCa (bottom) for various depolarization durations. Data are grand averages of the cells' means. Exocytic ΔCm was reduced in the knockout from 20 ms onwards, indicating reduced sustained exocytosis. Data from OtofPga/Pga animals were replotted for direct comparison from (Pangrsic et al, 2010).E. ΔCm/QCa2+ ratio indicates a lower efficacy of Ca2+ influx in driving exocytosis in Wrbfl/fl:CreA IHCs, for stimuli of 20 ms or longer. Data represents means ± SEM; *p<0.05; **p<0.01; ***p<0.001
Figure 7A. All SGNs of Wrbfl/fl:CreA mice had spontaneous firing rates (SR) below 20 Hz (p>0.05, Kolmogorow-Smirnov test).B. Representative tuning curves from Wrbfl/fl:CreA and Wrb+/+:CreA SGNs indicating preserved active cochlear amplification despite disruption of Wrb in IHCs.C. Normal thresholds at the characteristic frequency (CF) in Wrbfl/fl:CreA SGNs.D-D''. Mean poststimulus time histograms (PSTH) ± SEM of Wrbfl/fl:CreA and Wrb+/+:CreA SGNs in response to 50 ms tone bursts presented at CF, 30 dB above threshold at stimulus rates of 2 Hz (D), 5 Hz (D'), or 10 Hz (D''). While the general response pattern was preserved, spike rates were drastically reduced in Wrbfl/fl:CreA SGNs, especially at higher stimulus rates. Data from auditory neurons (SGN and cochlear nucleus) of OtofPga/Pga animals were replotted for direct comparison from (Pangrsic et al, 2010).E. Spike rates in response to sound onset (maximum rate in PSTH with 0.5 ms binwidth) and adapted rates (averaged between 35-45 ms after stimulus onset) were significantly lower in Wrbfl/fl:CreA SGNs (stimulus rate 5 Hz; p<0.001).F. In line with the reduction in spike rates, first spike latency (FSL) following stimulus onset was greatly increased in Wrbfl/fl:CreA SGNs compared to Wrb+/+:CreA (stimulus rate 5 Hz; p<0.001).G. Illustration of the stimulus paradigm for forward masking experiments: a 100 ms masker tone presented at CF, 30 dB above threshold was followed by a silent interval of variable duration and a 15 ms probe tone (CF, 30 dB above threshold). Bottom: in Wrbfl/fl:CreA SGNs, the response to the first 5 ms of the masker probe tone tone (shown as a fraction of the response to the first 5ms of the masker) was strongly reduced, and the half time of recovery (from normalized curves) was increased from 39.2 ± 12.2 ms in Wrb+/+:CreA to 215.7 ± 51.2 ms in Wrbfl/fl:CreA SGNs (p<0.001, Mann-Whitney U test). Data represent means ± SEM.
Figure 8C. Apparent decrease in insertion efficiency for the TMD mutant version. While the WT otoferlin glycosylation amounted to 3.84 ± 0.53, the TMD mutant had only 2.13 ± 0.65 (n=3, p<0.05). Data are represented as mean ± SEM.
Figure 9A. Visual reinforcement audiometry performed in the free field: a mild hearing loss.B. Distortion product otoacoustic emissions (DPOAEs) were detected in each ear, whereasC. Auditory brainstem responses (ABR) were lacking, together providing the signature of auditory synaptopathy or neuropathy.D-E. Cochlear microphonic potentials (CM), summating potential (SP) and compound action potential (CAP) recorded through transtympanic electrocochleography (ECochG) in response to clicks at decreasing stimulus intensities are superimposed on the corresponding potentials recorded from one normally-hearing control (range as measured at 120 dB 4.31-28.02 µV in 20 normally-hearing children). Means and standard errors of peak latency and amplitude are also reported for each potential category. The ECochG waveform resulting from CM cancellation in the control begins with an abrupt negative deflection, the SP, followed by a negative peak, the neural CAP. In the child with the OTOF TMD mutation, the ECochG responses begin with a rapid negative deflection that peaks at the same latency as the SP in the control and has comparable amplitude. This is followed by a low-amplitude negative potential that peaks at the same CAP in controls but shows a markedly prolonged duration. In all graphs time "0" refers to CM onset. R: right, L: left.F. Means and standard errors of peak latency and amplitude are reported for each potential category.
