EMBO/soda-nel huggingface dataset:
A KDM6A-KLF10 reinforcing feedback mechanism aggravates diabetic podocyte dysfunction (2019)
Figure 7

Figure 7. Elevated levels of KDM6A and KLF10 expression, along with decreased levels of nephrin (or WT-1) expression, are detected in kidney tissues and urinary exosomes of patients with diabetic nephropathy.

A Immunofluorescence analysis of kidney sections from diabetic nephropathy subjects and non-diabetic controls stained with KDM6A, KLF10, nephrin and WT-1. Scale bars, 50 μm. *P < 0.05 by Wilcoxon two-sample test (n = 6 for each group).

B Western blot analysis of kidney tissues from non-diabetic controls and diabetic nephropathy subjects. Expression levels of KDM6A, KLF10, nephrin and WT-1 in kidney tissues were determined by immunoblotting using the indicated antibodies. Relative protein levels in kidney tissues were normalized to actin. *P < 0.05 by Wilcoxon two-sample test (n = 6 for each group).

C Levels of KDM6A and KLF10, nephrin mRNAs in urine exosomes of diabetic nephropathy patients and non-diabetic controls. Relative mRNA levels in human urinary exosomes were normalized to 18S rRNA. Horizontal lines are medians. *P < 0.05, **P < 0.01, and ***P < 0.001 by Wilcoxon two-sample test (n = 12 for each group).

Data information: Data are expressed as mean ± SEM (A and B). See the exact P values for comparison tests in Appendix Table S7.

Expanded View Figure legends
Microtubule number and length determine cellular shape and function in Plasmodium (2019)
Figure 1

Figure 1 - α1-tubulin is essential for sporozoite formation

A. Cartoon of a simplified Plasmodium life cycle indicating in red the stages important for our work. Note that transfection is performed in blood stage parasites.

B. Schematic cartoon of a sporozoite with an indicated apical cross section. Subpellicular (cytoplasmic) microtubules (green) can be found below the three membrane layers (plasma membrane and inner membrane complex) of the pellicle (bottom).

C. Isolated sporozoites from the hemolymph (HL) and salivary glands (SG) of infected mosquitoes stained with Hoechst and SiR-tubulin. Note the similar staining of WT and α1-tubulin(-) complemented lines. Scale bars: 5 µm.

D. SiR-tubulin staining (green, I and II) and TEM (III) show that microtubules shrink after wild type sporozoite formation. During budding microtubules can be up to 15 µm long (I), while after formation they measure on average only 6 µm (graph). Inset in II shows sporozoite cross-section at the sporoblast membrane; note the SiR-tubulin fluorescence (green, arrowheads) next to the nuclear Hoechst stain (blue). (III) TEM longitudinal section of budding sporozoites with an indicated subpellicular microtubule (white line, length). Green arrowheads point to microtubules close to the sporoblast. Rh: rhoptry; N: nucleus; ER: endoplasmic reticulum; Mi: mitochondrion; Sb: sporoblast. **** indicate p<0.0001; Kruskal-Wallis-test. Scale bars: I&II: 5 µm, magnification box in II: 1 µm, III: 1 µm, magnification box in III: 0.1 µm.

E. Comparison of sporozoite development in oocysts of WT, α1-tubulin(-) and complemented lines. Hoechst (blue) and SiR-tubulin (green) staining reveal that the complemented line generates sporozoites indistinguishable from WT controls, while SiR tubulin staining is absent from α1-tubulin(-) oocysts. mCherry stains the cytoplasm of the parasite. Scale bars: 5 µm.

F. Transmission electron micrographs of developing sporozoites in WT and α1-tubulin(-) parasite lines. Note the slender sporozoites in the WT oocysts, which are absent in the mutant. Sb: sporoblast; scale bars: 5 µm. See also Fig EV4 and EV5.

