EMBO/soda-nel huggingface dataset:
Stable kinetochore-microtubule attachments restrict MTOC position and spindle elongation in oocytes (2021)
Figure 1

Fig 1: Establishment of 4D quantitative analysis for MTOC dynamics

(A) Live imaging of MTOC dynamics during meiosis I. Z-projection images (top) show MTOCs (mNG-Cep192, green) and chromosomes (H2B-mCherry, magenta). Central MTOCs are magnified (squares). 3D-reconstructed spindles are aligned with the spindle axis (middle). Central MTOCs are indicated with arrowheads. Time in h:mm. The kymograph (bottom) shows projected signals on the spindle axis for all time points.

(B) Density maps of MTOCs (left) and chromosomes (right) along the spindle axis in a representative oocyte. The color represents the percentage of MTOCs or chromosome volumes, respectively, coded from dark (0%) to white (50% or more of the total volume).

(C) Average density maps of 21 oocytes mapped as in (B). Data from 6 independent experiments are used.

(D) Dynamics of MTOC sorting and chromosome congression. Ratio of MTOCs (left) or chromosomes (right) in the polar region versus those in the middle region of the spindle are plotted over time.

(E) Dynamics of spindle elongation. The spindle was visualized with EGFP-Map4 (Fig EV1). The length/width ratio of the spindle was measured after 3D reconstruction (n=13 oocytes from 3 independent experiments).

(F) Percentage of oocytes carrying one or more MTOCs in the middle region of the spindle. Oocytes were fixed and stained for MTOCs (pericentrin, green), chromosomes (Hoechst33342, magenta) and microtubules (α-tubulin, grey). n=21, 28, 24 oocytes. A representative oocyte fixed at 5 hours after NEBD is shown. Arrowheads indicate central MTOCs.

Data information: Time after NEBD. Mean ± SD are shown. Scale bars, 10 μm. See also Movie EV1.
Stable kinetochore-microtubule attachments restrict MTOC position and spindle elongation in oocytes (2021)
Figure 2

Fig 2: Central MTOCs originate from spindle poles

(A) Live imaging of MTOC and kinetochore dynamics. MTOCs (mNG-Cep192, green) and kinetochores (tdTomato-CENP-C, magenta) in B6D2F1 oocytes are shown. Time in h:mm after NEBD. Circles indicate central MTOCs, which are positioned in the middle region of the spindle later than 4 hours after NEBD. Arrows indicate displacement of the MTOCs. Scale bar, 10 μm.

(B) Tracking analysis of central MTOCs. In the left graph, the positions of central MTOCs in (A) along the spindle axis are shown over time. MTOC positions were used to calculate distance to the spindle equator. In the right graph, lines showing temporal changes in MTOC-equator distance are aligned based on the time when the MTOC reached a position closest to the spindle equator. Horizontal lines at 7 and at −7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle.

(C) Central MTOCs originate from spindle poles and transiently stay in the middle region of the spindle. As in (B), the trajectories of central MTOCs (n=25 MTOCs of 8 oocytes from 3 independent experiments) were analyzed and their temporal changes in MTOC-equator distance are shown (Left graph). Central MTOCs were categorized into two groups: (1) ones that came from polar regions of the spindle (n=21, middle graph) and (2) others that stayed in the middle region throughout the period before reaching a position closest to the equator (n=4, right graph).

(D) Origin of central MTOCs. Origins were categorized based on the results shown in (C). Note that 7 central MTOCs came from outside of the spindle and are not included in (C).

(E) Destination of central MTOCs. The tracks of central MTOCs that originated from polar regions were used (n=21 MTOCs). The pole of origin was defined as Pole 1, while the other pole was defined as Pole 2. MTOC positions along the spindle axis (positive values for those closer to Pole 1) over time are shown. Horizontal lines at 7 and at −7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle. Note that central MTOCs moved back to the original pole (Pole 1) or switched their positions to the opposite pole (Pole 2).

(F) Destination of central MTOCs that originated from a polar region was categorized as in (E).

