EMBO/soda-nel huggingface dataset:
Stabilization of the metaphase spindle by Cdc14 is required for recombinational DNA repair (2016)
Figure 9

Figure 9. Constitutive Cdk-dependent Spc110 phosphorylation affects metaphase spindle stabilization, DSB-SPB tethering and DNA repair by homologous recombination.

A) DNA damage sensitivity to MMS and phleomycin of a Cdk phospho-mimic mutant of Spc110. Ten-fold serial dilutions from mid-log phase cultures of wild-type, spc110S36-91A and spc110S36-91D cells grown on solid rich media containing mock DMSO, MMS or Phleomycin at 30ºC.

B) Schematic representation of the genomic background and the approximation used to measure DSB-SPB tethering. Wild-type Spc110 and spc110S36-91D were labelled with the RFP. Ddc2 was tagged with the GFP to be used as DSB reporter. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. At least 100 cells from three independent experiments were scored using the maximum projection of nine z-planes. Graph shows the mean ± S.D. P values were calculated using a 2-tailed unpaired Student t test.

C) Time-lapse experiments of living cells after induction of the HO endonuclease in wild-type and spc110S36-91D cells. Three z-planes every 10-second intervals over a period of 5 minutes were captured to measure the average SPBs track length and velocity. Spc110 and spc110S36-91D were labelled with the RFP and used to measured SPBs kinetics. At least 100 cells from three independent experiments were scored.

D) Southern blot of wild-type and spc110S36-91D mutant bearing the inter-chromosomal gene conversion assay depicted. Mid-log phase cells were HO-induced at 32ºC by adding galactose to the media. Samples were collected at different time-points and the genomic DNA was extracted. Preps were digested with EcoRI and analysed by Southern blot. Blots were hybridized with MATa-only and actin probes. FACS profiles for DNA content are displayed. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.

Data information: DMSO, dimethyl sulfoxide. MMS, methyl-methanesulfonate. SPB, Spindle Pole Body. DSB, double-strand break. Raf, raffinose. Gal, galactose. GC, gene conversion. CO, crossover.
A unique binding mode of Nek2A to the APC/C allows its ubiquitination during prometaphase. (2020)
Figure 1

Figure 1. Contribution of Nek2A degrons in binding and ubiquitination by the APC/C and Nek2A MR tail binding site on Apc8.

Schematic representation of the domain and degron architecture of Nek2A.

The MR tail is the main contributor for Nek2A ubiquitination. Ubiquitination reactions performed with APC/CCdc20 for Nek2A wild type (Nek2AWT) and degron mutants. Nek2AK is the KEN box mutant (391KEN393/KAA), Nek2AK/D is the (KEN and D box mutant (391KEN393/KAA, 361RKFL364/AKFA), Nek2AMR is the 443∆MR mutant.

The APC/C requires the Cdc20 coactivator subunit to ubiquitinate Nek2A. Nek2A ubiquitination reactions performed with the APC/C in absence and in presence of increasing concentrations of Cdc20 using the E2 UbcH10.

The MR tail is essential for Nek2A binding to the APC/C whereas in contrast Cdc20 is dispensable. Size exclusion chromatography peak fractions of APC/C complexes with Nek2A. The input material (13% of total) is indicated (i). Peak fractions are numbered in respect of the chromatograms in Fig EV1.

Nek2A binding does not stabilize binding of the coactivator subunit Cdc20 to the APC/C and does not compete with cyclin A for binding to the APC/C. Size exclusion chromatography peak fractions of APC/C complexes with Nek2A.

Top: cylinder representation of the APC/C-Nek2A complex structure. The TPR lobe subunits are highlighted: Apc8 (cyan), Apc6 (pink), Apc3 (yellow) and Apc7 (green). The platform subunits and the small subunits from the TPR lobe are shown in transparency. Bottom: cryo-EM density of the Nek2AMR tail dipeptide and details of the Nek2AMR tail (orange) binding site on Apc8A.

Details of the IR tail of Cdc20MCC (orange) bound to Apc8A in APC/CMCC-closed. This is the same site as the Nek2AMR tail-binding site shown above. Shown is the cryo-EM density for the Cdc20MCC IR tail and contacting residues of Apc8A.
A unique binding mode of Nek2A to the APC/C allows its ubiquitination during prometaphase. (2020)
Figure 2

Figure 2. Nek2A MR tail binding site on Apc2 and 4.

