EMBO/soda-nel huggingface dataset:
NuMA assemblies organize microtubule asters to establish spindle bipolarity in acentrosomal human cells (2019)
Figure 1

Figure 1. Acentrosomal human cells form a specific structure of NuMA in the process of bipolar spindle assembly

(A-C) Distribution of spindle pole factors in centrosomal and acentrosomal spindles. DMSO-treated control mitotic spindles (2-centrosomes) and centrinone-treated acentrosomal spindles (0-centrosome) of HeLa cells. (A) Red, gray, green, and blue represent NuMA, α-tubulin, Cep152, and DNA, respectively. Z-projections of eight sections, every 0.3 μm. Scale bar, 5 μm. (B) Red, gray, and blue represent NuMA, Katanin p60, and DNA, respectively. Z-projections of five sections, every 0.3 μm. Scale bar, 5 μm. (C) Red, gray, and blue represent p150glued, ASPM, and DNA, respectively. Z-projections of 10 (2-centrosomes) or five (0-centrosome) sections, every 0.3 μm. Scale bar, 5 μm.

(D) The structure of NuMA and microtubules in centrinone-treated acentrosomal spindles of HeLa cells. Two-way arrows indicate the bipolarity of elongated NuMA structures. Gray, red, green and blue represent α-tubulin, NuMA, Cep152 and DNA, respectively. Scale bar, 5 μm.

(E) The localization of p150glued in centrinone-treated acentrosomal spindles of HeLa cells. Red, gray, green and blue represent NuMA, p150glued, Cep152 and DNA, respectively. Scale bar, 5 μm.

(F) NuMA distribution in 3D. Images in (D) of NuMA (Red) and chromosomes (blue) were reconstructed in 3D and surface-rendered.

(G) Representative images of NuMA (arrowheads) in centrosomal and acentrosomal cells before and after photobleaching. DMSO- or centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were observed. Time after photobleaching (sec) is indicated. Scale bar, 5 μm.

(H) Normalized fluorescence intensities of NuMA-mClover at centrosomal poles (control, 2-centrosomes) or at acentrosomal spindle pole (0-centrosome) during the FRAP time course (N=20 cells for each condition). The first three time points are before photobleaching. Error bars show standard deviation (SD).

(I) Mobile fractions from the normalized recovery curves of NuMA-mClover at centrosomal spindle poles (control, 2-centrosomes) or at acentrosomal spindle poles (0-centrosome). Mobile fractions (roughly corresponds to normalized fluorescence intensities at the last time point of (H)) were determined by single exponential fitting. Line and error bars represent the median and interquartile range (N=20 cells for each condition). The Mann-Whitney U test (two-tailed) was used to obtain a P value.
NuMA assemblies organize microtubule asters to establish spindle bipolarity in acentrosomal human cells (2019)
Figure 2

Figure 2. NuMA organizes small asters at the time of NEBD and assembles a small spindle before spindle elongation and chromosome congression

(A) Time-lapse observation of the establishment of bipolarity in acentrosomal cells. HeLa cells expressing EGFP-centrin1 and mCherry-NuMA were observed with a 63× objective. Magenta and green represent mCherry-NuMA and EGFP-centrin1, respectively. Arrowheads indicate the assembling NuMA after NEBD. Two-way arrows indicate the bipolarity. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD.

(B) Time-lapse observation of the dynamics of NuMA and chromosomes. Centrinone-treated HeLa cells expressing mCover-NuMA were observed with a 63× objective. Magenta and green represent SiR-DNA and mClover-NuMA, respectively. Arrowheads and two-way arrows indicate the assembling NuMA after NEBD and bipolarity, respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD.

(C) The timing of bipolarity establishment in the acentrosomal pole, chromosome alignment, and anaphase onset in (B). Each plot shows the cumulative percentage of each event at individual time points (N=49 cells from two independent experiments).