Data information: See also Movie EV2.
Stable kinetochore-microtubule attachments restrict MTOC position and spindle elongation in oocytes (2021)
Figure 3

Fig 3: Kinetochore-microtubule attachment stability is required to confine MTOCs at spindle poles

(A) Live imaging of Zp3-Cre Ndc80f/f oocytes injected with Ndc80-WT, Ndc80-9D or Ndc80-9A. Z-projection and 3D-reconstructed images show MTOCs (mNeonGreen-Cep192, green) and chromosomes (H2B-mCherry, magenta). Time in h:mm after NEBD. Scale bar, 10 μm.

(B) Ndc80-9D is defective for MTOC sorting. The density map of MTOCs along the spindle axis in Zp3-Cre Ndc80f/f oocytes expressing Ndc80-WT, Ndc80-9D or Ndc80-9A. The color represents the percentage of MTOC volume coded from dark (0%) to white (50% or more of the total volume).

(C) MTOCs do not accumulate at the spindle poles in Ndc80-9D-expressing oocytes. Ratio of MTOCs (green) or chromosomes (magenta) in the polar region versus those in the middle region of the spindle at 6 hours after NEBD (n=19, 19 and 22 oocytes, respectively, from at least 3 independent experiments). Boxes show 25-75 percentiles and whiskers encompass data points within 1.5 times the interquartile range.

(D) Ndc80-9A decreases central MTOCs. The number of central MTOCs (MTOCs that were positioned in the middle region of the spindle as shown in (C)), was determined at each time point (n=19, 21 oocytes, respectively, from at least 3 independent experiments). Two-tailed Student's t-test was performed.

Data information: Boxes and whiskers show 25-75 and 10-90 percentiles, respectively.

See also Movie EV6 and EV7.
Stable kinetochore-microtubule attachments restrict MTOC position and spindle elongation in oocytes (2021)
Figure 4

Fig 4: Kinetochore-microtubule attachment stability is not required for initiating but is required for terminating spindle elongation

(A) Live imaging of Zp3-Cre Ndc80f/f oocytes injected with Ndc80-WT, -9D or -9A. Z-projection and 3D-reconstructed images show microtubules (EGFP-Map4, green) and chromosomes (H2B-mCherry, magenta). Scale bar, 10 μm.

(B) Ndc80-9D is defective for limiting spindle elongation. The aspect ratio (length/width) of 3D-reconstructed spindles was measured over time (n=18, 18 and 18 oocytes, respectively, from 3 independent experiments).

(C) Ndc80-9D causes excessive spindle elongation. Spindle lengths measured after 3D reconstruction are shown (n=18, 18 and 18 oocytes, respectively, from 3 independent experiments). Two-tailed Student's t-test was performed. **p<0.01, ****p<0.0001.

Data information: Means ± SD are shown. See also Movie EV8.
Stable kinetochore-microtubule attachments restrict MTOC position and spindle elongation in oocytes (2021)
Figure 5

Fig 5: Model showing how kinetochore-microtubule attachments contribute to acentrosomal spindle bipolarization

(A) K-fibers are connected to MTOCs and extend to spindle poles. Oocytes 6 hours after NEBD were fixed following cold treatment. Microtubules (α-tubulin, green), MTOCs (pericentrin, magenta), kinetochores (ACA, red) and DNA (Hoechst333342) are shown. Five oocytes from 2 independent experiments were analyzed. Scale bar, 10 μm.

(B) A majority of MTOCs are attached to K-fibers. The volumes of individual MTOCs were measured after 3D reconstruction. The sum volume of MTOCs attached to K-fibers relative to the total volume of MTOCs was calculated for each oocyte (n=5 oocytes from 2 independent experiments). Means and SD are shown.

(C) K-fibers extend to spindle poles. The length of K-fibers and distance between kinetochores and a spindle pole (KT-to-Pole distance) were measured. K-fiber length relative to the KT-to-Pole distance was calculated (n=90 K-fibers of 5 oocytes from 2 independent experiments).

