EMBO/soda-nel huggingface dataset:
Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins (2020)
Figure 2

Figure 2. KIF4A drives MT-flux via its chromosome arm-based motor activity.

A. Representative spinning disk confocal live-cell imaging time series images of U2OS PA-GFP-α-tubulin cells conditionally co-expressing KIF4A shRNA and RNAi-resistant mCherry-Kif4A variants induced using doxycycline. Chromosomes were stained using SiR-DNA. White arrowheads highlight poleward flux of the photoactivated regions. Scale bar, 10 µm. Time, min:sec.

B. Representative immunoblot of cell lysates obtained before and after doxycycline induction to validate the efficiency of KIF4A shRNA construct and expression of the RNAi-resistant mCherry-KIF4A variants. The anti-KIF4A antibody was used, together with anti-vinculin antibody as a loading control.

C. Quantification of MT-flux upon shRNA-mediated depletion of KIF4A alone or in combination with conditional expression of the RNAi-resistant mCherry-KIF4A variants. The bars in graph represent mean ± SD. N (number of cells, number of independent experiments): uninduced shKIF4A (48, 4), shKIF4A (60, 4), uninduced KIF4A WT (36, 5), shKIF4A + KIF4A WT (43, 5), uninduced KIF4A K94A (42, 7), shKIF4A + KIF4A K94A (38, 6), uninduced KIF4A ΔZip1 (32, 4), shKIF4A + ΔZip1 (30, 4). P-values were calculated using Student's t-test and Mann-Whitney U test. n.s. - not significant, **** P ≤ 0.0001.

D. Model illustrating chromosome arms-localized KIF4A driving MT-flux.
Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins (2020)
Figure 3

Figure 3. Bipolar spindles with expelled chromatin in MUGs exhibit reduced MT-flux.

A. Representative spinning disk confocal time series of MT-flux in bipolar spindles in control cells and cells undergoing mitosis with unreplicated genomes (MUGs). U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), labeled for chromosomes with SiR-DNA (grey) are shown, with 5 mM caffeine used as a control. Scale bar, 10 µm. Time, min:sec.

B. Quantification of the MT-flux rates from indicated conditions. MT-flux values with mean ± SD are plotted. N (number of cells, number of independent experiments): Caffeine-only control (30, 3) and MUGs (34, 3). P-values from Mann-Whitney U test. **** P ≤ 0.0001.

C. Representative point-scanning confocal maximum-intensity projected images of mitotic spindles in U2OS cells subjected to indicated conditions, immunostained with antibodies against KIF4A and α-tubulin. DNA was counterstained with DAPI. KIF4A in cyan and α-tubulin in red in merged image. Scale bar, 10 µm.
Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins (2020)
Figure 4

Figure 4. The MT-crosslinking proteins HSET and NuMA facilitate the distribution of MT-flux associated spindle forces on metaphase chromosomes.

A. Representative spinning disk confocal live-cell image series of MT-flux in U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red) treated with indicated siRNAs. White arrowheads highlight poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 µm. Time, min:sec.

B. Quantification of the impact of the MT-crosslinking proteins on MT-flux in U2OS PA-GFP/mCherry-α-tubulin cells transfected with respective siRNAs. Graph represent MT-flux values with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siPRC1 (45, 3), siNuMA (40, 3), siHSET (37, 3), siHSET + siNuMA (39, 3). P-values were calculated against control using one-way ANOVA. n.s., not significant, ** P ≤ 0.01, **** P ≤ 0.0001.

C. Quantification of the impact of the motor activity of HSET on MT-flux in U2OS cells stably expressing mEOS-α-tubulin treated with control or HSET 3′UTR siRNAs in presence or absence of the respective RNAi-resistant GFP-HSET constructs. Graph represent MT-flux values with mean ± SD. N (number of cells, number of independent experiments): siControl (23, 3), siHSET 3′UTR (33, 3), siHSET 3′UTR + HSET WT (29, 5), siHSET 3′UTR + HSET N593K (30, 4). P-values were calculated against control using one-way ANOVA. n.s., not significant, **** P ≤ 0.0001.

D. Representative kymographs of the photoactivated spindles from (B) displaying asynchronous flux motion (split of the two red dashed lines towards individual poles) upon RNAi-mediated depletion of MT-crosslinkers. Scale bars, 30 sec.