G. TEM cross sections of WT and α1-tubulin(-) parasites showing the aberrant structure of the latter. Subpellicular and centriolar plaque (spindle pole body) associated (hemispindle) microtubules are readily recognizable in the WT sporozoites (green arrowheads in insert) but absent in the mutant, while the inner membrane complex is formed below the plasma membrane of both parasites (white arrowheads). Note the different scale chosen to highlight the malformed shapes in the mutant. N: nucleus; scale bars: 0.2 µm, magnification boxes: 0.1 µm,.
Microtubule number and length determine cellular shape and function in Plasmodium (2019)
Figure 2

Figure 2 - Sporozoite budding is impaired in α1-tubulin(-) parasites

A. Electron micrographs showing different stages of development in WT and α1-tubulin(-) parasites. Key structural elements are highlighted in the following colors: blue: plasma membrane; yellow: inner membrane complex; green: microtubules; light blue: micronemes; lilac: rhoptries; brown: rootlet fiber; cyan: nucleus. Note the undulating shapes of the elongated mutants and that micronemes are hard to distinguish from other small vesicles in the α1-tubulin(-) parasites. The rootlet fiber in panel II starts at the apical tip and in panel V is associated with the centriolar plaque (dark structure in nuclear envelope). Panel III corresponds to Fig 1D. Scale bars: 200 nm.

B. 3D reconstruction from serial sectioning reveals that growing α1-tubulin(-) 'sporozoites' lack apical polarity and fail to pull in nuclei. Microtubuli: green; rootlet fiber (connecting apical pole to centriolar plaque): red; rhoptries: purple; endomembraneous vesicles (Golgi, premicronemes): brown; nucleus: cyan; sporozoite plasma membrane and inner membrane complex: blue; dotted lines show location of sporoblast membrane. 17 and 10 sections of 100 nm and 300 nm thickness were processed for the tomograms in WT and α1-tubulin(-) parasites, respectively. Scale bar: 1 µm. See also Movie EV1.

C. Tomography of serial sections from budding α1-tubulin(-) parasites at day 13 (same as in panel B) and day 17 to highlight the increase of surface area (blue) with time. The oocyst wall at day 17 is highlighted by cyan lines (top), the sporoblast membrane is highlighted by red lines (bottom). The sporoblast membrane at day 13 is indicated by a dashed line (red). 10 and 11 sections of 300 nm thickness were processed for the tomograms at day 13 and day 17, respectively. A single continuous parasite was highlight at day 17. Scale bar: 1 µm.

D. α1-tubulin(-) parasites show multiple attachments (arrowheads) with the sporoblast membrane at day 17 post infection. Note the highly branched nature of the budding parasite (same parasite as in panel C) shown in two views in I and II. Right: slices through the tomogram in grey scale with the outlines used for the 3D model indicated. Two connections with the sporoblast are indicated by 1 and 2. Scale bars: 1 µm. See also Movie EV2.
Microtubule number and length determine cellular shape and function in Plasmodium (2019)
Figure 3

Figure 3 - Genome replication and nuclear multiplication is proceeding in the absence of microtubules

A-D Life imaging of wild-type (WT) and α1-tubulin(-) oocysts expressing ef1α:GFP (early and late oocysts) and csp:mCherry (late oocysts) to locate the oocyst cytoplasm. Microtubules were labeled with SiR-tubulin (green) and DNA with Hoechst (blue). Oocysts are shown in a chronological order from early (day 4) to late (day 12) development. (A, B) Early oocyst development with remaining subpellicular microtubules of the preceding ookinete stage (A) and subsequent DNA replication (B), where some but not all DNA is co-stained by microtubules in the mutant. (C) Nuclear alignments to the invaginated plasma membrane and budding of sporozoites. Note the strong SiR-tubulin signal only seen in sporozoites of WT oocysts and complete absence from the mutant. Sb: sporoblast. (D) Oocyst ready to burst with fully formed sporozoites in WT oocysts. Note that α1-tubulin(-) oocysts retained sporozoite nuclei predominantly within the sporoblast (Sb) during budding. Scale bars: 5 μm, magnification box in B: 1 µm,.