Left: cylinder representation of the APC/C-Nek2A complex structure. Apc2 (light blue) and Apc4 (light brown) are highlighted, the rest of the APC/C is shown in transparency. (i) Two different views of cryo-EM density for the Apc2 and Apc4 subunits. Secondary structure elements of the Apc2WHB domain are highlighted. (ii) Cryo-EM density for the second MR tail of Nek2A.

Details of the second MR tail binding site formed by the Apc2 (including the repositioned Apc2WHB) and Apc4 subunits. Nek2AMR2 is shown in orange.

Nek2A ubiquitination reactions performed with either APC/CCdc20 wild type (APC/CWT) or mutants. APC/C2m is the N392A/E395A mutant of Apc2. APC/C2/4m is the N392A/E395A/R48A/H53A/S51A mutant of Apc2 and Apc4.

Securin ubiquitination reactions performed with either APC/CCdc20 wild type (APC/CWT) or mutants.
A unique binding mode of Nek2A to the APC/C allows its ubiquitination during prometaphase. (2020)
Figure 3

Figure 3. Nek2A binds the APC/CMCC complex when the APC/CMCC closed conformation is destabilized.

Ubiquitination reactions of either Nek2A or securin by the APC/CCdc20 and the effect of increasing concentrations of the MCC.

B-F. Size exclusion chromatography peak fractions of APC/C complexes with either Nek2A (B-E) or securin (F). APC/CΔApc15 is the Apc15 deleted mutant APC/C, MCCΔIR is the Cdc20 IR tail deleted mutant MCC. MCC-BubR1Wm is the MCC mutant at the APC2WHB binding surface on BubR1 [14]. Chromatograms are shown in Fig EV1.
A unique binding mode of Nek2A to the APC/C allows its ubiquitination during prometaphase. (2020)
Figure 4

Figure 4. Substrate-dependent E2 preferences in APC/C ubiquitination.

Bottom: overview of the APC/C-Nek2A structure is shown coloured as in Fig 2. Top: close up of the Apc2 and Apc4 binding site for Nek2A in the APC/C-Nek2A structure (this study, left), APC/CUbcH10-Hsl1 structure [27] and APC/CMCC structure [14].

Ubiquitination reactions of cyclin A by the APC/CCdc20 in presence of increasing concentrations of either UbcH10 or UbcH5.

Ubiquitination reactions of Nek2A by the APC/CCdc20 in presence of increasing concentrations of either UbcH10 or UbcH5.

Western blot of Hek293 cells showing the siRNA-mediated depletion of UbcH5, UbcH10, or both E2 enzymes together. Tubulin serves a loading control.

Exemplary still images from time courses between NEBD and anaphase of eGFP-Nek2A degradation in HEK cells. Cells were either treated with siGL2 as control or depleted of the indicated E2 enzymes. The chromosomes are coloured in cyan and eGFP-Nek2A in green, with the outline of the cells are indicated with dashed yellow lines. Time is given as hh:mm. Scale bar 10 μm. See also Movie EV1-4 and Appendix Fig S1.

Degradation profiles of eGFP-cyclin A2 (top) and eGFP-Nek2A (bottom) in HEK cells during mitosis. The timepoint of NEBD is marked at 0 minutes in the graphs. Asterisks indicate values that are significantly different from the same time point of the siGL2 control as determined by a Mann-Whitney U-test (the statistics are listed in Table EV2). Mean ± s.d. is shown. The number of cells analysed are N = 66 (Cyclin A2 siGL2), 52 (Cyclin A2 siUbcH5), 28 (Cyclin A2 siUbcH10), 27 (Cyclin A2 siU5/siU10), 80 (Nek2A siGL2), 64 (Nek2A siUbcH5), 21 (Nek2A siUbcH10), 32 (Nek2A siU5/siU10). All data are from at least two biological replicates. See also Movie EV5-8.
Rab35 controls cilium length, function and membrane composition (2019)
Figure 1

Figure 1. Rab35 localises to the ciliary membrane

A Localisation of transiently expressed GFP-Rab35 in IMCD3 cells after 48h serum starvation and staining for acetylated tubulin (acetyl. tub.) and DNA. Insets show higher magnification images of the cilia region. Scale bars, 10 µm.

B Localisation of transiently expressed GFP-RAB35 in hTERT-RPE1 cells after 24 h serum starvation and staining for polyglutamylated tubulin (polyglu. tub.) and DNA. Insets show higher magnification images of the cilia region. Scale bars, 10 µm.

