EMBO/soda-nel huggingface dataset:
SCFFbxw5 targets kinesin-13 proteins to facilitate ciliogenesis (2021)
Figure 2

Figure 2: Fbxw5 interacts with kinesin-13 family members

A. Co-IP of HA-MCAK with Flag-Fbxw5. Indicated proteins were expressed in Hek293T cells, extracted and subjected to anti-Flag IP followed by western blot analysis.

B. In vitro pull-down experiment. Indicated proteins (purified from Sf21 cells, 5-fold molar excess of MCAK over Fbxw5/Skp1) were mixed with competing E.coli lysate and precipitated via Ni-NTA agarose. Proteins were washed, eluted in SDS sample buffer and analysed by SDS-PAGE.

C. NanoBRET™ (Nano-bioluminescence resonance energy transfer) assay. Mdm2, p53 and Fbxw7 serve as controls. Mdm2 or MCAK were tagged with NanoLuc®-luciferase (Nluc), p53, Fbxw7 and Fbxw5 with HaloTag® (HT) and expressed in HeLa cells. After incubation with ligand overnight, substrate was added and plates were directly measured. Left: Quantification of 4 independent experiments. Error bars indicate standard deviation, asterisks the p-value of a two-tailed unpaired Student's t-test comparing each control with the MCAK/Fbxw5 pair (***P < 0.001). Right: Cartoon showing the NanoBRET™ principal.

D. Endogenous co-IP of MCAK with Fbxw5. RPE-1 cells were either grown asynchronously or arrested for 24 hours with 2 mM HU (S phase), 10 µM RO-3306 (G2) or 75 ng/ml nocodazole (M). Protein extracts were subjected to unspecific rabbit IgG (-) or anti-Fbxw5 (+) IP and analysed by western blotting. Asterisk indicates an unspecific band detected by the Fbxw5 antibody.

E. Co-IP of HA-Kif2a, HA-Kif2b or HA-MCAK with Flag-Fbxw5 as in A.

F. In vitro pull-down experiment as in B. Asterisk indicates an unspecific protein from E.coli. Note: Kif2b purification from Sf21 cells was much less efficient and it was therefore not included in the experiment.

Data information: Source data for C are presented in Source Data for Figure 2.
SCFFbxw5 targets kinesin-13 proteins to facilitate ciliogenesis (2021)
Figure 3

Figure 3: SCFFbxw5 ubiquitylates MCAK in a highly efficient and specific manner

A. Coomassie-stained SDS-PAGE of different proteins used in the in vitro ubiquitylation assay. Cul1 was obtained as a full-length protein (and therefore runs at around 85 kDa in contrast to the split version of Fig 1) from Sf21 cells and in vitro neddylated (see Fig EV2A and B). SCFFbxw5 complexes for all ubiquitylation experiments were prepared by mixing equimolar amounts of Fbxw5/Skp1 and Cul1~Nedd8/Rbx1 sub-complexes.

B. Drop-out ubiquitylation experiment. 0.2 µM MCAK was incubated with 75 µM ubiquitin (untagged), 170 nM E1, 0.25 µM of UbcH5b and/or Cdc34 and 0.05 µM SCFFbxw5 for 15 min at 30°C followed by western blotting using anti-MCAK antibodies for detection. Top labelling indicates component(s) that have been included (+) or omitted (-) from the reaction.

C. in vitro ubiquitylation experiment as in B using Cdc34 as E2, stopped at the indicated time points by adding SDS sample buffer.

D. In vitro ubiquitylation experiment as in B. Blots 1&2: Plk1 or RanGAP1 used as substrates instead of MCAK. Blot 3: Left: Experiment as in B. Asterisks indicate signals from Plk1 antibody (same species as anti-Fbxw5). Right: Same reaction except that Cul1~Nedd8/Rbx1 complexes were replaced by Cul4A~Nedd8/DDB1/Rbx1 complexes (see Fig EV2A and B). Bottom blots: Same blots as above incubated with anti-Fbxw5 antibodies. Autoubiquitylation of Fbxw5 indicates activity of the E3 ligase complex.