Figure 1(Top) PCR detection of the recombined BrafLox−V600E allele (Lox-V600E) in BVE mouse tissues at 8 weeks of age without tamoxifen induction. Substantial recombination is observed in the lung, while weaker recombination is also detected in the liver. No recombination was detected in hematopoietic tissues (bone marrow and spleen) even after 40 cycles of amplification (right). (Bottom) H&amp;amp;E staining of lung sections from wild-type (WT, 10 weeks of age), BVE (1-10 weeks of age) and Braf+/LSL−V600E mouse 8 weeks after nasal delivery of AdCre is indicated below. V600EBRAF expression induced by two different methods causes similar pathology showing papillary adenomas accompanied by stroma development. Scale bars, 100 μm.Ki67 immunostaining of lung sections from BVE mice at 3-10 weeks of age. Scale bars, 25 μm. The right bar graph summarises %Ki67+ cells in tumours at different ages post-partum. Four to six mice were analysed for each age group as indicated, and more than 3,500 tumour cells per mouse were evaluated. The data represent mean ± SD.Representative H&amp;amp;E staining and Mac2 immunohistochemistry of serial lung sections from 10-week-old BVE mice at low (upper panels) and high (lower panels) magnifications. Scale bars, 100 μm (upper panels) or 25 μm (lower panels).
Figure 2Giemsa staining of isolated stroma IMCs. These cells show round or oval-shaped nuclei that lack indentations, and have an abundant basophilic cytoplasm without showing prolonged cytoplasmic processes, suggestive of immature myeloid-lineage cells that are morphologically distinct from monocytes/DCs. Scale bar, 25 μm.Cell surface marker analysis of IMCs showing lack of common hematopoietic marker expression except for CD11b, which is expressed at low levels (overlaid with isotype control histograms shown in grey).Cell surface marker analysis (dot plots) of IMCs demonstrating CD11c+ IMCs do not express macrophage (F4/80), DC (CD24) or M1-polarisation (CD86) markers, but weakly express the M2 marker CD206. Insets show isotype control staining.Morphological evaluation of IMCs cultured for 2 weeks without additional cytokines. The cells grown on culture plates were imaged by phase-contrast microscopy (left). Then, the cells were trypsinised and smeared on glass slides for Giemsa staining (right). These cells are intermediate to large sized, round or amoeboid cells with cytoplasmic vacuolation, consistent with a macrophage morphology. Scale bars, 50 μm.Cell surface marker analysis of cultured IMCs showing differentiation into F4/80+ macrophages with CD11bhighCD11c+CD86+CD206low surface phenotype (overlaid with isotype control histograms shown in light grey).PCR detection of Braf recombination in purified IMCs, AT2 cells and tumour cell aggregates.