C Super resolution (FV-OSR) imaging of hTERT-RPE1 stably expressing GFP-RAB35 after 24 h serum starvation and staining for GFP, ARL13B (cilia membrane), IFT88 (axoneme), acetylated tubulin (acetyl. tub.; axoneme), and γ-tubulin (γ-tub; centrosome). Top panels show representative images and bottom panel shows line profile plots of fluorescence intensity (arbitrary units; a. u.) in the distal (dotted line) and proximal (dashed line) cilia regions. Data are mean ± S.E.M. (n = 5 cilia), with the solid line in the line profile plots indicating mean and the dotted lines indicating S.E.M. values. Scale bars, 1 µm.

D Representative FV-OSR images of 24 h serum-starved hTERT-RPE1 stably expressing GFP-RAB35 and stained for GFP, ARL13B and acetylated tubulin. Right panels show a cilium with proximal enrichment of GFP-RAB35 and left panels a cilium with even GFP-RAB35 localisation along the full length. Scale bars; 1 µm.

E Representative FV-OSR images of 24 h serum-starved hTERT-RPE1 stably expressing GFP-RAB35 and stained for GFP, EHD1 and ARL13B. Line profile plots of fluorescence intensity in the proximal cilia region (dashed line) are shown to the right. Data are mean ± S.E.M. (n = 6 cilia), with the solid line in the line profile plots indicating mean and the dotted lines indicating S.E.M. values. Scale bars; 1 µm.
Rab35 controls cilium length, function and membrane composition (2019)
Figure 3

Figure 3. Rab35 ciliary localisation and function depends on its nucleotide-bound state

A Representative images of hTERT-RPE1 cells transiently expressing wild type (WT), GDP-bound (S22N) or GTP-bound (Q67L) GFP-tagged RAB35. Cells were serum-starved for 24 h and stained for polyglutamylated tubulin (polyglu. tub.) and DNA. Higher magnification images of the cilia region shown in smaller panels. Scale bars; 10 µm.

B-D Quantification of GFP-RAB35 ciliary localisation (B), ciliation (C), and ciliary length in hTERT-RPE1 cells transiently expressing indicated GFP-RAB35 constructs or GFP. Data in (B, C) are mean ± S.E.M. of 3 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test ( ** P < 0.01; P-values: (B) P = 0.0015, (C) P = 0.0062). Cilia length quantification in (D) is shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n ≥ 50 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (* P < 0.05, ** P < 0.01; P-values: GFP vs. GFP-RAB35-S22N P = 0.0015, GFP vs. GFP-RAB35-Q67L P = 0.0158)

E, F Analysis of cilia length in hTERT-RPE1 cells depleted of GEF (DENND1A, DENND1B) and GAP (TBC1D10A, TBC1D10B) regulators of RAB35. Cells transfected with indicated siRNAs were serum-starved for 48 h and stained for acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (E). Regions within white boxes shown at higher magnifications to the right. Scale bars; 10 µm. Cilia length quantification in (F) is shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n ≥ 50 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (** P < 0.01, *** P < 0.001; P-values: Neg vs. DENND1B P = 0.0033, Neg vs. TBC1D10A P = 0.0008)

G Quantification of ciliary localisation of GFP-RAB35 in hTERT-RPE1 cells stably expressing GFP-RAB35 treated as in (E). Data are mean ± S.E.M. of 5 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test (* P < 0.05, *** P < 0.001; P-values: Neg vs. DENND1B P < 0.0001, Neg vs. TBC1D10A P = 0.0322).

H-J Localisation of transiently expressed GFP-DENND1B (H) or GFP-TBC1D10A (I) in hTERT-RPE1 cells after 24 h serum starvation and staining with indicated antibodies. Higher magnification images of the cilia region are shown in smaller panels. Scale bars; 5 µm. Quantification of localisation to ciliary base or along axoneme in (J). Data are mean ± S.E.M. of 3 independent experiments (n ≥ 30 cilia per experimental condition).
Rab35 controls cilium length, function and membrane composition (2019)
Figure 4

Figure 4. Zebrafish Rab35 regulates KV cilia length and left-right organ patterning

A Immunostaining of representative images of KV embryos at 8 somite stage in which mRNA encoding for mCherry or mCherry-Rab35 WT was injected. Staining of acetylated tubulin was used as cilia marker. Inset shows a higher magnification image of the cilia region, displaying colocalization of mCherry-Rab35 and acetylated tubulin. Scale bar, 10 µm.