E. Percentage of cells with asynchronous flux movements calculated from (B).

F. Model illustrating the role of MT-crosslinking activities of NuMA and HSET in uniform distribution of poleward forces across the mitotic spindle.
Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins (2020)
Figure 5

Figure 5. CENP-E is the predominant driver of MT-flux in early prometaphase.

A. Representative spinning disk confocal live-cell time series of MT-flux in U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red) treated with indicated conditions. White arrowheads follow the poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 µm. Time, min:sec.

B. Quantification of the impact of stable end-on KT-MT attachments on MT-flux in NDC80-depleted cells under indicated conditions and in early prometaphase cells with and without the CENP-E inhibitor GSK923295. MT-flux values are plotted with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siNDC80 (59, 6), siNDC80 + siKIF4A (41, 3), siNDC80 + STLC (bipol.) (28, 3), siNDC80 + siKIF15 (36, 4), siNDC80 + siCLASPs (33, 3), siNDC80 + siKIF2A (38, 4), siNDC80 + GSK923295 (38, 3), early prometaphase (23, 3), early prometaphase + GSK923295 (28, 3). P-values were calculated using Kruskal-Wallis H test compared to siNDC80, except for early prometaphase data for which Student's t-test was used. n.s., not significant, **** P ≤ 0.0001.

C. Kymograph profiles (left) highlighting that the photoactivated regions in prometaphase (upper panel) split and move towards both poles, whereas metaphase spindles (lower panel) flux uniformly towards individual poles (indicated by red dotted lines). Illustration of the photoactivated regions (right) in prometaphase and metaphase spindles, highlighting that interpolar MTs overlap over a broader region in early mitosis, becoming focused at the spindle equator in metaphase. Scale bar, 30 sec.

D. Percentage of cells with parallel and antiparallel flux movements during prometaphase and metaphase. N (number of cells, number of independent experiments): prometaphase (23, 3), metaphase (22, 3).
Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins (2020)
Figure 6

Figure 6. CENP-E localizes to KTs and MT bundles, including overlapping anti-parallel MTs during early mitosis.

A. Representative maximum-intensity projected coherent-hybrid STED (CH-STED) images of mitotic spindles in U2OS cells treated with control and NDC80 siRNAs and stained for DNA (DAPI), KTs (CENP-C, magenta), α-tubulin (red) and CENP-E (green). Scale bar, 10 µm.

B. Model illustrating potential mechanisms for CENP-E mediated MT-flux forces either through its KT- or antiparallel MT- based localization and sliding. Zoomed insets from (A) highlight CENP-E localized at KTs laterally interacting with MTs (white arrowheads in 1, 2), and CENP-E localized at interpolar MTs (white arrows in 3, 4), in control and NDC80-depleted mitotic cells. Scale bars, 2 µm.

C. CH-STED analyses of HeLa cells stably expressing CENP-E-GFP illustrating bipolar spindles in prometaphase, metaphase and just after sister-chromatid separation at anaphase onset. Approximate x-y resolution is 70 nm. The expected/described localization of CENP-E-GFP (green) at KTs is shown. In addition, CENP-E-GFP was found associated with interpolar MTs (ipMTs), including regions of overlapping anti-parallel MTs, and k-fibers. Chromosomes (confocal mode only) were revealed in the larger panels with DAPI (cyan) and MTs (magenta) were detected with an anti-α-tubulin antibody. Arrows indicate examples of clear MT bundles, including regions of overlapping anti-parallel MTs. Scale bar in all panels is 5 μm.

D. Representative spinning disk confocal live-cell time series of HeLa cells stably expressing CENP-E-GFP. White arrowheads highlight CENP-E-GFP at KTs, white arrows highlight CENP-E-GFP on interpolar MTs. Scale bar, 10 µm. Time, hour:min.
Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins (2020)
Figure 7

Figure 7. Combined action of EG5 and KIF15 support MT-flux driving activities of CENP-E and KIF4A.

A. Representative spinning disk confocal time series of U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), with indicated treatments (left). White arrowheads highlight poleward flux of the photoactivated regions. Scale bar, 10 µm. Time, min:sec. Right panels display corresponding kymographs of the photoactivated spindles used for quantification of the flux rates (red dotted lines highlight MT-flux slopes). Scale bars, 2 µm (horizontal) and 30 sec (vertical).

