EMBO/soda-nel huggingface dataset:
Neonatal AAV gene therapy rescues hearing in a mouse model of SYNE4 deafness (2020)
Figure 6

Figure 6. AAV.Syne4 promotes long term survival of OHC in Syne4-/- mice

A Whole mount immunofluorescence at 12w of the organ of Corti of a Syne4-/- mouse injected with AAV.Syne4 mouse injected with AAV.Syne4.

B Whole mount immunofluorescence at 12w of the 12 kHz region of WT, Syne4-/- mouse, and Syne4-/- mouse injected with AAV.Syne4.

C IHC and OHC counts, n=3 for WT, n=4 for Mut, n=6 for Mut + AAV.Syne4.

D Correlation between HC count and ABR threshold at 12kHz.

E 3D surface projection of an OHC at 12w from the 12kHz region of WT and Syne4-/- mice injected with AAV.Syne4. Open arrows denote basal end, white arrows denote cuticular plate.

F Image analysis of nucleus position of OHC.

G Quantification of nuclear distance from the cuticular plate. A total of 30 OHC from 3 WT mice and 38 OHC from 4 Syne4-/- mice injected with AAV.Syne4 were measured.

Data information: Scale bars = 100 μm for (A), 10 μm for (B), and 5 μm for (E). Statistical tests were Pearson correlation with two-tailed P values for (D) and unpaired Student's t-test for (G). Plots show mean ± SD.
Neonatal AAV gene therapy rescues hearing in a mouse model of SYNE4 deafness (2020)
Figure 7

Figure 7. AAV.Syne4 rescues behavioral response to sound in Syne4-/- mice

A Schematic representation of cued-fear conditioning experimental procedure.

B, C Quantification of activity level and cumulative freezing duration at 12w during seconds 90-210 of day 2 in WT, Syne4-/- mice, Syne4-/- mice injected with AAV.Syne4, and Syne4-/- mice injected with AAV.GFP, n=14 for WT, n=16 for Syne4-/-, n=9 for Syne4-/- + AAV.Syne4, n=5 for Syne4-/- + AAV.GFP. (C) Activity level. (B) Cumulative freezing duration. Statistical test was Repeated-Measures ANOVA with Holm-Sidak correction for multiple comparisons, comparing the scores of each individual mouse before and after the appearance of the tone. Plots show mean ± SD. ***P<0.001, ****P<0.0001. ns= not significant.

Expanded View Figure legends
Structural basis of transport and inhibition of the Plasmodium falciparum transporter PfFNT (2020)
Figure 5

Figure 5. Relative rates for transport of lactic acid via PfFNT.

(A) Stopped-flow assay measuring relative rates of lactic acid transport into reconstituted vesicles containing wild-type or mutant transporter (green, control with empty vesicles; red, wild-type; blue, K35A; cyan, F90A; magenta, F94A; yellow, K177A; olive green, H230A; and gray, H230N).

(B) Relative rates of lactic acid transport based on reswelling rates of proteoliposomes. Each data point is an average of five different light scattering data sets. Error bars depict standard deviation for these five light scattering data. All of the transport rates from the mutant transporters are significantly different compare from that of wild-type PfFNT (K35A, P = 2.8 x 10-3; F90A, P = 4.4 x 10-7; F94A, P = 4.9 x 10-6; K177A, P = 1.2 x 10-3; H230A, P = 4.5 x 10-7; H230N, P = 2.0 x 10-6; student's t-test) (green, control with empty vesicles; red, wild-type; blue, K35A; cyan, F90A; magenta, F94A; yellow, K177A; olive green, H230A; and gray, H230N).
Clarin-2 is essential for hearing by maintaining stereocilia integrity and function (2019)
Figure 1