B,C Immunoblot analysis with anti-Rab35 and anti-GAPDH antibodies of 40 µg of total lysates of zebrafish embryos injected with Rab35 morpholino (MO) at 8 somite stage, compared with non-injected embryos. The plot in (C) shows Rab35 protein level in non-injected and Rab35 morphants, normalized to GAPDH protein level. Data are mean ± S.E.M. of 3 independent experiments (n > 50 embryos per condition).

D Organ situs was determined by observing the heart position at 30 hours post-fertilisation (hpf), and the liver position at 53 hpf, by in situ hybridisation with a probe for foxa3, in controls (Mismatch MO and non-injected [WT]) and Rab35 MO. Situs solitus means left heart and liver; situs inversus means right heart and liver; and heterotaxia means any other possible combination of the heart and liver position.

E Whole-mount in situ hybridization with the dand5 probe at 8 somite stage (13 hpf) in wild type non-injected embryos and mismatch morphants, both presenting right-sided distribution of dand5, and Rab35 morphants presenting bilateral localization of the dand5 probe. Scale bar, 50 µm. Graph shows quantification of embryos (%) with bilateral or right-sided dand5 localisation from both conditions. Statistical significance according to Fisher Exact test (** P = 0.0002)

F-I Immunostaining of representative images of KV embryos at 8 somite stage in which: (F) Mismatch or Rab35 MO was injected, as well as a co-injection of Rab35 MO with mRNA encoding mCherry-Rab35 WT [Rescue]; (H) mRNA encoding mCherry or mCherry-tagged Rab35 WT, Rab35-Q67L or Rab35-S22N were injected. Acetylated tubulin is shown in green as cilia marker. Quantification of KV cilia length in (F) and (H) is shown respectively in (G) and (I) as box-and-whisker plots of 3 independent experiments. N ≥ 8 embryos and n > 300 cilia per experimental condition. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (** P < 0.01, *** P < 0.001, ns. not significant; Mismatch vs Rab35 MO, P < 0.001; Cherry vs Rab35-SN, P = 0.0063; ). Scale bars, 10 µm.
Rab35 controls cilium length, function and membrane composition (2019)
Figure 6

Figure 6. Rab35 interacts with Arl13b

A HEK293T cells were transiently co-transfected with ARL13B-FLAG and GFP or GFP-RAB35. Cells were cultured either in full growth medium (+serum) or serum-starved for the final 8 h before harvesting. Immunoprecipitations (IP) were performed using anti-GFP beads. Cell lysates (2.5% of input) and immunoprecipitates were loaded on the same gel, subjected to immunoblot (I) analysis with indicated antibodies and detected with the same exposure.

B Schematic representation of the human ARL13B structure depicting the guanine nucleotide-binding (G), coiled coil (CC) and proline-rich (PR) domains as well as the point mutations affecting N-terminal palmitoylation or C-terminal ciliary targeting RVEP motif.

C HEK293T cells were transiently co-transfected with mCherry-RAB35 together with GFP or indicated ARL13B-GFP wild type, mutant or truncation constructs. Cells were serum-starved for the final 8 h before harvesting. IPs were performed using anti-GFP beads and interacting proteins detected by immunoblotting (IB).

D HEK293T cells were transiently transfected with ARL13B-FLAG and serum-starved for the final 8 h before harvesting. HEK293T cell lysates expressing ARL13B-FLAG were subjected to pull-down with GFP-RAB35 bound to anti-GFP beads and preloaded with either no nucleotide, GTPγS or GDP. Bound proteins and cell lysates (1% of input) were analysed by immunoblotting. The graph shows the ratio of precipitated ARL13B and RAB35, normalized to the no nucleotide control. Data are mean ± S.E.M. of 3 independent experiments. Statistical significance according ANOVA followed by Tukey post-hoc test (* P =0.0333).