(D) Model summarizing the results of this study. In early phases of acentrosomal spindle assembly, spindle bipolarizers, including Kif11, HURP, NuMA, HSET, and the kinetochore Ndc80 complex that recruits Prc1, establish spindle bipolarity by promoting MTOC sorting and spindle elongation. Meanwhile, the stability of kinetochore-microtubule attachments gradually increases, depending on the dephosphorylation of kinetochore components including Ndc80. As spindle bipolarity is established, stable kinetochore-microtubule attachments spatially confine MTOCs at spindle poles and limit spindle elongation.
Base excision repair AP endonucleases and mismatch repair act together to induce checkpoint-mediated autophagy (2013)
f1

(a) Cartoons showing schemes of the (i) BER and (ii) MMR pathways. F1 survival (b) in N2 wild type (black diamonds) and ung-1 mutants (red squares), (c) following the depletion of EXO-3 (red triangles), APN-1 (blue circles) or control RNAi (L4440, black diamonds) in the N2 wild-type background, and (d) in msh-2(ok2410) (orange triangles), msh-6(pk2504) (green circles) and mlh-1(ok1917) (grey squares) mutants. (e) Western blottings showing expression of MSH-6 protein in the wild type strain grown on E. coli expressing control (N2) or the indicated RNAi, as well as in msh-6(pk2504) and msh-2(ok2410) mutants (top panel), and expression of MSH-2::GFP fusion protein in transgenic worms grown on E. coli expressing RNAi for MSH-2, MSH-6 or empty vector control as indicated (lower panel). Actin was used as the loading control. (f) F1 survival after depletion of MSH-2 and MSH-6 in the mlh-1(ok1917) mutant. (g,h) F1 survival after depletion of the AP endonucleases EXO-3 and APN-1 in (g) the msh-6(pk2504) or (h) msh-2(ok2410) mutant background. Kruskal–Wallis one-way analysis on ranks (P=0.963 and P=0.985 in h and g, respectively). Mann–Whitney rank-sum test failed to identify a difference in median survival after depleting EXO-3 in the msh-6 mutant (g, P=0.929). (i) F1 survival following depletion of the indicated genes in exo-3(tm4374) mutants. (j) F1 survival in msh-6(pk2504), exo-3(tm4374) and exo-3;msh-6 double mutants. (k) F1 survival in exo-3(tm4374), mlh-1(ok1917) and exo-3;mlh-1 double mutants. All survival curves (b–d,f–k) show the mean±s.d. for each data point from three independent experiments.
Base excision repair AP endonucleases and mismatch repair act together to induce checkpoint-mediated autophagy (2013)
f2

(a) Immunofluorescence images showing anti-RPA-1-positive foci in response to 5-FU after depleting the indicated genes by RNAi or empty vector control RNAi (L4440; scale bars, 5 μm). (b) Quantification of the fraction (%) of 5-FU-treated embryos that contain RPA-1-positive foci. (c) Quantification of the fraction (%) of 5-FU-treated embryos that contain RPA-1-positive foci in N2, mlh-1(ok1917) and exo-1(tm1842) mutants fed control (L4440) or RNAi targeting APN-1 (apn-1). (d) Immunofluorescence showing CHK-1 phosphorylation at Ser139 in response to 5-FU in N2 but not in msh-6(pk2504) mutants or in N2 after apn-1(RNAi) (scale bars, 5 μm). (e) Quantification of the fraction (%) of 5-FU-treated embryos with phospho-CHK-1 foci. (f) Western blottings showing H1X.101 levels in N2 and msh-6 mutants, treated (+) or not (−) with 5-FU. Actin was used as loading control. (g) Quantification of H1X.101 levels relative to actin in control versus 5-FU-treated worms. (b,c,e,g) Bar graphs show the mean±s.d. from three independent experiments.
Base excision repair AP endonucleases and mismatch repair act together to induce checkpoint-mediated autophagy (2013)
f3