E. 3D reconstructions from spinning disc confocal microscopy z-stacks across an early oocyst labelled with the DNA dye Hoechst (left). 50 and 19 images with a z-distance of 0.5 µm and 1 µm were collated for WT and α1-tubulin(-) oocysts, respectively. Right: 3D reconstructions from late WT and α1-tubulin(-) oocysts labelled with Hoechst (blue) and SiR-tubulin (green). Note that the mutant does not form proper sporozoite shapes during budding and the few peripheral nuclei that were uptaken into budding sporozoites. 10 and 28 images with a z-distance of 1,5 µm and 1 µm were collated for WT and α1-tubulin(-) oocysts, respectively. Sb: sporoblast; scale bars: 5 µm.

F. TEM cross sections of WT and α1-tubulin(-) oocysts during nuclear division. Spindle microtubules (arrowheads) emerging from centriolar plaques (M) were seen in WT oocysts. Although individual nuclei (N) were visible in α1(-) oocysts, microtubules were absent even in dividing centriolar plaques (M). Scale bars: 0.2 μm.
Microtubule number and length determine cellular shape and function in Plasmodium (2019)
Figure 4

Figure 4 - Lowering expression of α1-tubulin leads to fewer microtubules and aberrant sporozoite shapes

A. Complementation of α1-tubulin(-) parasites with different α1-tubulin constructs lacking introns (α1∆introns) or featuring only codon-modified exons (α1cm&∆introns). Scanning electron micrographs show the aberrant shape of the α1cm&∆introns-expressing sporozoites. See also Fig EV5. Scale bar: 1 μm.

B. Fluorescence images showing the SiR-tubulin stained microtubules, nuclei (Hoechst) and cytoplasmic shape (mCherry) of the parasite lines. Note the small amount of DNA (black arrowhead) and the reduced level of SiR-tubulin fluorescence in the α1cm&∆introns-expressing sporozoites (lower graph). See also Fig S6. Quantitative data was derived from 96-120 sporozoites per line. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. Scale bar: 5 μm. **** indicates p<0.0001, Kruskal-Wallis-test. Note that α1cm&∆introns-expressing sporozoites do not express mCherry; their autofluorescence (auto-fluo) indicates an aberrant shape.

C. qRT-PCR analysis of the three parasite lines at day 7 and 12 after infection. Error bars correspond to the standard deviation from two biological replicates.

D. TEM reveals α1cm&∆introns-expressing sporozoites with smaller (A and B) and larger (C) diameters than wild type sporozoites. See also Fig S7. Scale bar: 0.2 µm.

E. α1cm&∆introns-expressing sporozoites show a reduced number of subpellicular microtubules (green arrowheads) by TEM. **** indicates p<0.0001, ns: not significant; Kruskal-Wallis-test. Scale bar: 0.2 µm.

F. Cartoon showing that sporozoites cannot form in the absence of microtubules, while lower numbers allowed formation of aberrantly shaped parasites. Note that subpellicular microtubules are exclusively polymerized from the apical polar ring.
Microtubule number and length determine cellular shape and function in Plasmodium (2019)
Figure 5

Figure 5 - α1-tubulin specific amino acids modulate microtubule length while modulating expression level tunes microtubule numbers

A. Expression of different α1-α2 chimeric α-tubulins instead of α1-tubulin leads to sporozoite shape changes as revealed by SEM. See also Fig EV5. Scale bar: 1 µm. α1∆c-term lacks the last 3 amino acids of α1-tubulin. α2+ parasites contain an α1-tubulin with all amino acids that are different between the two α-tubulins changed into those of α2-tubulin; see also Fig S4. α2++ parasites contain α2-tubulin (exons, introns, 3'UTR) and α2+++ parasites express the exons of α2-tubulin and introns of α1-tubulin from the α1-tubulin promoter.