E. in vitro ubiquitylation experiment as in B except that different F-box/Skp1 sub-complexes (Fbxw5/Skp1, Fbxl2/Skp1 or Fbxw7/Skp1) were used (see Fig EV2H).

F. In vitro ubiquitylation experiment as in B using E.coli-derived MCAK, Kif2a or Kif2b as substrates and Cdc34 as E2.

G. In vitro ubiquitylation experiment as in B using either wild type, K48R, K63R, KK48,63RR or methylated ubiquitin (Kmethyl, see Fig EV2K). Top: Cdc34 used as E2. Bottom: UbcH5b used as E2.
SCFFbxw5 targets kinesin-13 proteins to facilitate ciliogenesis (2021)
Figure 4

Figure 4: SCFFbxw5 affects centrosomal MCAK levels in G0

A. Extracts of RPE-1 cells treated for 72 hours with either non-targeting or Fbxw5-directed siRNAs (mix of siFw5-1&3) either grown asynchronously or arrested using 2 mM HU (S-phase), 75 ng/ml nocodazole (M), 10 µM RO-3306 (G2) for 18 hours or via serum-starvation for 24 hours (G0). Indicated antibodies were used for detection. Asterisk indicates an unspecific band detected by the Fbxw5 antibody. Uba2 served as a loading control (same for all following blots).

B. Quantification of the MCAK/Uba2 signal ratio normalised to each non-targeting control of 4 independent experiments. Error bars indicate standard deviation, asterisks the p-value of a two-tailed unpaired Student's t-test comparing each Fbxw5-directed siRNA sample with the according non-targeting control (*P < 0.05, **P < 0.01, ***P < 0.001).

C. RPE-1 cells were transfected with the indicated siRNAs (72 hours total time), split on coverslips 24 hours later and serum-starved for the last 24 hours. Cells were fixed in methanol and analysed via immunofluorescence using the indicated antibodies together with Hoechst staining. Left: Maximum intensity projections (same for all following microscopy images) of representative images. Arrows indicate ODF2 signal. Scale bar = 5 µm. Note: Middle panel shows an example image of siFw5-3. Right: Quantification of MCAK signals co-localising with ODF2. Long black line shows mean intensity and error bars indicate standard error of the mean of 3 independent experiments covering in total more than 280 cells. Asterisks indicate p-value of a Mann Whitney test comparing each sample set with the non-targeting control (**P < 0.01, ***P < 0.001).

Data information: Source data for B and C are presented in Source Data for Figure 4.
SCFFbxw5 targets kinesin-13 proteins to facilitate ciliogenesis (2021)
Figure 5

Figure 5: The APC/C regulates MCAK after mitosis

A. Fluorescence-based pulse-chase experiment using a monoclonal RPE-1 cell line expressing mNG-MCAK under a doxycycline-inducible promoter. Cells were transfected with the indicated siRNAs (siFw5-1&3 together) and split 24 hours later on Ibidi 8 Well Glass Bottom µ-slides while simultaneously inducing mNG-MCAK expression with 6 ng/ml doxycycline (pulse). 24 hours later, doxycycline was washed out and cells were released into serum-free medium. Cells that have just finished or were about to finish mitosis (distinguishable for example by the midbody signal of mNG-MCAK (arrow heads)) were selected and imaged over 24 hours, taking an image every 20 min (chase). Left: representative images of selected time points. Scale bar = 10 µm. Arrows indicate centrosomal mNG-MCAK signals, arrowheads mNG-MCAK signals at the midbody. See also Movie EV2. Right: Quantification of centrosomal mNG-MCAK signals. Error bars show standard error of the mean of 3 independent experiments with 25 cells in total. P-value of a two-tailed unpaired Students t-test comparing signal intensity of Fbxw5 knockdown over non-targeting control was mostly below 0.01 for time points 0 to 8 hours and below 0.05 for time points 9h to 20h. Dark and light blue lines indicate the time points at which mNG-MCAK signals are reduced by 50% compared to time point 0 for siNT and siFbxw5 samples, respectively.