(a) Autophagy induction in response to 5-FU measured in a GFP::LGG-1 reporter strain fed control RNAi (L4440) or RNAi targeting the indicated genes (scale bars, 10 μm). (b) Immunofluorescence showing anti-VPS-34 staining in dissected embryos in the absence or presence of 5-FU (scale bar, 5 μm). (c) Western blot analysis of embryonic extracts with and without the addition of 5-FU or the autophagy inhibitor 3-MA, detecting the ~40-kDa GFP::LGG-1 fusion protein and the cleaved ~25-kDa product. (d) F1 survival measured after depletion of BEC-1 (red triangles) and ATG-7 (blue squares) as compared with worms fed control RNAi (black diamonds). The survival curve shows the mean±s.d. for each data point from three independent experiments. (e) Induction of autophagy (% GFP-positive embryos) following depletion of the indicated genes. (f) Immunofluorescence showing anti-VPS-34 staining in N2 and atl-1(tm853) embryos in the absence or presence of 5-FU (scale bar, 5 μm). (g) The fraction (as % of untreated control) of embryos with VPS-34-positive foci following 5-FU treatment. (h) Induction of autophagy (% GFP-positive embryos) in control (GFP::LGG-1) and atm-1(gk186) mutants (atm-1; GFP::LGG-1). (e,g,h) Bar graphs represent mean±s.d. from three independent experiments.
Base excision repair AP endonucleases and mismatch repair act together to induce checkpoint-mediated autophagy (2013)
f4

(a) Induction of autophagy in response to 5-FU in the GFP::LGG-1 reporter strain fed on RNAi targeting the indicated DNA repair genes (scale bars, 10 μm). (b) The fraction (as % of untreated control) of hermaphrodites having embryos with excessive GFP expression following 5-FU treatment. Autophagy induction in independent experiments (circles) and the mean (line) are presented. (c) F1 survival after control (black diamonds) or bec-1 RNAi (red squares) in N2 and msh-6(pk2504) mutants. The survival curve shows the mean±s.d. for each data point from three independent experiments.
Base excision repair AP endonucleases and mismatch repair act together to induce checkpoint-mediated autophagy (2013)
f5

(a) Expression of ATF-2::GFP in 5-FU-treated hermaphrodites fed control or RNAi targeting the indicated genes (scale bars, 20 μm). (b) The number of animals harbouring embryos with excessive ATF-2::GFP expression was scored in at least 150 animals and given as the fraction (%) of the total. (c) N2 or msh-6(pk2504) embryos were continuously monitored under differential interference contrast microscopy (scale bars, 5 μm) from the three-cell stage until completion of the four-cell stage. Magnified images of representative nuclei are shown. (d) The fraction of embryos with visible, enlarged nucleoli (arrows) after 5-FU treatment was scored. Bar graphs represent mean±s.d. from three independent experiments.
Base excision repair AP endonucleases and mismatch repair act together to induce checkpoint-mediated autophagy (2013)
f6

(a) Western blots showing autophagy induction by monitoring LC3 levels in whole-cell extracts from U2OS cells treated or not with 5-FU alone or in combination with Mx for the indicated time. Staurosporin was used as a positive control for apoptosis induction shown by the appearance of cleaved fragments of caspase-3 (17 and 19 kDa) and poly (ADP-ribose) polymerase 1 (PARP1; ΔPARP, 85 kDa). α−Actin was used as loading control. (b) Quantification of LC3-II levels in cells treated as in a. Intensities of the lower LC3 band (LC3-II) was normalized to those of actin. (c) Autophagy induction demonstrated by accumulation of LC3-positive puncta in U2OS cells treated or not with 5-FU alone or in combination with Mx for 48 h. The cells were fixed and prepared for immunofluorescence of LC3 (green). DAPI staining is shown in blue. Scale bar, 20 μm. (d) Quantification of LC3-positive puncta in cells treated as in c. In each separate experiment, 200–1,600 cells were scored per condition. (e) Western blots showing absence of LC3-II accumulation in U2OS cells transfected with siRNA against MSH-2. The same blot probed with anti-MSH-2 antibodies confirm efficient knockdown. (f) Quantification of LC3-II levels in cells transfected with control or MSH-2 siRNA with or without FU treatment for 24 h. (g) Immunofluorescence showing reduced accumulation of LC3 puncta in U2OS cells transfected with siRNA against MSH-2. (h) Quantification of LC3-positive puncta in cells treated as in g. (i) Quantification of normalized RPS3 protein level (black bars) or RPS3 mRNA level (white bars) in U2OS cells after 48 h 5-FU treatment. RPS3 mRNA levels were determined by quantitative real-time reverse transcriptase–PCR. Graphs (b,d,f,h,i) show mean values±s.e.m. quantified from at least three independent experiments. Student’s t-tests were performed to assess the significance between treatment groups in all panels as indicated; *P0.05; **P0.01.
Metabolic Energy Sensing by Mammalian CLC Anion/Proton Exchangers (2020)
Figure 1