Figure 1. Clrn2 is essential for mammalian hearing

(A) Identification of the ENU-induced hearing loss pedigree MPC169, subsequently named clarinet. ABR phenotyping of pedigree Muta-Ped-C3PDE-169 at 3-months of age identified 8 mice with elevated hearing thresholds (red triangles) compared to their normal hearing colony mates (n=61, black triangles). Indeed, all eight affected mice were found to not respond to the highest intensity stimulus (90 dB SPL) at the three frequencies tested, or the click stimulus, and so their thresholds are shown as 95 dB SPL. (B-D) The genomic structure of mouse Clrn2 (ENSMUST00000053250), and domains of the encoded tetraspan-like glycoprotein (232 amino acids). Black filled boxes represent untranslated region of Clrn2. The positions of the transmembrane (TM) domains (blue) and the structure of Clrn2clarinet (C) and Clrn2del629 (D) alleles are indicated. The clarinet mutation, Clrn2clarinet, (c.12G>A) is predicted to lead to a premature stop codon at position 4 (p.Trp4*) (C), whereas the Clrn2del629 allele consists of a CRISPR/Cas9-mediated 629 nucleotide deletion encompassing exon 2, leading to splicing of exon 1 to exon 3, which if translated would produce a protein lacking 2 (TM2 and TM3) of the 4 transmembrane domains (D). (E) Averaged ABR thresholds for Clrn2clarinet/del629 compound heterozygotes at P21, showing significantly elevated thresholds compared to Clrn2+/+, Clrn2clarinet/+ and Clrn2del629/+ control colony mates. All five Clrn2clarinet/del629 mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least one frequency/click stimulus. Data shown are mean ± s.d. ***p<0.001, one-way ANOVA (Please see Appendix supplemental table 1 for exact P-values). (F) Regional plot of p values for SNP association with hearing difficulty around the CLRN2 gene locus. The genes within the region are annotated, and the direction of the transcripts is shown by arrows. Colouring is based on linkage disequilibrium (LD) across the region with the most associated SNP, rs35414371, shown in purple.
Clarin-2 is essential for hearing by maintaining stereocilia integrity and function (2019)
Figure 2

Figure 2. Clarin-2 is required for hearing function

(A) RT-PCR analysis in P30 mice showing the presence of Clrn2 transcripts in the inner ear, eye, but not in brain or muscle. β-actin was used as a positive control. (B) Clrn2 transcripts could be detected in both inner (IHCs) and outer (OHCs) hair cells of P15 wild-type mice. Otoferlin (Otof) and oncomodulin (Ocm) transcripts were used as positive controls for IHCs and OHCs, respectively. Ocm transcripts were only present in the OHC lysate, demonstrating that the IHC sample had not been contaminated with OHCs. (C-F) Auditory phenotyping of clarinet mice at P16 (C), P21 (D), P28 (E) and P42 (F). ABR threshold measurements show that Clrn2clarinet/clarinet mice (red) exhibit a severe-to-profound hearing loss affecting all frequencies tested. At 16 kHz in Clrn2clarinet/clarinet mice, the mean ABR hearing thresholds vary from 55-65 dB SPL at P16, 60-90 dB SPL at P21, and to 80-100 dB SPL at P28 and P42. Age-matched Clrn2+/+ (black) and Clrn2clarinet/+ (grey) controls display thresholds within the expected range (15-45 dB SPL) at all frequencies and time-points tested. At P16, all eight Clrn2clarinet/clarinet mice exhibited recordable ABR responses for each frequency tested and the click stimulus. For the longitudinal ABR study, at P21 and P28 three of the seven Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least one frequency-specific/click stimulus. By P42, five of the Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least two frequency-specific/click stimuli. ABR data shown are mean ± s.d. ***p<0.001, one-way ANOVA. (G) Averaged DPOAE responses for clarinet mice at P28, showing significantly reduced responses in Clrn2clarinet/clarinet mutants at all frequencies tested. DPOAE data shown are mean ± s.d. *p<0.02, **p<0.01, one-way ANOVA. Please see Appendix supplemental table 1 for exact P-values.
Clarin-2 is essential for hearing by maintaining stereocilia integrity and function (2019)
Figure 3