B. Extracts of RPE-1 cells treated with the indicated siRNA for 72 hours, serum starved for the last 24 hours and treated either with DMSO (-) or 10 µM MG-132 (+) for the very last 6 hours. Left: Representative blot. Right: Quantification of MCAK/Uba2 signal ratio normalised to non-targeting control with DMSO of 5 independent experiments. Error bars indicate standard deviation, asterisks the p-value of a two-tailed unpaired Student's t-test (**P < 0.01, ***P < 0.001).

C. RPE-1 cells were treated with 75 ng/ml nocodazole for 4 hours. Mitotic cells were shaken off, washed 2 times with phosphate-buffered saline (PBS) and released in serum free medium. After 2 hours, cells were either directly harvested (2 hour sample) or treated with the indicated APC/C inhibitor for another 18 hours before harvesting. Left: Representative blot. Right: Quantification of MCAK/Uba2 signal ratio normalised to the untreated, 2 hour release sample Error bars show standard deviation of 3 independent experiments, asterisks the p-value of a two-tailed unpaired Student's t-test (*P < 0.05).

D. Same samples as in C, except that cells grown on coverslips were fixed in methanol and analysed by immunofluorescence. Left: Representative images. Arrows indicate ODF2 signal. Scale bar = 5 µm. Right: Quantification. Error bars show standard error of the mean of 3 independent experiments, covering in total more than 150 cells. Asterisks indicate the p-value of a Mann Whitney test (***P < 0.001).

Data information: Source data for A, B, C and D are presented in Source Data for Figure 5.
SCFFbxw5 targets kinesin-13 proteins to facilitate ciliogenesis (2021)
Figure 6

Figure 6: SCFFbxw5 regulates MCAK in G2/M

A. Cycloheximide (CHX) chase experiments of RPE-1 cells treated with the indicated siRNA for 48 hours and then either grown further asynchronously or arrested in S phase with 2 mM hydroxyurea (HU) or in G2 with 10 µM RO-3306 for 18 hours. 50 µg/ml CHX was added to the cells, samples were harvested at the indicated time points and analysed via SDS-PAGE and western blotting using the indicated antibodies for detection. Bottom: Representative blots. Asterisks indicate an unspecific band detected by the Fbxw5 antibody. Top: Quantification of MCAK/Uba2 signal intensity ratios normalised to time point zero. Error bars show standard deviation of 4 independent experiments and asterisks indicate p-value of a two-tailed unpaired Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001). B. CHX chase experiment as in A, except that cells were treated with the indicated siRNAs for 48h and arrested with 200 mM thymidine for 16h, released for 9 hours and again arrested for 16h. Following the 2nd release, 50 µg/ml CHX was added as indicated and samples were analysed as in A. Left: representative blots. Asterisks indicate unspecific bands detected by the Fbxw5 antibody. Right: Quantification of MCAK/Uba2 signal intensity ratios normalised to time point zero. Error bars show standard deviation of 6 independent experiments and asterisks indicate p-value of a two-tailed unpaired Student's t-test (**P < 0.01). C. Extracts of RPE-1 cells treated with the indicated siRNAs for 72 hours, grown asynchronously or arrested with the indicated compounds for the last 24 hours and treated either with DMSO (-) or 10 µM MG-132 (+) for the very last 6 hours. Left: Representative blots. Asterisk indicates an unspecific band detected by the Fbxw5 antibody. Right: Quantification of MCAK/Uba2 signal ratio normalised to non-targeting control with DMSO of 6 independent experiments. Error bars indicate standard deviation, asterisks the p-value of a two-tailed unpaired Student's t-test (**P < 0.01).