(D) The distribution of kinetochores during acentrosomal spindle pole separation was confirmed in fixed cells. Two-way arrow indicates the bipolarity of elongated NuMA structures. Red, gray, green, and blue represent NuMA, CENP-C, GT335, and DNA, respectively. Scale bar, 5 μm.

(E, F) Time-lapse observation of the structure of NuMA and microtubules upon centrosome removal. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were observed with a 60× objective. Red and green represent NuMA and SiR-tubulin, respectively. Z-projections of 17 sections, every 1 μm. Scale bar, 5 μm. Time zero corresponds to NEBD. (F) An example of NuMA formed several asters (arrowheads) at the time of NEBD.
NuMA assemblies organize microtubule asters to establish spindle bipolarity in acentrosomal human cells (2019)
Figure 3

Figure 3. Microtubule, Dynein and clustering activity of NuMA are required for the assembly of NuMA aggregates in acentrosomal human cells

(A) NuMA structure upon microtubule depolymerization. Nocodazole-treated HCT116 CMV-OsTIR1 CEP152-mClover-mAID cells were treated with 100 ng/ml nocodazole. Gray, red, green and blue represent α-tubulin, NuMA, CEP152 (mClover) and DNA, respectively. Scale bar, 5 μm.

(B) Immunostaining of DHC1 and NuMA in centrinone-treated HCT116 TetOsTIR1 DHC1-3X-mAID-mClover cells. Gray, green, red and blue represent DHC1 (mClover), GT335, NuMA, and DNA, respectively. Scale bar, 5 μm.

(C) Schematic illustration of target protein depletion using the AID system to analyze the requirement of dynein for acentrosomal spindle pole formation.

(D) NuMA structure upon DHC1 depletion. Centrinone-treated HCT116 TetOsTIR1 DHC1-3X-mAID-mClover cells were treated with 1 μg/ml doxycycline (Dox) and 500 μM indole-3-acetic acid (IAA). Arrowhead indicates the spindle pole. Green, red, gray and blue represent DHC1 (mClover), NuMA, GT335 and DNA, respectively. Scale bar, 5 μm.

(E) Frequency of acentrosomal pole patterns upon DHC1 depletion in (D). Values are mean percentages ± SD. from three independent experiments (N≧24 spindles in each experiment).

(F) The distribution of NuMA upon DHC1 depletion. Arrowheads indicate the presence of NuMA on microtubules. Green, red, and blue represent α-tubulin, NuMA, and DNA, respectively. Scale bars, 5 μm.

(G) Structure of acentrosomal spindle poles upon replacement of endogenous NuMA-mAID-mClover-FLAG with either mCherry-NuMA WT or 5A-3. Arrowheads indicate the assembled NuMA structure. Green, red, gray and blue represent endogenous NuMA (mClover), expressed NuMA (mCherry), GT335 and DNA, respectively. Z-projections of 21 sections, every 1 μm. Scale bar, 5 μm.

(H) The number of NuMA structure in (G). Values are mean percentages ± SD. from four independent experiments (N≧15 spindles in each experiment). One-way ANOVA with Tukey's multiple comparisons test was used to obtain a P value.
NuMA assemblies organize microtubule asters to establish spindle bipolarity in acentrosomal human cells (2019)
Figure 4

Figure 4. NuMA promotes the establishment of spindle bipolarity in acentrosomal human cells

(A, B) Structure of the spindle upon NuMA depletion. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with 1 μg/ml doxycycline (Dox) and 500 μM IAA. Two-way arrow indicates the bipolarity. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), GT335, and DNA, respectively. Z-projections of 10 sections, every 0.3 μm. Scale bar, 5 μm.

(C) Frequency of spindle structure patterns upon NuMA depletion in (A, B). Values are mean percentages ± SD. from three independent experiments (N=30 spindles in each experiment).

(D) The localization of Eg5 in centrinone-treated acentrosomal HeLa cells. Red, gray, green and blue represent NuMA, Eg5, GT335 and DNA, respectively. Scale bar, 5 μm.