B. SiR-tubulin staining reveals differences in microtubule lengths and intensity. Between 69 and 156 sporozoites were analysed per line. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. ***, and **** indicate p<0.001 and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.

C. qRT-PCR analysis of the parasite lines at day 12 after infection shows different expression level. Note the subtly higher expression of α2++ compared to α2+++. Error bars show standard deviation from the mean of two biological replicates.

D. Schematic cartoon of sporozoite indicating the apical, central and nuclear sections as analysed in panels (E-G).

E. TEM along sporozoites shows a decrease of microtubule numbers towards the nucleus in parasites expressing α-tubulin chimeras. Green arrowheads point to microtubules. See also Fig S8 and S9. Note that the WT control panel is shown already in Figure 4E to indicate that here we perform an extended analysis to the dataset generated earlier. Scale bars: 0.2 µm.

F. TEM reveals different numbers of microtubules at the apical end of sporozoites for the different lines. Each dot represents the numbers of microtubules counted per section. Red lines indicate median from 23 to 40 analysed sections per line. **** indicate p<0.0001; ns: not significant; Kruskal-Wallis-test.

G. Microtubule quantification along the sporozoite reveals the presence of fewer microtubules in central and nuclear sections of α2+ and α1∆c-term parasites compared to wild type sporozoites. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median and + the mean. *, **, and **** indicate p<0.05, p<0.01 and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.

H. Cartoon summarizing the effect of α2-tubulin expression on microtubule numbers and lengths. Numbers indicate the median number of observed microtubules per line.

I. Semilog correlation of gene expression as determined by qRT-PCR with microtubule numbers as determine by TEM. Bars show range of values in x and standard deviation in y around the mean (dots) of two biological replicates (x) and all determined values (y).
Microtubule number and length determine cellular shape and function in Plasmodium (2019)
Figure 6

Figure 6 - Mathematical model explains average number and length of microtubules at different tubulin expression levels

A. A cartoon depicting four possible states of a microtubule nucleation site considered in the mathematical model, namely unbound, bound, nucleating and microtubule growing, as well as the transition between them. The transition rates depend on the dimer concentration c. An unbound nucleation site transitions to a bound state when a tubulin dimer binds to it with rate kmcp. A bound nucleation site starts nucleation with rate kncn and adds tubulin dimer sequentially with rate kmc to grow the microtubule. Here p and n (where p+n=12 being the size of the critical nucleus, see Methods) represents the cooperativity of tubulin dimer binding to a free and a bound nucleation site, respectively.

B. The minimum value of MSE in MT number (shown by black points) increases with the increase in binding cooperativity (in curves from left to right).

C. The MSE in MT length at binding rates corresponding to MSE minimum in MT number (shown by open black circles) decreases with increase in binding cooperativity. The parameter set shown by star symbol is selected for comparing the modelling results with the experiments. The x-values are multiplied by a factor of (10 − 5 )p to present the plots in a common range for easy comparisons. The rate of microtubule growth is fixed to km=66 s-1 per dimer in all the simulations (see Appendix Text).

D. The microtubule number increases with the increase in the concentration of tubulin dimers. Microtubule number scales with tubulin concentration at low expression. At high expression, microtubule number is limited by the number of nucleation sites. Shown are mean +/- standard deviation from all determined values.

E. The average microtubule length remains close to the maximum length 6 μm. The circles are obtained by averaging results from 200 stochastic simulations of the model equations and stars are the experimental data (blue stars are for (α2+, α2++, α2+++) strains). The error bar shows the standard deviation in the data and simulation results.

F. For the WT expression level (c0 = 36.6 (m0+p+n)), the rate of nucleation is faster than the microtubule growth. Then the microtubule number is determined by the number of nucleation sites. The average microtubule length is around 1 μm (shown by dashed arrow) when the last nucleus forms.