Figure 3. Clarin-2 is dispensable for balance and vision

(A) Vestibular behavioural tests (swim tests, contact-righting, trunk-curl, and platform). The Clrn2clarinet/clarinet mice (red, P28, n=5, and P60, n=7) have no vestibular dysfunction, displaying similar performances to age-matched control Clrn2clarinet/+ mice (grey, P28, n=3, and P60, n=7). Being similar, the values at P28 and P60 were combined. (B-F) Electroretinogram (ERG) measurements from control Clrn2clarinet/+ (grey) and mutant Clrn2clarinet/clarinet (red) mice. Each trace in B and C is representative of an ERG response from the eye of age-matched Clrn2clarinet/+ and Clrn2clarinet/clarinet mice, showing no significant difference in a- or b-wave amplitudes. (D-F) The lack of change in ERG amplitude responses in Clrn2clarinet/clarinet mice (aged 3 or 6-7 months), regardless of the test conditions: scotopic (D,E), or photopic (F), indicate normal photoreceptor kinetics and no change in the sensitivity of photoreceptor cells.
Clarin-2 is essential for hearing by maintaining stereocilia integrity and function (2019)
Figure 4

Figure 4. Normal architecture of cochlear hair bundles in clarinet mice

(A,B) Confocal microscopy images of whole-mount preparations of mid-basal cochlear sensory epithelia from Clrn2clarinet/+ (A) and Clrn2clarinet/clarinet (B) P6 mice immunostained for actin. Despite lack of clarin-2, the developing sensory epithelium of mutants is similar to that of heterozygous controls. (C-E) Confocal microscopy images of whole-mount preparations of cochlear sensory epithelia from Clrn2clarinet/+ (C), Clrn1-/- (D), and Clrn2clarinet/clarinet (E) P6 mice immunostained for stereocilin (green) and actin (red). Unlike the fragmented immunostaining in Clrn1-/- mice (mirroring the fragmentation of the hair bundle), stereocilin immunostaining in Clrn2clarinet/clarinet mice reflects the normally V-shaped bundles of OHCs, similar to Clrn2clarinet/+ OHCs. (F,G) Representative scanning electron micrographs of the sensory epithelium of Clrn2clarinet/+ (F) and Clrn2clainet/clarinet (G) P8 mice, showing no apparent differences in the gross patterning of IHCs and OHCs. Arrowheads in a,b (white),f and g (black) illustrate the convex shape of the apical circumference of OHCs. Scale - 2 µm (A-E), 10 µm (F, G).
Clarin-2 is essential for hearing by maintaining stereocilia integrity and function (2019)
Figure 5

Figure 5. Sensory hair cell bundle patterning and measurements in clarinet mice

(A,B) Pseudo-coloured scanning electron micrographs of individual outer and inner hair cell bundles from clarinet mice at P8 and P16, showing that gross morphology of OHC and IHC bundles is similar between Clrn2+/+ and Clrn2clarinet/clarinet mice. Representative images from the mid region of the cochlear spiral are shown and close-up views illustrate the three full rows, tallest (red), middle (blue) and short (yellow), of stereocilia in IHC and OHC hair bundles. At P8 and P16 (upper panels in A and B), the three stereocilia rows are observed in Clrn2clarinet/clarinet OHCs (A) and IHCs (B), although the shortest and middle row of IHC stereocilia appear less prolate compared to controls. (C,D) Distribution of individual stereocilia measures across genotypes at P8 and P16. (C) The OHC stereocilia measurements at both P8 and P16 (C) show a significant difference in height of Clrn2+/+ stereocilia compared to Clrn2clarinet/clarinet in: e.g. for the shortest row at P8: Clrn2+/+: 0.6188 ± 0.0944 p.µm (n=27); Clrn2clarinet/clarinet: 0.2948 ± 0.1029 p.µm (n=27) (p<0.0001, Mann-Whitney ranks comparison), and at P16: Clrn2+/+: 0.579 ± 0.06179 p.µm (n=27); Clrn2clarinet/clarinet: 0.07282 ± 0.1626 p.µm (n=27) (p<0.0001, Mann-Whitney ranks comparison). Note the amount of value points equal to zero at P16. (D) Conversely, IHC stereocilia measurements at P8 and P16 did not show a difference between Clrn2+t/+ and Clrn2clarinet/clarinet in: for the shortest row at P8: Clrn2+/+: 0.8808 ± 0.1753 p.µm (n=27); Clrn2clarinet/clarinet: 0.9313 ± 0.1217 p.µm (n=27) (p=0.2244, unpaired t-test); and at P16: Clrn2+/+: 0.6784 ± 0.1171 p.µm (n=23); Clrn2clarinet/clarinet: 0.6063 ± 0.1855 p.µm (n=26) (p=0.2436, Mann-Whitney ranks comparison). (E) Pseudo-coloured scanning electron micrographs of individual OHC (left panels) and IHC (right panels) hair bundles from clarinet mice at P28. Clrn2clarinet/clarinet mutants have only two rows of OHC stereocilia, and the middle row of stereocilia is less uniform in height compared to controls. The middle and short rows of stereocilia in Clrn2clarinet/clarinet IHC bundles appear fewer in number, and heterogeneous in height. Scale - 2 µm.
Clarin-2 is essential for hearing by maintaining stereocilia integrity and function (2019)
Figure 6