(E) The localization of Eg5 in the spindle upon NuMA depletion. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with Dox and 500 μM IAA. Gray, green, red, and blue represent Eg5, NuMA (mClover), CP110, and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm.

(F) Structure of the spindle upon Eg5 inhibition. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with 30 μM monastrol. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), GT335, and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm.

(G) Frequency of spindle structure patterns upon Eg5 inhibition in (F). Values are mean percentages ± SD. from three independent experiments (N≧23 spindles in each experiment).
NuMA assemblies organize microtubule asters to establish spindle bipolarity in acentrosomal human cells (2019)
Figure 5

Figure 5. Contribution of Eg5 and stable KT-MT attachment to the initial establishment of bipolarity and subsequent separation of acentrosomal spindle poles

(A) Time-lapse observation of the establishment of bipolarity within the acentrosomal spindle pole upon inhibition of Eg5. HeLa cells expressing EGFP-centrin1 and mCherry-NuMA were observed in the presence of 50 μM of monastrol with a 63× objective. Magenta and green represent mCherry-NuMA and EGFP-centrin1, respectively. Arrowheads and two-way arrows indicate the monopolar and bipolar states, respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD.

(B) The time of bipolarity establishment and being stuck in the monopolar-like state of acentrosomal spindle poles in (A). Each plot shows the cumulative percentage of each event at each time point (N=total 75 cells from two independent experiments).

(C) Time-lapse observation of the dynamics of mClover-NuMA and chromosomes. HeLa cells expressing mClover-NuMA were observed in the presence of SiR-DNA with a 63× objective. Magenta and green represent SiR-DNA and mClover-NuMA, respectively. Arrowheads indicate initial bipolarity in the acentrosomal spindle pole (8 min), dispersed chromosomes (328 min), and the monopolar-like state of the acentrosomal spindle pole (332 min), respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD.

(D) The time of bipolarity establishment and being stuck in the monopolar-like state of acentrosomal spindle poles in (C). Each plot shows the cumulative percentage of each event at each time point (N=total 35 cells from three independent experiments).
NuMA assemblies organize microtubule asters to establish spindle bipolarity in acentrosomal human cells (2019)
Figure 6

Figure 6. NuMA promotes the initial step of spindle bipolarization in cells with centrosomes

(A) Structure of the spindle upon NuMA and Eg5 inhibition. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml doxycycline (Dox), 500 μM IAA and monastrol. Green, red, and blue represent NuMA (mClover), α-tubulin, and DNA, respectively. Z-projections of 10 sections, every 0.3 μm. Scale bar, 5 μm.

(B) Frequency of spindle structure patterns upon NuMA depletion and Eg5 inhibition in (A). Values are mean percentages ± SD. from three independent experiments (N≧22 spindles in each experiment).

(C) The localization of Eg5 in the spindle upon NuMA depletion. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml doxycycline Dox and 500 μM IAA. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), Eg5 and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm.

(D) Time-lapse observation of the establishment of spindle bipolarity upon NuMA depletion. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml Dox and 500 μM IAA and observed with a 40× objective. Red and green represent SiR-tubulin and NuMA, respectively. Z-projections of 14 sections, every 2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD.

(E) The time required for the initial establishment of spindle bipolarity in (D). Line and error bars represent the mean and SD. (N≧60 cells from two independent experiments). The Mann-Whitney U test (two-tailed) was used to obtain a P value.
GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis (2014)
Figure 1