G. At intermediate tubulin numbers (c0 = 16 (m0+p+n)), corresponding to the α1c-term mutant, the microtubule formation rate is comparable to nucleation rate. The average microtubule length is around 4 μm (shown by dashed arrow) when the last nucleus forms.
Microtubule number and length determine cellular shape and function in Plasmodium (2019)
Figure 7

Figure 7 - Microtubule length and numbers determine sporozoite curvature and infectivity

A. Quantitative analysis of salivary gland sporozoite lengths from the different lines. n indicates numbers of investigated sporozoites. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. *** and **** indicate p<0.001, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test; scale bar: 1 µm.

B. Quantification of SiR-tubulin fluorescence from 69-127 salivary gland derived sporozoites. Note the decrease in the fluorescence intensity of sporozoites expressing α1-tubulin without introns as well as the decreased intensity and length of the chimeras. Linear correlation of sporozoite length and microtubule length reveals a R2=0.94. *, **, and **** indicate p<0.05, p<0.01, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.

C. Shorter sporozoites exhibit stronger curvatures and hence tighter circular motility on glass. Scale bar: 20 µm. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. * and **** indicate p<0.05, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.

D. Sporozoites with fewest microtubules (α2+++) show a motility defect. n indicates numbers of investigated sporozoites; Numbers on grey bars indicate percentage of partially and persistently moving sporozoites.

E. Sporozoites from lines with fewer microtubules move more frequently in apparent clockwise (CW) direction. Numbers on black bars indicate percentage of CW moving sporozoites; numbers in movie stills indicate time in seconds. CW moving sporozoites move at lower speed than CCW moving ones.

F. Salivary gland sporozoites contain at least 10 microtubules as revealed by TEM of α2++ infected salivary glands. Green arrowheads point to microtubules. Scale bar: 0.2 µm. Error bars show the full range and boxes 50% of values. Red crosses indicate the mean, red lines the median. Fisher's exact test (two-tailed).

G. Infectivity of sporozoites to mice decreases with decreasing numbers of microtubules and shorter microtubules. The prepatency is prolonged in parasites with a reduced sporozoite length and gliding circle diameter. See also Table EV 2. NMRI mice were exposed to ten mosquito bites of the respective parasite line. Numbers indicate infected mice / mice exposed to mosquito bites (left) and infected mice / mice injected with 10.000 sporozoites (right).

H. Cartoon illustrating the various consequences of different microtubule numbers and lengths during Plasmodium sporozoite formation and tissue invasion. Sporozoites with more than 10 microtubules can enter salivary glands while salivary glands sporozoites with shorter microtubules are least infectious.

Expanded View Figure legends
Neonatal AAV gene therapy rescues hearing in a mouse model of SYNE4 deafness (2020)
Figure 2

Figure 2. Syne4-/- hair cells mature normally and then degenerate between P12 and P14

A Whole mount immunofluorescence of a P8 Syne4-/- organ of Corti showing intact hair cells, as labelled with myosin VIIa.

B Whole mount immunofluorescence of WT and Syne4-/- organ of Corti from the 8, 16, and 32 kHz regions at P8 and P14.

C Inner and outer hair cell counts of Syne4-/- organ of Corti at P8, P10, P12, and P14.

D FM1-43 uptake performed on P8+1 DIV (days-in-vitro) WT and Syne4-/- organ of Corti. Top shows OHC plane and bottom shows IHC plane.

Data information: Scale bars = 100 μm for (A) and 10 μm for (B and D).
Neonatal AAV gene therapy rescues hearing in a mouse model of SYNE4 deafness (2020)
Figure 3

Figure 3. AAV9-PHP.B transduction and Syne4 expression

A Schematic representation of AAV.Syne4 and AAV.GFP constructs.

B Whole mount immunofluorescence of a P9 organ of Corti of a mouse injected with AAV.GFP at P1 showing complete transduction of both inner and outer HC. Myosin VIIa was used to label the hair cells.