B, C. Quantification of MT-flux in prometaphase (B) and metaphase (C) bipolar spindles subjected to indicated treatments. Graphs represent MT flux of individual cells with mean ± SD. siNDC80, siNDC80 + GSK923295, early prometaphase, early prometaphase + GSK923295, siControl, and siKIF4A data are from Figure 1E and 5B. N (number of cells, number of independent experiments): siNDC80 + KIF15 + STLC (33, 5), siNDC80 + KIF15 + STLC + GSK923295 (25, 3), early prometaphase + KIF15 + STLC + GSK923295 (11, 2), siKIF15 + STLC (29, 3), siKIF4A + siKIF15 + STLC (10, 3). P-values were calculated using Student's t-test and Mann-Whitney U test. ** P ≤ 0.01, *** P ≤ 0.001 **** P ≤ 0.0001.

D. Representative spinning disk confocal time series of U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), with indicated treatments (left) and corresponding kymographs (right) as described in (A). Scale bars, 2 µm (horizontal) and 30 sec (vertical).
Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins (2020)
Figure 8

Figure 8. MT poleward flux regulates spindle length in response to MCAK-mediated depolymerization of KT-MTs.

A-C. Graph depicting a direct correlation between MT-flux and mitotic spindle where mean spindle length is plotted over mean MT-flux rates for all indicated conditions (A), mitotic conditions containing stable end-on attachments (B), and NDC80 depleted conditions lacking stable end-on attachments (C). Solid and dotted lines represent the linear regression and 95% confidence interval, respectively. Pearson correlation coefficient (r), coefficient of determination (r2) and corresponding p values are indicated. The error bars over x and y axis represent the standard deviation of MT-flux and spindle length, respectively.

D. Representative images from spinning disk confocal live-cell time series of U2OS PA-GFP-α-tubulin/mCherry-α-tubulin cells under indicated treatments, with SiR-DNA-labelled DNA. Microtubules are shown in red and DNA in cyan in the merged image. Scale bar: 10 µm.

E. Quantification of spindle lengths at metaphase following indicated treatments. Graph represents the spindle length of individual cells with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siMCAK (34,3), siKIF4A + siKIF15 + STLC (46, 6), siKIF4A + siKIF15 + STLC + siMCAK (43, 3), siKIF4A + siKIF15 + STLC + siKIF2A (22, 3), siCLASPs (64, 6), siCLASPs + siMCAK (33, 3), siCLASPs + siKIF2A (32, 3). P-values were calculated using Student's t-test and Mann-Whitney U test. n.s. - not significant, **** P ≤ 0.0001.
The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1 (2010)
figf1

(a) Diagrams of the deletion–mutation constructs of Keap1 (left) and the corresponding immunoprecipitation assays (right). Each Flag-tagged mouse Keap1 and mutant was expressed in HEK293T cells. At 22 h after transfection, lysates were prepared and immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS–PAGE and analysed by immunoblotting with anti-Flag, anti-p62, anti-Nrf2 and anti-actin antibodies. Data are representative of three individual experiments. (b) Diagrams of the deletion–mutation constructs of p62 (left) and the corresponding input (upper right) and pull-down assay (lower right). The MBP-tagged mouse p62 deletion mutants conjugated to amylose (AM) resins were incubated with purified GST-tagged mouse Keap1-DC. The pulled-down complexes with the MBP–p62 mutants were subjected to SDS–PAGE and revealed by staining with Coomassie brilliant blue. The bands corresponding to MBP–p62 and its mutants are indicated by black dots. Red arrowheads indicate the band corresponding to GST–Keap1-DC. For details of construct 14 see Supplementary Information, Fig. S3. (c) Alignment of the Keap1-interacting regions (KIR; red line) and the LC3-recognition sequences (LRS; green line) of p62 homologues in various species. Black and grey boxes indicate identical amino acid residues with complete and partial conservation, respectively. (d) Immunoprecipitation assays. Flag-tagged p62, KIR-deleted p62 (p62 ΔKIR) and a p62 mutant defective in oligomerization (p62 K7AD69A) were expressed in primary mouse hepatocytes by the adenovirus system (left) or in HEK293T cells by transfection (right). Cell lysates were immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS–PAGE and analysed by immunoblotting with anti-p62 and anti-Keap1 antibodies. The bands corresponding to Flag–p62, endogenous p62, Keap1 and actin are indicated. The data shown are representative of three separate experiments. (e) Interaction of endogenous p62 with Keap1. Lysates prepared from the human hepatocellular carcinoma cell line Huh-1 were immunoprecipitated with anti-p62 antibody or anti-Flag antibody (negative control) followed by immunoblotting with antibodies against p62 and Keap1. The bands corresponding to endogenous p62, Keap1 and IgG heavy chain (IgG H.C.) are indicated. The data shown are representative of three separate experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.
The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1 (2010)
figf3