The LRR RLK PRK5 is required for GRI‐peptide‐induced ion leakage

Infiltration of GRIp65–84 induced cell death similar to GRIp31–96. Bacterially produced 37 nM GST, GST‐GRIp31–96 or biochemically pure GRI‐peptides (GRIp31–96, GRIp31–51, GRIp47–68, GRIp65–84, GRIp80–96) were infiltrated into leaves of Col‐0 plants.
Infiltration of increasing concentrations of GRIp65–84 into Col‐0 leaves, electrolyte leakage was measured after 12 h. Background (red line) shows ion leakage from infiltration of leaves with increasing concentrations of (inactive) GRIp80–96.
Infiltration of leaves with 37 nM GRIp31–96 induced elevated ion leakage in Col‐0 and prk4, but not in prk5‐1 or prk5‐2. Infiltration with GST caused the same background effect for all lines.
Genomic complementation of prk5 rescues the insensitivity to induction of elevated ion leakage by GRIp31–96.
Enzymatic superoxide production from xanthine/xanthine oxidase (XXO) induced more electrolyte leakage in gri compared to Col‐0 or prk5‐1, prk5‐2 and prk4 after infiltration into leaves. Infiltration with xanthine buffer (X) was used as a control.

Data information: All panels show average ± standard deviation (SD) of four replicates consisting of four leaf disks each. Asterisks indicate statistically significant differences from GST infiltration (A, C, D), from infiltration with (inactive) GRIp80–96 (B) or from Col‐0 (E) according to Sidak's test (P 0.05). All experiments were repeated at least four times with similar results.
GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis (2014)
Figure 2

PRK5 is an atypical, enzymatically inactive RLK

A PRK5 domain structure: SP signal peptide (aa 1–39), LRR leucine‐rich repeat, RPT internal repeat, LC region of low complexity, TM transmembrane domain (aa 282–304), EM Y‐based sorting/endocytosis motif (YSSM; aa 670–673).
B PRK5‐CFP localized to the cell periphery in Col‐0 mesophyll protoplasts.
C–F Co‐localization of PRK5‐CFP and PM localized CAAX‐YFP in Col‐0 mesophyll protoplasts. (C) overlay; (D) PRK5‐CFP (465–510 nm); (E) CAAX‐YFP (521–587 nm); (F) chloroplast (636–711 nm).
G Alignment of subdomains VIb and VII of the catalytic core of the kinase domains of active (BRI1, FLS2, EFR, CRK7) and inactive RLKs (PRK4, PRK5, BIR2, SUB). Residues marked in green highlight conservation of the consensus of active protein kinases while residues highlighted in red indicate deviations from the consensus sequence. An alignment of the full kinase domains for the RLKs used in this figure can be found in Supplementary Fig S11A.
H Kinase activity of PRK5 in in vitro phosphorylation assays using γ32P‐ATP and myelin‐basic protein (MBP) as a substrate in the presence of 10 mM MnCl2. GST‐PRK5 and GST‐PRK4 did not show kinase activity. Mutation of conserved residues in kinase subdomains VIb and VII to reconstitute the consensus kinase domain motif restored GST‐PRK5H500DA520G kinase activity. GST‐CRK7 was used as a positive control. Upper panel shows autoradiograph, and lower panel shows the Coomassie‐stained 15% SDS–polyacrylamide gel.

Data information: Experiments in (B–F and H) were repeated three times with similar results. Source data are available online for this figure.
GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis (2014)
Figure 3