Data information: Source data for A, B and C are presented in Source Data for Figure 6.
SCFFbxw5 targets kinesin-13 proteins to facilitate ciliogenesis (2021)
Figure 7

Figure 7: Fbxw5 is required for ciliogenesis in an MCAK-dependent manner

A. Polyclonal RPE-1 cells expressing mNG-MCAK under a doxycycline-inducible promoter were seeded on coverslips, induced with 20 ng/ml doxycycline and grown for 24 hours. Subsequently, cells were washed twice with PBS and incubated for another 24 hours in serum-free medium without doxycycline. Cells were then fixed in methanol and subjected to IF using the indicated antibodies together with Hoechst staining. Left: Representative images. Scale bar = 10 µm. Right: Quantification of ciliated cells. Error bars show standard deviation of 5 independent experiments covering more than 200 cells and asterisks indicate p-value of a two-tailed unpaired Student's t-test (**P < 0.01).

B. Immunoblot of extracts of cells used in A with the indicated antibodies for detection.

C. RPE-1 cells were treated with the indicated siRNA for 48 hours followed by serum-starvation for another 24 hours. Cells were then fixed in methanol and subjected to IF using the indicated antibodies together with Hoechst staining. Left: Representative images. Note: Left panel shows an example image of siFw5-1. Scale bar = 10 µm. Right: Quantification of ciliated cells. Error bars show standard deviation of 4 independent experiments covering more than 200 cells and asterisks indicate p-value of a two-tailed unpaired Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

D. Immunoblot of RPE-1 cell extracts. Left: Representative blots. Asterisk indicates an unspecific band detected by the Fbxw5 antibody. Right: Quantification of MCAK/Uba2 signal ratio. Error bars show standard deviation of 6 independent experiments and asterisks the p-value of a two-tailed unpaired Student's t-test comparing each knockdown with the non-targeting control (**P < 0.01, ***P < 0.001).

E. Model: MCAK is regulated by two distinct E3 ligases at different time points. While SCFFbxw5 targets MCAK for degradation during G2/M, the APC/C takes over after mitotic exit. Via the SCFFbxw5 pathway, cells ensure that levels of MCAK and its orthologs are kept low upon entry into G0 and thus permit ciliogenesis in the following cell cycle.

Data information: Source data for A, C and D are presented in Source Data for Figure 7.

Expanded View Figure legends
Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease (2008)
figf1

(a) Wild-type (WT), atg17Δ, atg7Δ and atg19Δ strains containing RPL25–GFP were grown to mid log phase, starved in SD-N for 48 hours and examined under a fluorescence microscope (left panel). The arrows indicate vacuolar accumulation of the GFP signal. CVT, cytoplasm-to-vacuole-targeting pathway. After the times indicated, at least 100 cells per sample were scored for the presence of Rpl25p–GFP in the vacuole (right panel). Scale bar, 5 μm. (b) Wild-type and atg7Δ strains containing RPL25–GFP or GFP–ATG8 were grown and starved as in a. Samples were taken at indicated time points and GFP cleavage was analysed by western blotting. (c) The levels of endogenous Rps3p (top) and Rpl3p (middle) were analysed by immunblotting in wild-type and atg7Δ cells. Ponceau staining (bottom) controlled for equal loading. Uncropped images of blots are shown in Supplementary Information, Fig. S6. Starv, starvation. (d) Wild-type and pep4Δ cells expressing RPL25–GFP were analysed as in b; 0.1 mM of PMSF was added to pep4Δ cells at the time of starvation to block all vacuolar proteases completely. The LI-COR imaging system was used to quantify the ratio between cleaved GFP and full-length protein as described in Methods. The last time point of wild-type cells containing RPL25–GFP was set to 1 arbitrary unit (a.u.). One of two independent experiments is shown. (e) Wild-type cells containing either RPL25–GFP or control GFP (HOG1–GFP) were analysed as in d. The average of four independent experiments is shown, and the standard deviation was less than 10% (Supplementary Information, Table S3).
Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease (2008)
figf2