(a) A representative ITC profile of the titration of Keap1-DC with p62M7 (residues 168–391). The upper panel shows the raw ITC thermograms and the lower panel shows the fitted binding isotherms. (b) Immunoprecipitation assays. Flag-tagged Keap1 was co-expressed with increasing concentrations of green fluorescent protein (GFP)–p62 (lanes 3–6) in HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS–PAGE and analysed by immunoblotting with anti-Flag, anti-p62 and anti-Nrf2 antibodies. The bands corresponding to Flag–Keap1, endogenous p62, Nrf2 and actin are indicated. Data are representative of three independent experiments. (c) The competitive p62 activity against Keap1 was measured by luciferase assay. The expression plasmids for Nrf2, Keap1 and p62 wild-type (w.t.) or its mutants were transfected into Hepa1 cells along with pNqo1-ARE reporter plasmid and pRL-TK as an internal control. At 36 h after transfection, the luciferase activity was measured in accordance with the instructions provided by the manufacturer. Assays were performed twice in triplicate. Data are means and s.d. for six determinations. (d) Immunoblot analysis. Flag-tagged p62 and its mutants defective in interacting with Keap1 were overproduced in wild-type and p62−/− primary mouse hepatocytes by the adenovirus system. At 48 h after infection, total cell lysates and nuclear fractions were prepared and subjected to immunoblot analysis with the antibodies specified. The bands corresponding to Flag–p62, endogenous p62, Keap1, Nrf2, Nqo1, actin and Lamin B are shown. Data are representative of three independent experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11. (e) Quantification of mRNA levels of the detoxification enzymes Nqo1, Gstm1 and Cyp2a5 in hepatocytes overexpressing Flag–p62 and its mutants. Total RNAs were prepared from non-infected or infected hepatocytes and reverse transcribed into their respective cDNAs, which were used as templates in real-time PCR analysis. Values were normalized to the amount of each mRNA in the non-infected hepatocytes. The experiments were performed three times. Data are means ± s.d. for three experiments.
The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1 (2010)
figf4

(a) Insolubilization of Keap1 in Atg7-deficient hepatocytes. Liver homogenates from Atg7F/F:Mx1 mice on various days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions with 0.5% Triton X-100. Each fraction was subjected to SDS–PAGE and analysed by immunoblotting with the indicated antibodies. The data displayed are representative of three separate experiments. (b) Immunoblot analysis of Atg7-deficient (Atg7F/F:Mx1; Atg7F/F shown here as F/F) and Atg7 p62-deficient (Atg7F/F:Mx1:p62−/−) livers. Liver homogenates from mice of the stated genotypes at 12 days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions. Each fraction was subjected to SDS–PAGE and analysed by immunoblotting with the indicated antibodies. Atg7F/F mice in which Atg7 is efficiently expressed at a level similar to that in the wild-type mice were used as control. Data shown are representative of three separate experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11. (c) Quantitative real-time PCR analyses of Nqo1 and Gstm1 in mouse livers. Total RNAs were prepared from livers of the indicated genotypes at 12 days after injection of poly(I)•poly(C). Values were normalized to the amount of mRNA in Atg7F/F liver. Data are means ± s.d. for three experiments. (d) Immunofluorescence analysis of the cellular localization of p62 and Keap1. Liver sections from mice of the indicated genotypes at 28 days after injection of poly(I)•poly(C) were immunostained with anti-Keap1 (top) and anti-p62 (middle) antibodies. Bottom: merged images of Keap1 (green) and p62 (red). Each inset in the Atg7-deficient liver panels is a magnified image of the boxed region. Scale bars, 20 μm.
The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1 (2010)
figf5