A 20‐aa peptide binds to the extracellular domain of PRK5

In vitro‐produced 35S‐labeled ectodomains of PRK5 (PRK540–281) or PRK4 (PRK440–279) were incubated with bacterially produced GST, GST‐GRI or GST‐GRI31–96 and purified. GST‐GRI31–96 but not GST directly bound to the ectodomain of PRK5. Binding of GST‐GRI to PRK5 ectodomain and binding of GST‐GRI31–96 and GST‐GRI to the ectodomain of PRK4 were strongly reduced. Upper part: autoradiograph, lower part: Coomassie‐stained 12% SDS–polyacrylamide gel. Asterisks in the Coomassie‐stained gel indicate GST, GST‐GRI and GST‐GRI31–96, respectively. Supplementary Fig S12 shows a Western blot of the GST‐tagged proteins.
Infiltration of 37 nM GST, GRIp31–96, GRIp65–84 or Y‐GRIp65–84 into Col‐0 or prk5‐2 leaves. Tyrosine‐labeled GRIp65–84 still induced cell death in Col‐0 but not in prk5‐2 plants.
125I‐labeled Y‐GRIp65–84 (0.46 nM) bound specifically to Col‐0 membrane fractions (light gray bars), the binding was significantly reduced in prk5 plants. Excess of non‐radioactive Y‐GRIp65–84 (10 μM) reduced binding to background levels (dark gray bars; all bars show the average of three samples, triangles show individual data points).
Immunoprecipitation of PRK5‐c‐myc, PRK4‐c‐myc or YFP expressed in protoplasts with rabbit polyclonal anti‐c‐myc antibody, blotted with mouse monoclonal anti‐c‐myc antibody.
Binding of 125I‐Y‐GRIp65–84 (0.46 nM) to immunoprecipitates (using anti‐c‐myc antibodies) from prk5‐2 protoplasts transfected with PRK5‐c‐myc, PRK4‐c‐myc or YFP, respectively. Binding was competed out with 10 μM unlabeled Y‐GRIp65–84. Bars show the average of two samples, and triangles show individual data points. Western blot is shown in panel (D).
Analysis of 125I‐Y‐GRIp65–84 (0.46 nM) binding competed out with increasing amounts of unlabeled Y‐GRIp65–84 to Col‐0 membrane extracts. Fifty percent inhibition (IC50) occurred at 25.2 nM. Red line shows binding average competition according to a sigmoid curve, dotted lines show 95% confidence intervals, and circles show data points.
Excess of GRIp65–84 (10 μM) but not of GRIp31–96 or other peptides (GRIp31–51, GRIp47–68, GRIp80–96) competed the binding of 0.46 nM 125I‐Y‐GRIp65–84 to membrane extracts from Col‐0 (all bars show the average of two samples, triangles show individual data points).

Data information: Data in (B) are shown as average ± SD of four replicates consisting of four leaf disks each. Asterisks in (B) mark statistically significant differences from infiltration with GST according to Sidak's test (P 0.05). All experiments were repeated three times with similar results.Source data are available online for this figure.
MCL‐1 is a stress sensor that regulates autophagy in a developmentally regulated manner (2010)
Figure 1

MCL‐1 is degraded following nutrient deprivation. (A) HeLa cells were incubated for 4 h in either EBSS or media without glucose/pyruvate. Lysates were then analysed by western blot for the presence of MCL‐1, as well as actin as a loading control. (B) CGNs were subjected to 17 h hypoxia after 7 days in vitro. Protein lysates were collected at indicated times after reoxygenation and blotted for MCL‐1 expression and for actin as a loading control. (C) H1299 cells stably expressing GFP–LC3 were incubated for 2 h in EBSS, and expression of various BCL‐2 homologues was analysed by western blot. Alternatively, primary cortical neurons were incubated for 4 h in media without glucose/pyruvate. (D) H1299 cells stably expressing GFP–LC3 were incubated for 3 h in media without glucose/pyruvate, followed by re‐addition of glucose and pyruvate for the indicated times. (E) HeLa cells were infected with 5 p.f.u. per cell of either Ad HA‐tBID or Ad LacZ for 7 h, the media being changed to EBSS for the last 3 h where indicated. Alternatively, cells were treated with 50 μM camptothecin for 3 h. (F, G) HeLa cells were treated as in E in the presence of 50 μM zVAD‐FMK and analysed by immunofluorescence for active BAX (6A7 epitope; F) or cyt c (G). Data are expressed as the average of three experiments±s.d. *P0.01.
MCL‐1 is a stress sensor that regulates autophagy in a developmentally regulated manner (2010)
Figure 2