Figure 1. Cytosolic adenine nucleotides regulate the function of ClC-3, ClC-4, and ClC-5. (A) Homology model depicting the ClC-5 dimeric structure with bound ATP molecules represented as spheres. Nucleotide-coordinating residues in the CBS domains are represented as sticks. Dashed lines indicate the hydrocarbon boundaries of the lipid bilayer. (B) Representative leak-subtracted ClC-3, ClC-4 and ClC-5 whole-cell currents measured with (upper row) or without (lower row) 5 mM ATP added to the pipette solution. Currents were elicited every 10 s by voltage steps from 0 mV to +140 mV, followed by a step to -100 mV to maximize the off-gating currents visible as sharp peaks at the end of the pulses. The recording started shortly (10s - 30s) after obtaining a whole-cell configuration. The first and last recorded currents in a series are depicted in green and red, respectively. NMDG-based solutions were used to characterize ClC-4. (C) Effects of various adenine nucleotides or their washout on the ionic transport of ClC-3, ClC-4 and ClC-5. The current amplitudes were measured at the end of the test pulses from experiments as shown in (B) and normalized to the initial current amplitudes obtained after establishing the whole-cell configuration. Some of the experimental data points have been omitted for clarity. Monoexponential fits to the data are depicted as red lines. Asterisks indicate statistical significances evaluated by two-sample t-test analysis of the residual currents determined at the end of each 10-minute measurement (*, p < 0.05; ***, p < 0.001, n.s., not significant; n= 5-11). (D), Left panel: residual currents determined at the end of 10-minute measurements as shown in (C). (D), Right panel: time constants (τ) of the current rundown obtained by fitting monoexponential functions to individual measurements as depicted in (C), (n= 5-11). Asterisks indicate statistical significances evaluated using two-sample t-test analysis (*, p < 0.05; ***, p < 0.001, n.s., not significant).

Data information: Error bars in all figures represent SEM.
Metabolic Energy Sensing by Mammalian CLC Anion/Proton Exchangers (2020)
Figure 2

Figure 2. Adenine nucleotides regulate the CLC voltage dependence and relative gating charge amplitudes. (A) Schematic representation of the gating charge analysis. The gating charge Q was obtained by calculating the surface beneath the off-gating currents (enlarged in gray and denoted by "charge"). Relative gating charge amplitudes in (B) and (C) were obtained by dividing the gating charge Q by the ion current amplitude I ("Ampl.") at +165 mV. (B) Voltage dependence of the WT ClC-5 off-gating charge normalized to the ionic current at +165 mV in the absence or presence of adenine nucleotides (n=7-12). Error bars represent SEM. Red lines depict Boltzmann fits to the data; fit parameters are provided in Appendix Table S1. (C) Voltage dependence of the WT ClC-3 off-gating charge normalized to the ionic current at +165 mV in the absence or presence of adenine nucleotides (n=4-5, Error bars represent SEM.). Red lines represent Boltzmann fits to the data; fit parameters are provided in Appendix Table S2.
Metabolic Energy Sensing by Mammalian CLC Anion/Proton Exchangers (2020)
Figure 3