Figure 6. Expression of clarin-2 in the hair bundle of auditory hair cells

(A) Schematic representation of the auditory sensory organ, illustrating the positioning of the electrode used for injectoporation of GFP-tagged clarin-2 construct into cochlear supporting and hair cells. (B-F) Top and side views of representative images of supporting cells (asterisk) and hair cells expressing clarin-2. In supporting cells, GFP-tagged clarin-2 (green) was distributed diffusely throughout the cytoplasm (A,B). By contrast, in all injectoporated hair cells, the majority of clarin-2 was observed in the apical stereocilia (C-E). Scale - 2 µm.
Clarin-2 is essential for hearing by maintaining stereocilia integrity and function (2019)
Figure 7

Figure 7. The distribution of hair bundle and synaptic proteins in clarinet mice

(A,B) Confocal images of IHC and OHC hair bundles of Clrn2clarinet/clarinet mice and heterozygous littermates at P6 immunostained for PDZD7 (A, green) and whirlin (B, green) and actin (red in both figures). The PDZD7 immunostaining is normally restricted to the base of stereocilia in both Clrn2clarinet/+ and Clrn2clarinet/clarinet P6 mice (A). Whirlin immunostaining is properly located at the stereocilia tips of IHC and OHC hair bundles (B). C-F Harmonin-b immunostaining in IHC hair bundles. In Clrn2clarinet/clarinet mice (D), the harmonin-b immunoreactive puncta (green) were still observable on the stereocilia, but unlike in age-matched heterozygote littermates (C), were located much closer to the tip of stereocilia. The two diagrams in (C) and (D) illustrate the position of harmonin-b immunostaining (green) corresponding to the site of the upper attachment point of the tip link (UAPTL), facing the tip link. The bright green signal outside stereociliary bundles are non-specific. The change of harmonin-b localization along the stereocilium in Clrn2clarinet/clarinet mice is illustrated further by line scan (E) and quantification (F) analyses. The insets in (E) show images of individual stereocilia used for the line scan signal analysis. The harmonin-b immunoreactive puncta were located within 850 ± 28 nm distance from the tip of the tallest stereocilia in Clrn2clarine/+ mice (n=31 hair bundles from 5 mice) (F), and within 575 ± 23 nm in Clrn2clarinet/clarinet mice (n=35 hair bundles from 5 mice). Data shown are mean ± s.e.m. ****p<0.0001, Student t-test. G Cochlear whole-mounts from P21 clarinet mice, labelled with the IHC pre-synaptic ribbon marker Ribeye (red) and the post-synaptic density marker GluR2 (green), showing a similar number of total and matched Ribeye-positive and GluR2-postive puncta in Clrn2clarinet/clarinet mutant cochleae compared to Clrn2clarinet/+ littermates, which is reflected in puncta counted per five hair cells (H) n = 4 per genotype. Data shown are mean ± s.d.., one-way ANOVA. Scale - 2µm.
Clarin-2 is essential for hearing by maintaining stereocilia integrity and function (2019)
Figure 8

Figure 8. Clrn2 is required for the acquisition of normal mechano-electrical transducer (MET) function in immature cochlear hair cells