(a) Immunoblotting of Atg7-deficient (Atg7F/F:Mx1; Atg7F/F shown here as F/F) and Atg7 Nrf2-deficient (Atg7F/F:Mx1:Nrf2−/−) livers. Liver homogenates from mice of the assigned genotypes at 28 days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions. Total, soluble and insoluble fractions were subjected to SDS–PAGE and analysed by immunoblotting with the indicated antibodies (top section). Total lysates were subjected to SDS–PAGE and analysed by immunoblotting with antibodies against Nqo1, Gstm1 and actin (bottom left section). Nuclear fractions were prepared from the livers of the indicated genotypes at 28 days after injection of poly(I)•poly(C), subjected to SDS–PAGE and analysed by immunoblotting with antibodies against Nrf2 and Lamin B (as control) (bottom right section). Data were obtained from three independent experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11. (b) Immunofluorescence analysis of the cellular localization of p62 and Keap1. Liver sections from mice of the indicated genotypes at 28 days after injection of poly(I)•poly(C) were immunostained with anti-Keap1 (left) and anti-p62 (middle) antibodies. Right: merged images of Keap1 (green) and p62 (red). Each inset in the Atg7-deficient and Atg7 Nrf2-deficient liver panels is a magnified image of the boxed region. Scale bars, 20 μm. (c) Quantitative real-time PCR analyses of Nqo1, Gstm1 and Cy2a5 in mouse livers. Total RNAs were prepared from livers of the indicated genotypes at 28 days after injection of poly(I)•poly(C). Values were normalized to the amount of mRNA in the Atg7F/F liver. Data are means ± s.d. for three experiments. (d) Liver weight. The weights of the mouse livers of the different genotypes shown at 28 days after injection of poly(I)•poly(C) were measured. Data are means ± s.d. for five mice from each group. Three asterisks, P 0.001 (Student's t-test). (e) Histological analysis of the mouse liver of the indicated genotypes. At 28 days after injection of poly(I)•poly(C), the livers were processed for haematoxylin/eosin staining. Higher-magnification views are shown in the bottom panels. CV, central vein; P, portal vein. Scale bars, 100 μm. (f) Liver function tests of the mice used in d. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were measured. (IU/L, international unit per liter). Data are means ± s.d. for seven mice from each group. Asterisk, P 0.05; three asterisks, P 0.001.
The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1 (2010)
figf6

(a) Immunoblotting of Keap1-deficient (Keap1F/F:Alb) and Keap1 Atg7-deficient (Keap1F/F:Atg7F/F:Alb) livers. Liver homogenates from 8-week-old mice of these genotypes were separated into detergent-soluble and detergent-insoluble fractions. Total, soluble and insoluble fractions were subjected to SDS–PAGE and analysed by immunoblotting with the indicated antibodies (top section). Total lysates were subjected to SDS–PAGE and analysed by immunoblotting with antibodies against Nqo1 and actin (bottom section). Data were obtained from three independent experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11. (b) Quantitative real-time PCR analyses of Nqo1, Mrp4, p62 and Ho-1 in mouse livers. Total RNAs were prepared from livers of 9-week-old indicated genotypes. Values were normalized to the amount of mRNA in the Atg7F/F liver (control). Data are means ± s.d. for three experiments. (c) Kaplan–Meier curves of survival of Atg7F/F:Alb and Keap1F/F:Atg7F/F:Alb mice. The survival analysis of control (n = 33), Keap1F/F:Alb (n = 15), Atg7F/F:Alb (n = 12) and Keap1F/F:Atg7F/F:Alb mice (n = 14) was based on a 16-week follow-up period. (d) Liver weight relative to body weight was measured for the different genotypes. Data are means ± s.d. for Atg7F/F (control) (n = 24), Keap1F/F:Alb (n = 13), Atg7F/F:Alb (n = 9) and Keap1F/F:Atg7F/F:Alb mice (n = 7). Two asterisks, P 0.01 (Student's t-test). (e) Histological analysis of mouse liver of the indicated genotypes. The livers from 9-week-old mice were processed for haematoxylin/eosin staining. Scale bar, 50 μm (left bottom panel); 20 μm (other panels). (f) Liver function tests of the mice used in d. The serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. Data are means ± s.d. for Atg7F/F (control) (n = 24), Keap1F/F:Alb (n = 13), Atg7F/F:Alb (n = 9) and Keap1F/F:Atg7F/F:Alb mice (n = 7). Two asterisks, P 0.01. (g) Immunohistochemical analysis of cellular localization of p62 and Keap1. Paraffin sections of the liver from 9-week-old mice of the indicated genotypes were immunolabelled with anti-p62 (left) and anti-Keap1 (right) antibodies. Scale bars, 20 μm.