C Examples of 8, 16, and 32 kHz regions of an organ of Corti of a mouse injected with AAV.GFP. Top shows OHC plane, bottom shows IHC plane, right shows YZ orthogonal projection. Black asterisks show bright Deiters cells.

D Quantification of GFP intensity of inner and outer HC from 3 injected mice, normalized to the average intensity of HC in a control, un-injected mouse.

E Transduction rates of AAV9-PHP.B at 8, 16, and 32kHz regions based on GFP fluorescence. A total of 162 IHC and 841 OHC were analyzed from 3 injected mice and 1 control littermate.

F Staining for FLAG at at P14 of the organ of Corti of a mouse injected at P1 with AAV.Syne4.

G Quantification of FLAG and DAPI fluorescence intensity along a line centered at the nuclear envelope. Eight OHC were measured.

H 3D surface projection of two adjacent OHC from a mouse injected with AAV.Syne4. In the left cell myosin VIIa and DAPI signals were removed to only show FLAG staining.

Data information: Scale bars = 100 μm for (B), 10 μm for (C and F), 5 μm for (H). Plots show mean ± SD.
Neonatal AAV gene therapy rescues hearing in a mouse model of SYNE4 deafness (2020)
Figure 4

Figure 4. AAV.Syne4 rescues OHC morphology in Syne4-/- mice

A Whole-mount immunofluorescence of the 8 kHz region from P14 organ of Corti from WT, Syne4-/-, and Syne4-/- mice injected with AAV.Syne4.

B 3D surface projection of IHC and OHC at P14 from the 8kHz region of WT, Syne4-/- mice and Syne4-/- mice injected with AAV.Syne4. Open arrows denote basal end, white arrows denote cuticular plate.

C Image analysis of nucleus position of IHC and OHC. Images show an arc fitted through the apical surface, nucleus, and basal end of the cell in 3D. X, Y, and Z axes show position in μm.

D Quantification of nuclear distance from the cuticular plate. A total of 45 IHC and 135 OHC from 3 WT mice, 45 IHC and 81 OHC from 3 Syne4-/- mice, 30 IHC and 90 OHC from 2 Syne4-/- mice injected with AAV.Syne4 were measured. Statistical test was 2-way ANOVA with Holm-Sidak correction for multiple comparisons. Plots show mean ± SD. ns=not significant, ****P<0.0001.

Data information: Scale bars = 10 μm.
Neonatal AAV gene therapy rescues hearing in a mouse model of SYNE4 deafness (2020)
Figure 5

Figure 5. AAV.Syne4 rescues auditory function in Syne4-/- mice

A Representative example of ABR traces obtained at 4w from a WT, Syne4-/- mouse, and Syne4-/- mouse injected with AAV.Syne4 in response to an 18 kHz stimulus.

B ABR thresholds at 4w of WT, Syne4-/-, Syne4-/- mice injected with AAV.Syne4, and Syne4-/- mice injected with AAV.GFP, n=7 for WT, n=8 for Syne4-/-, n=20 for Syne4-/- + AAV.Syne4, n=6 for Syne4-/- + AAV.GFP.

C Quantification of P1-N1 amplitude delta from (B), n=4 for WT, n=4 for Syne4-/-, n=7 for Syne4-/- + AAV.Syne4.

D Quantification of P1 latency from (B), n=4 for WT, n=2 for Syne4-/-, n=7 for Syne4-/- + AAV.Syne4.

E DPOAE thresholds at 4w, n=6 for WT, n=5 for Syne4-/-, n=6 for Syne4-/- + AAV.Syne4.

F ABR threshold at 12w, n=6 for WT, n=8 for Syne4-/-, n=16 for Syne4-/- + AAV.Syne4, n=5 for Syne4-/- + AAV.GFP.

Data information: Statistical test was 2-way ANOVA with Holm-Sidak correction for multiple comparisons. Plots show mean ± SD. ns=not significant, *P<0.05, **P<0.01 ****P<0.0001.