(A,B) Saturating MET currents recorded from P6 Clrn2clarinet/+ (A) and Clrn2clarinet/clarinet (B) apical-coil OHCs by applying sinusoidal force stimuli of 50 Hz to the hair bundles at −121 mV and +99 mV. The driver voltage (DV) signal of ± 40 V to the fluid jet is shown above the traces (positive deflections of the DV are excitatory). The holding potential was −81 mV. Extracellular Ca2+ concentration was 1.3 mM. Arrows and arrowheads indicate the closure of the MET currents (i.e. resting MET current) elicited during inhibitory bundle displacements at hyperpolarized and depolarized membrane potentials, respectively. Dashed lines indicate the holding current, which is the current at the holding membrane potential. (C) Average peak-to-peak current-voltage curves recorded from Clrn2clarinet/+ (grey, P6, n=7) and Clrn2clarinet/clarinet (red, P6-7, n=9) apical coil OHCs. (D,E) Resting open probability (Po) of the MET current at the membrane potential of −121 mV and + 99 mV from apical- (D) and basal-coil (E) OHCs. Number of OHCs investigated is shown above the columns. (F,G) Saturating MET currents recorded from a P7 Clrn2clarinet/+ (F) and a P8 Clrn2clarinet/clarinet (G) apical-coil IHC using the same experimental protocol described above. (H) Average Po of the MET current measured in apical coil IHCs at the membrane potential of −121 mV and + 99 mV from Clrn2clarinet/+ (P7, n=3) and Clrn2clarinet/clarinet (P7-8, n=6) apical-coil IHCs.
Clarin-2 is essential for hearing by maintaining stereocilia integrity and function (2019)
Figure 9

Figure 9. IHCs, but not OHCs, fail to acquire adult-like basolateral currents in clarinet mice

(A,B) Potassium currents recorded from mature Clrn2clarinet/+ (A, P15) and Clrn2clarinet/clarinet (B, P16) apical-coil OHCs. Currents were elicited by depolarizing voltage steps (10 mV nominal increments) from -144 mV to more depolarized values from the holding potential of -84 mV. Note that the current characteristic of mature OHCs, The size of IK,n, measured in isolation as the deactivating tail currents (difference between instantaneous and steady-state inward currents) for voltage steps from the holding potential to −124 mV, was 545 ± 115 pA, (n=5) in Clrn2clarinet/+ and 595 ± 67 pA, (n=7) in Clrn2clarinet/clarinet OHCs. (C) Average peak current-voltage relation for the total K+ current recorded from Clrn2clarinet/+ (P15-16, n=7) and Clrn2clarinet/clarinet (P15-16, n=5) OHCs. (D,E) Potassium currents recorded from P22 mature Clrn2clarinet/+ and Clrn2clarinet/clarinet apical-coil IHCs, respectively, using the same voltage protocol described above. Holding potential of -64 mV. (F) Peak current-voltage relation for the total K+ current recorded from the IHCs shown in panel (A) and (B). (G-I) Voltage responses from P22 Clrn2clarinet/+ (G) and Clrn2clarinet/clarinet (H,I) IHCs. Note that Ca2+-dependent action potentials could be induced in mature IHCs (I).

Expanded View Figure Legends
Phytohormone cytokinin guides microtubule dynamics during cell progression from proliferative to differentiated stage (2020)
Figure 2

Fig. 2 | Cytokinin modulates CMT patterns and dynamics.

A. Arabidopsis root expressing the dual reporter TCSn::dndT-tNOS DR5v2:3nGFP that is sensitive to cytokinin (CK) and auxin. CK (red), auxin (green) and overlay of both signals detected in nuclei of cells at the root tip (top, scale bar 50 µm). Green, pink, and blue arrowheads point at epidermal cells located in the meristematic (MZ), transition (TZ) and elongation (EZ) zones, respectively. Magnification of the TZ shown (bottom, scale bar 10 µm).

B. Relative fluorescence intensity of TCSn::ntdT:tNOS signal (red) and DR5v2:3nGFP signal (green) measured in epidermal cells along the longitudinal root growth axis. Cell number 1 corresponds to a meristematic cell placed at position -4 before the beginning of TZ (as marked by green arrow at 2A). Mean ± s.d., * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by Student's t-test, n= 18 roots.

C-F. CMTs visualized with MAP4-GFP in root epidermal cells of the TZ and the EZ at time point 0 and 60 min after CK (C), CK and oryzalin (D), auxin (E) and CK and auxin (F) treatments, and in DZ cells at time point 0 and 60 min after auxin treatment (E). As CK and auxin sources 10 µM BAP and 0.1 µM NAA were used, respectively, and 1 µM oryzalin. For the double (CK/Oryzalin and CK/Auxin) treatments, roots were pretreated with CK for 60 min prior to transfer to medium supplemented with both compounds. Scale bar 10 µm.