MCL‐1 degradation is an early event following starvation‐induced autophagy. (A) H1299 cells stably expressing GFP–LC3 were incubated for the indicated times in EBSS in the presence of 50 μM zVAD‐FMK and analysed by immunofluorescence for cyt c or active BAX (6A7 epitope). As a positive control, cells were infected with 5 p.f.u. per cell Ad HA‐tBID for 7 h. Data are expressed as the average of three experiments±s.d. (B) H1299 cells stably expressing GFP–LC3 were incubated for 4 h in either EBSS or media without glucose/pyruvate. Cells were then fixed, stained with an antibody against cyt c and analysed by immunofluorescence. Scale bars=50 μm. Alternatively (C), the cells were analysed by western blot for the presence of MCL‐1 and the autophagic markers GFP–LC3 (anti‐GFP antibody) and p62. (D) H1299 cells stably expressing GFP–LC3 were incubated for the indicated times in either EBSS or media without glucose/pyruvate and analysed by western blot for the presence of MCL‐1 and autophagic markers. (E, F) H1299 cells stably expressing GFP–LC3 were pre‐incubated for 1 h with 5 mM 3MA (E) or 200 nM bafilomycin (F), and then incubated for 2 h in complete media or EBSS in the presence of 3MA or bafilomycin. Lysates were then analysed for the presence of MCL‐1, the autophagy marker p62 and actin as a loading control.
MCL‐1 is a stress sensor that regulates autophagy in a developmentally regulated manner (2010)
Figure 3

MCL‐1 levels regulate autophagy. (A) H1299 cells stably expressing GFP–LC3 were infected with 10 p.f.u. per cell of either Ad LacZ or Ad Myc‐MCL‐1 for 24 h then incubated in EBSS for 4 h. Alternatively, cells were transfected with control or MCL‐1 siRNA 24 h before EBSS treatment. Cells were stained with an antibody against cyt c and analysed by immunofluorescence. Representative images are shown. Scale bars=50 μm. (B) The percent of the cell surface area covered by GFP–LC3 vesicles was quantified using the Image J software. n>70 cells in three experiment, *P0.01. (C, D) H1299 cells stably expressing GFP–LC3 were transfected as in A and incubated with 200 nM bafilomycin for 1 h where indicated. The levels of endogenous LC3‐II relative to actin were quantified by western blot in C. Data are expressed as the average of three experiments±s.d, *P0.05. (E, F) H1299 cells stably expressing GFP–LC3 were treated as in A and analysed by western blot for the presence of p62 and GFP–LC3. (G) Primary cortical neurons of the indicated genotypes were infected with a lentivirus expressing Cre at the time of plating and analysed for the presence of MCL‐1 and p62 by western blot 3 days later. (H) Transformed wild‐type (WT) and MCL‐1Δ/Δ (KO) MEFs were analysed by western blot for the expression of endogenous LC3. (I, J) Colocalization of GFP–LC3 vesicles with the lysosomal marker LAMP1 was analysed by immunofluorescence in cells treated as in A. The number of LC3‐positive vesicles that colocalised with LAMP1 was quantified in I. Data are expressed as the percentage of LC3 vesicles per cell that are LAMP1‐positive±s.e.m., with at least 14 cells per condition. Representative images are shown in J. Arrows indicate LC3‐positive vesicles colocalizing with LAMP1. Scale bars=10 μm.
MCL‐1 is a stress sensor that regulates autophagy in a developmentally regulated manner (2010)
Figure 4

MCL‐1 interacts with Beclin‐1 at the mitochondria. (A) MCL‐1 interacts with Beclin‐1. Lysates from H1299 stably expressing GFP–LC3 were immunoprecipitated using control or MCL‐1‐specific antibodies and analysed for the presence of Beclin‐1 by western blot. (B) H1299 cells stably expressing GFP–LC3 were fractionated into HM (containing mitochondria), and LM (containing ER). Fractions were analysed for the presence of MCL‐1, BCL‐2, Beclin‐1, the mitochondrial marker mtHSP70 and the ER marker calreticulin. (C) MCL‐1 was immunoprecipitated from HM and LM before analysing the presence of MCL‐1 and Beclin‐1.