(a) Wild-type, ubp3Δ or bre5Δ strains containing RPL25–GFP and either a control plasmid (pRS315), or plasmids expressing Ubp3p or catalytically inactive Ubp3p-C469A from the endogenous promoter, were grown and starved as described in Fig. 1a. Cells were revealed by phase contrast (right), and the localization of Rpl25p–GFP was analysed by GFP microscopy (left). At least 100 cells per sample were analysed. Scale bar, 5 μm. (b) Ribosomes from wild-type and ubp3Δ cells grown as indicated for 16 h under rich or starvation (Starv) conditions were enriched through a high-salt cushion (pellet) and the levels of endogenous Rpl3p were analysed by immunoblotting. Ponceau staining of the blot revealed the total amount of proteins (bottom panel). Uncropped images of blots are shown in Supplementary Information, Fig. S6. (c) Wild-type and ubp3Δ strains containing RPL25–GFP were grown and starved as in Fig. 1a, and GFP cleavage was analysed by western blotting at the times indicated (hours). (d, e) GFP cleavage of Rpl25p–GFP was analysed in wild-type, ubp3Δ, atg9Δ and bre5Δ strains containing the indicated plasmids, and quantified at the times indicated as described in Fig. 1d. One of three representative experiments is shown for d. The average of three independent experiments is shown in e, and the statistical analysis showed that the standard deviation was less than 13% (Supplementary Information, Table S3).
Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease (2008)
figf3

(a) The vacuolar localization of GFP–Atg8p was analysed in more than 100 wild-type, ubp3Δ, bre5Δ and atg7Δ cells containing GFP–ATG8 after 10–15 h of nitrogen starvation (right panel). Corresponding GFP and phase contrast images of representative cells are shown in the left panel. (b) Vacuolar cleavage of GFP–Atg8p was examined at the indicated time points (hours) by anti-GFP western blotting. (c) Wild-type, ubp3Δ and bre5Δ strains containing APE1–RFP were analysed by RFP microscopy (left) and phase contrast (right) 15 h after nitrogen starvation. Scale bars, 5 μm.
Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease (2008)
figf4

(a) Wild-type, ubp3Δ or bre5Δ strains containing either a control plasmid (pRS315) or plasmids expressing Ubp3p or catalytically inactive Ubp3p-C469A from the endogenous promoter were spotted in serial dilutions onto YPD plates with (left) or without (right) 5 nM rapamycin. The plates were photographed after 2 days at 30 °C. (b) Induction of a GCN4-LacZ reporter was measured 0, 2 and 4 h after amino-acid starvation, and is plotted as Miller units. (c) Polysome profiles of exponentially growing wild-type and ubp3Δ cells were determined in rich medium or 3 h after nitrogen starvation. The arrows mark the 60S peak. (d) Wild-type, atg7Δ and ubp3Δ cells containing either RPL25–GFP, control GFP (HOG1–GFP) or RPS2–GFP were analysed as in Fig. 1c. One of three representative experiments is shown. (e) Ribosomes and associated proteins were immunopurified from starved wild-type and ubp3Δ cells containing (RPL25–GFP) and (9 × MYC–UBI) as indicated. The GFP immunoprecipitates (IP; left) and total extracts (TCA, right) were analysed by immunoblotting with anti-Myc antibodies to reveal ubiquitinated proteins (top). Ub, ubiquitin. Bottom: immunoblotting with anti-GFP antibodies controls for the presence of Rpl25p–GFP, and anti-Rpl1p antibodies confirm equal loading of the extract. The asterisk marks protein G on the beads recognized by the secondary antibody.
Reflux of Endoplasmic Reticulum proteins to the cytosol inactivates tumor suppressors (2021)
Figure 4

Figure 4: PDI proteins are redistributed to the cytosol during ER stress.

(A, B) Representative transmission electron microscopy images of the gold particles distribution (after immunogold labelling with PDIA3 antibodies) in A549 cells treated with DMSO or Tm. In the inserts, gold particles in the ER were surrounded by red circles and those in the rest of the cytoplasm by blue circles. (Scale bar 500 nm). n represents the nucleus and m represents the mitochondria. (C) violin plots of the gold particles distribution from the electron microscopy experiment (immunogold labelling of PDIA3) as shown in Figure 4B and 4C. *P-value=0.0486. n=8 for DMSO and n=8 for Tm. Thick horizontal lines represent mean ± SD -lighter dashed lines. Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad),
Reflux of Endoplasmic Reticulum proteins to the cytosol inactivates tumor suppressors (2021)
Figure 5

Figure 5: AGR2 reflux from the ER to the cytosol in A549 cells results in non-genetic inactivation of p53.