G. Histograms of CMT orientation distributions in epidermal cells of the TZ and the EZ at time point 0 and 60 min after hormonal treatments as described above (C, E and F). n = 15-20 cells per root growth zone with 5-8 roots per condition in 4 independent replicates.

H. Analysis of the CMT plus-end growth with the EB1b-GFP reporter. Z-stack maximum image projections of EB1b-GFP tracked over 30 sec in epidermal cells without (mock, DMSO) and with CK, auxin or CK and auxin treatment (60 min). Scale bars 10 µm.

I. CMT plus-end growth rates (µm/min) measured by tracking of EB1b-GFP reporter over 20-30 min in epidermal cells of different growth zones treated for 60 min with mock (DMSO), or media supplemented with CK (30 or 60 min), auxin and CK plus auxin. In the boxplots, the center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software; whiskers span minimum to maximum values, individual data points are represented by dots. The number of CMT plus-end events (EB1b-GFP) tracked per cell was 20-100/min, of which the growth rate average is represented as a single dot. Statistical significance evaluated by two-way ANOVA (n = 3-5 cells per root growth zone with 3-5 roots per replicate in 3 independent experiments. On the right, single trajectories of EB1b-GFP signal tracked over 60 sec in root epidermal cells at different growth zones and treatment conditions. Scale bar 0.5 µm. Hormone concentrations and treatment conditions (H, I) were as described (C, E and F).
Phytohormone cytokinin guides microtubule dynamics during cell progression from proliferative to differentiated stage (2020)
Figure 4

Fig. 4 | The ARR1 cytokinin signalling component is involved in fine-tuning the CMTs network.

A. Relative root growth of wild-type (Col-0) (grey lines) and 35S::ARR1∆DDK-GR (purple lines) seedlings grown on mock (DMSO; continuous line) or with dexamethasone (DEX 10 µM; dashed line) supplemented medium. Five days old seedlings (Day 0) were monitored for 3 days. Mean ± s.d.; ** P < 0.01, **** P < 0.0001 by Student's t-test, referred to wild-type - mock. n = 12-14 roots.

B. Immunostaining of α-tubulin in epidermal cells of the transition (TZ) and the elongation (EZ) zone of 35S::ARR1∆DDK-GR roots after non-, 3h or 16h of treatment with dexamethasone (DEX 10 µM). Scale bar 10 µm.

C. Histograms present the CMT orientation distributions (%) in epidermal cells of the TZ and the EZ treated as indicated in (B). Mean ± s.d., (* P < 0.05, ** P < 0.01, and **** P < 0.0001 by Student's t-test, referred to same root zone without DEX induction). n = 16-24 cells per growth zone in 8-9 roots per condition were analyzed.

D, E. Analysis of root sensitivity to oryzalin in wild-type (Col-0) and 35S::ARR1∆DDK-GR. Seedlings were grown for 5 days on mock (Murashige and Skoog) medium and then transferred to medium supplemented with 1 µM oryzalin, CK with oryzalin (10 µM BAP and 1 µM oryzalin), with or without DEX 10 µM, for 3 days. For the double CK and oryzalin treatment, seedlings were pretreated with 10 µM BAP for 60 min prior to transfer to medium supplemented with both compounds. Representative images; white arrowheads indicate root length at day of transfer. Scale bar 1 mm (D). Quantifications calculated as percentage (%) of root tips exhibiting swelling (dark grey bars), and weak and strong resistance to oryzalin (grey and white bars, respectively). On the right representative images of root phenotype categories. n=10-25 roots per treatment were evaluated. (E).

F. Relative root growth of wild-type (Col-0) (grey lines) and 35S::ARR1∆DDK-GR (purple lines) seedlings grown on oryzalin 1µM (continuous line) or on oryzalin 1µM and DEX 10 µM (dashed line) supplemented media monitored for 3 days. Day 0, day of transfer. Mean ± s.d.; n = 12-14 roots.