Figure 3CMT64 cells (1 × 104/well in 12-well plates) were co-cultured with 1.5-3 × 105 IMCs for 4 days. Cells were collected by trypsinisation to exclude most of the IMCs since they are resistant to trypsin due to their strong adhesion to the culture plate. Harvested cells were subjected to counting (left). Any contaminating IMCs were separately counted according to their morphological distinction from CMT64 cells. The bar graph (left) shows CMT64 growth (cell counting) data pooled from two independent experiments. Phase-contrast images of CMT64 cells cultured without IMCs (CMT64 only) or co-cultured with IMCs (CMT64/IMC co-culture) are also indicated in the middle and right. A higher magnification image of co-cultured CMT64 cells (right) highlights IMCs migrating into CMT64 cells (arrow heads). Scale bars are 200 μm, except for the high magnification image (right) in which the scale bar is 100 μm.(Left) Growth and EMT-like morphological changes of CMT64 cells cultured with 25% IMC-CM. CMT64 cells cultured for 4 days at low-density in DMEM/10%FCS without IMC-CM (-IMC-CM) or with 25% IMC-CM (+IMC-CM) were counted and normalised to the average of control as 100%. The data in the bar chart represent mean + SD of five cultures utilising IMC-CM obtained from three independent sources. (Middle) Phase-contrast images to indicate representative morphologies of CMT64 cells maintained without IMC-CM (control) or cultured with 25% IMC-CM (+IMC-CM) for 2 weeks. (Right) E-cadherin immunofluorescence of CMT64 cells cultured as above. Scale bars are 200 μm (phase contrast) and 10 μm (immunofluorescence).Co-culture of AT2 cells and autologous IMCs from BVE mice (8- to 10-week-old) for 48 h using the Transwell® culture system. In vitro BrdU incorporation (flow cytometry, left and middle) and EMT marker expression (RT-PCR, right) in co-cultured AT2 cells are indicated. Representative flow cytometry plots are indicated on the left, and the bar chart in the middle indicates % BrdU+ AT2 cell numbers normalised to control cultures without IMCs (n = 4, mean + SD). Gapdh serves as a loading control for the RT-PCR on the right.Morphological alterations and intracellular signalling in AT2 cells cultured with IMC-CM. On the left and middle, primary AT2 cells from 3-week-old BVE mice were cultured for 48 h and then incubated in serum-free IMC-CM (AT2 with IMC-CM) or DMEM (AT2 w/o IMC-CM, serving as a control) for another 48 h, followed by immunostaining for E-cadherin (red) and vimentin (green) and confocal laser scanning microscopy (CLSM) imaging. The 3× zoomed image in the middle right highlights dividing vimentin+ cells that show internalised E-cadherin (arrows). Scale bars, 50 μm. On the right, primary AT2 cells from 10-week-old BVE mice were cultured for 48 h, serum starved for 5 h and treated with or without serum-free IMC-CM for 30 min. Phosphorylation of AKT, SMAD3, MEK and ERK were analysed by immunoblotting.
Figure 4CCR1 immunofluorescence of primary IMCs (left, co-stained with Mac2) and AT2 cells (right, co-stained with E-cadherin) detected by CLSM. Scale bars, 10 μm. CCR1 staining was mainly detected in cytoplasmic vesicles within IMCs, but some CCR1 fluorescence was also detected at the plasma membrane along with Mac2 (inset in the left microphotograph).CCR1 immunohistochemistry of lung sections from BVE mice showing CCR1 expression in stroma IMCs. Scale bar, 50 μm.CT imaging of lungs of AdCre-infected Braf+/LSL−V600E mice treated with vehicle (top) or CCR1 inhibitor (bottom). H: heart; * indicates a tumour region accompanied by atelectasis.H&amp;amp;E staining of lung sections from vehicle (left) or CCR1 inhibitor (right) treated AdCre-infected Braf+/LSL−V600E mice. Scale bars, 250 μm (top) or 50 μm (bottom).Flow cytometry analysis of CD11c+ (top, green) and SPC+ (bottom, red) cells in the lung of AdCre-infected Braf+/LSL−V600Emice treated with vehicle (left) or CCR1 inhibitor (right).LungCD11c+ (left) and SPC+ (right) cell numbers (per left lobe) were quantitated in AdCre-infected Braf+/LSL−V600Emice treated with CCR1 inhibitor or vehicle (V) (n = 3, mean + SD).% LungCD4+ (left) and CD8+ (right) T lymphocytes were quantitated in AdCre-infected Braf+/LSL−V600E mice treated with CCR1 inhibitor or vehicle (V) (n = 3, mean + SD).