(A) Quantification of the subcellular protein fractionation of AGR2 in A549 cells treated with Tunicamycin (Tm), Thapsigargin (Tg) and Brefeldin-A (BFA) for different time points as shown in Appendix Figure S3A-C. n=3, biological replicates ﻿(mean ± SD. ﻿Differences were analyzed using Prism 9 (GraphPad), except when otherwise indicated. (B) Immunoprecipitation of p53 and AGR2 in the digitonin fraction of A549 cells. A549 cells were treated with Tunicamycin (Tm 100ng/ml), Thapsigargin (Tg 25nM) and Brefeldin A (BFA 0,1 and 0,25nM). Endogenous p53 was immunoprecipitated from the cytoplasmic fraction (digitonin fraction) of A549 cells. Coprecipitated endogenous AGR2 was detected by western blot. (C) A549 were treated with Etoposide (Eto) for 2 hours to induce p53 pathway. Then cells have been challenged with Tm, Tg and BFA at the indicated concentrations for 16 hours. Luciferase experiments were performed after 24 hours of transfection. Graph shows the fold induction of p53 luciferase construct. Western blot experiments for phospho-p53, pan-p53, p21 and HSP90 were performed as control. n=3, biological replicates ﻿(mean ± SD; ﻿Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad), except when otherwise indicated. *p<0.05, ***p < 0.001. (D) A549 were transfected with control siRNA (siC) and AGR2-tergetd siRNA (siAGR2). After 24 hours, cells were transfected with p53-luciferase construct. Cells were then treated as in (C) and luciferase experiments were performed. n=4, biological replicates ﻿(mean ± SD. ﻿Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad), except when otherwise indicated. (E) Left: Western blot experiments for phospho-p53, pan-p53, and p21 in AGR2-silenced A549 cells exposed to ER stressors: 100ng/ml Tunicamycin ( Tm), 25nM Thapsigargin (Tg) and 0.25 nM Brefeldin A (BFA), in the presence and absence of etoposide. Right: Quantification of the western blot. n=3, biological replicates, mean ±SD. (F) A549 were transfected with the indicated constructs of differently targeted nanobodies, cells were then treated with Tunicamycin (Tm 100 ng/ml and 250 ng/ml), Thapsigargin (Tg 25 nM) and Brefeldin A (BFA 0,25 nM) for 16 hours at the indicated concentration. Western blot experiments were performed and HSP90 were used as loading control. n=3, biological replicates ﻿(mean ± SD of n=3 independent experiments (***p<0,001 and **p<0,01). Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad), except when otherwise indicated.
Quantifying persistence in the T cell signaling network using an optically controllable antigen receptor (2021)
Figure 3

Figure 3. ERK activation remains sensitive to the state of proximal signaling

(A) Schematic of the OptoCAR‑T cell experiment to follow the dynamics of ERK phosphorylation. Dimerizer was added to cell conjugates at 0 min, with samples (denoted by triangles) taken at defined points. Where sample illumination was used, this is shown by blue region. Samples were rapidly fixed and stained for doubly phosphorylated ERK (ppERK) before measuring the distribution of ppERK levels in cell conjugates.
(B) OptoCAR‑T cells were conjugated with ligand-presenting cells and activated as in the upper regime shown in (A). The median of ppERK intensity distribution was plotted with time, after subtraction of background intensity. Bars show mean ± SEM (n=3).
(C) An equivalent experiment was performed as in (B) but following the middle regime shown in (A), with sample illumination after 5 minutes for the remainder of the dataset. Indicated half-life was calculated from the time taken for the median ppERK to decrease to 50% of maximum. Bars show mean ± SEM (n=4).
(D) An equivalent experiment was performed as in (B) but following the lower regime shown in (A), with sample illumination after 5 minutes for 3 minutes. Bars show mean ± SEM (n=3).
(E-G) A subset of ppERK distributions from individual datasets used to create plots in (B-D), respectively. Blue boxes denote samples that were illuminated.