Figure 3. Mutating the Gluext E211 does not abolish ATP effects. (A) Representative whole-cell recordings of cells expressing E211G ClC-5 upon voltage pulses to +140 mV from a -60-mV holding potential applied in 10-s intervals starting shortly after obtaining the whole-cell configuration with (left) or without (right) 5 mM ATP in the pipette solution. Leak subtraction was not applied. Asymmetric [Cl-] was used (110 mM internal NaCl substituted by TrisSO4) to achieve a stable negative reversal potential of <-50 mV allowing to recognize recordings with high unspecific leak current contamination. (B) Normalized time course of the steady-state E211G ClC-5 current amplitudes measured with (n=5) or without (n=4) internal ATP. Error bars show SEM. Red line represents a monoexponential fit to the data with a time constant of 108±8 s (SEM); currents measured with ATP-free pipette solution declined to 57.9±2.6% (SEM) of the initial amplitude.
Metabolic Energy Sensing by Mammalian CLC Anion/Proton Exchangers (2020)
Figure 4

Figure 4. Nucleotide chemistry and Mg2+ are important for the regulation of CLC transport. (A) Effects of different adenine nucleotides or their washout on the ClC-5 current amplitudes (n=4-7). The data were normalized to the initial current amplitude determined shortly after establishing whole-cell configuration. Some of the data points are omitted for clarity. For comparison, the ADP data set from Figure 1 is also depicted. Red lines indicate monoexponential fits to the averaged data. (B) Averaged residual currents determined after 600-s cell dialysis as shown in (A), and normalized to the current at the start of the measurements (n=4-7). Asterisks indicate statistically significant differences from a two-sample t-test analysis with p < 0.001; n.d. - not defined. (C) Averaged rundown time constants obtained from fits with monoexponential decay functions as shown in (A).

Data information: Error bars in all figures represent SEM.

Acknowledgments

We thank Birgit Begemann, Petra Killian, and Toni Becher for technical assistance.

Funding Information

This work was supported by the DFG Research Unit FOR 2612, FA 301/13-1

Author contributions

MG, KL, RG, ChF, and AKA contributed to the design of the work. MG, KL, RG, and AKA, and AKA contributed to acquisition and analysis of the data. AKA drafted the manuscript. MG, KL, RG, ChF, and AKA revised the paper critically for important intellectual content.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Dietary magnesium supplementation improves lifespan in a mouse model of progeria (2020)
Figure 4

Fig. 4. Oral magnesium treatment improves the longevity of LmnaG609G/+ mice.

(A) Magnesium intake and (B) Plasma magnesium concentration in 34-week-old untreated and treated LmnaG609G/+ mice (n=16).

(C) Calcium content of aortas obtained from 34-week-old wild-type mice and untreated and treated LmnaG609G/+ mice (n=20).

(D) Body masses of 34-week-old untreated and treated LmnaG609G/+ mice.

(E) Kaplan-Meier graph for untreated and treated LmnaG609G/+ mice (n=16).

(F) Representative photographs of 40-wk-old wild-type, untreated and treated LmnaG609G/+ mice.

Data information: Results are presented as the mean ± S.D. Statistical analyses were performed using Student's t-test (A, B, D), logrank test (E) or one-way ANOVA and Tukey's multiple comparison post-hoc test (C). ** p<0.05, ** p<0.01, *** p<0.001.
Dietary magnesium supplementation improves lifespan in a mouse model of progeria (2020)
Figure 5

Fig. 5. Magnesium improves mitochondrial ATP synthesis in LmnaG609G/+ mice.

(A) ATP concentration in liver homogenates obtained from 34-week-old wild-type, untreated or treated LmnaG609G/+ mice.

(B) Mitochondrial calcium measured in liver mitochondria isolated from the indicated experimental mouse groups.

(C) Magnesium concentration in isolated mitochondria.

(D) Activities of the indicated mitochondrial complexes (I, II, III, IV, and V) in the absence or presence of rotenone (2 µmol/L), 2-thenoyltrifluoroacetone (TTFA; 1 mmol/L), antimycin A (10 µmol/L), potassium cyanide (KCN; 1 mmol/L), or oligomycin (10 µmol/L).

(E) The boxed scheme shows the five mitochondrial complexes involved in the electron transport chain and their known inhibitors.Data information: Results are presented as mean ± S.D. (n=16). One-way ANOVA and Tukey's multiple comparisons post-hoc test were used for statistical analysis. *p<0.05; **p<0.01; ***p<0.001.