E HEK293T cells were transiently transfected with mCherry-RAB35 and serum-starved for the final 8 h before harvesting. HEK293T cell lysates expressing ARL13B-FLAG were subjected to pull-down with ARL13B-GFP bound to anti-GFP beads and preloaded with either no nucleotide, GTPγS or GDP. Bound proteins and cell lysates (1% of input) were analysed by immunoblotting. The graph shows the ratio of precipitated ARL13B and RAB35, normalized to the no nucleotide control. Data are mean ± S.E.M. of 3 independent experiments. Statistical significance according ANOVA followed by Tukey post-hoc test (* P =0.0466)
Rab35 controls cilium length, function and membrane composition (2019)
Figure 7

Figure 7. Rab35 depletion disrupts the ciliary levels of INPP5E and PIP(4,5)P2

A, B hTERT-RPE1 cells transfected with non-targeting siRNA control (Neg) or RAB35 siRNA, were serum-starved for 48 h and stained for INPP5E, ARL13B, acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (A). Regions within white boxes shown at higher magnifications to the right. Scale bars, 10 µm. (B) Box-and-whisker plots show quantification of ciliary INPP5E intensity in arbitrary units (a. u.). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 50 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (*** P < 0.001; P-values: Neg vs. RAB35-1 P < 0.0001, Neg vs. RAB35-2 P = 0.0002).

C For co-depletion experiments, hTERT-RPE1 cells were first transfected with Neg or RAB35 siRNA. After 24h, a second transfection with Neg or ARL13B siRNA was performed and cells were serum starved for 48 h and stained as in (A). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 40 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (** P = 0.0017).

D, E NIH3T3 wild type (WT) and Rab35 knockout (KO) cell lines were serum-starved for 24h and stained for Innpp5e, acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (D). Higher magnifications of regions within white boxes shown at the bottom. Scale bars; 10 µm. (E) Box-and-whisker plots show quantification of ciliary Inpp5e intensity in arbitrary units (a. u.). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 25 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (* P < 0.05, *** P < 0.001; NIH3T3 WT vs. Rab35 KO#1 P < 0.0001, NIH3T3 WT vs. Rab35 KO#2 P = 0.0345).

F, G Localisation of the PI(4,5)P2 sensor PH-PLCδ1-GFP to cilia in live NIH3T3 WT and Rab35 KO cells. Cilia are marked with HTR6-RFP. Representative images are shown in (F). Higher magnification images of the cilia region shown to the right. Scale bars; 10 µm. Quantification of localisation in (G). Data are mean ± S.E.M. of 3 independent experiments (n > 100 cilia per experimental condition). Statistical significance according to two-way ANOVA (* P = 0.0242).
Astrogenesis in the murine dentate gyrus is a life-long and dynamic process (2022)
Figure 1

Figure 1: Astrogenesis in the postnatal dentate gyrus (DG).

A Scheme illustrates developmental fate-mapping experiment using NestinCreERT2; CAG CAT GFP (from now on referred to as NestinCreERT2; GFP) mice to monitor radial glia-like neural stem cells (rNSCs) and their generated progeny.

B Experimental scheme of tamoxifen injection paradigm: perinatal mice were injected twice (12 h apart) to label early perinatal rNSCs and killed at postnatal day (P) 3, 7, 10, or 14 to analyse the rNSC-generated progeny within the DG layers hilus, granular zone (GZ), and molecular layer (ML).

C Confocal images of rNSC-derived astrocytes expressing GFP (green) and astrocyte-specific markers (ACSBG1, GFAP, S100β; magenta) at different timepoints (P7, P10, P14); lower pictures depict single-channel images the astrocyte markers (grey) used above.

D Representative pictures of recombined astrocytes (GFP+/ACSBG1+; arrowheads) during DG development (P3, P7, P10, P14).

E, F Quantification of total numbers of recombined astrocytes (GFP+/ACSBG1+) per area at postnatal timepoints in the total DG (E) or in distinct DG compartments (F), i.e. hilus, GZ, and ML indicated by different colours.

G Representative images of proliferating astrocytes (ACSBG1+, cyan; arrowheads) in combination with cell cycle markers KI67 or MCM2 (magenta; P3, P7: KI67; P10, P14: MCM2).

H Confocal image of proliferating astrocytes at P10 (ACSBG1+, cyan; MCM2+, magenta) that did not express NESTIN (white).

I, J Quantification of total proliferating astrocytes (ACSBG1+/KI67+ for P3/P7; ACSBG1+/MCM2+ for P10/P14) per area at distinct timepoints per total DG (I) or hilus, GZ and ML (J).

K Confocal images of proliferating rNSC-derived astrocytes (GFP+/ACSBG1+/MCM2+; arrowheads) at P10.

Data information: All data are represented as mean ± SEM; number of experimental animals throughout figure: n = 5 (P3) and n = 6 (P7, P10, P14), (E,I) indicated by red dots; all values are indicated in Table EV1; dotted lines mark GZ borders; scale bars (C,H,K) = 10 µm, (D,G) = 50 µm.