EMBO/soda-nel huggingface dataset:
PRC1-labeled microtubule bundles and kinetochore pairs show one-to-one association in metaphase (2016)
Figure 2

Figure 2. Dynamic properties of PRC1-labeled bundles and kinetochores in vertical spindles confirm their association.

A. Central plane of the spindle in a live HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented vertically with respect to the imaging plane.

B. Examples of trajectories, with respect to the spindle’s center of the mass, of individual PRC1-GFP (green) and corresponding mRFP-CENP-B (magenta) signals from the spindle in A that moved together for at least 200 s. Dots represent starting points of trajectories, t=0 s. Trajectories finish at t=200 s. Grey circle represents the center of mass of the spindle.

C. Pie chart showing the fraction of PRC1-labeled fibers and sister kinetochores that moved together for at least 60 s (blue, n=226 pairs in 5 cells). A small fraction of PRC1-labeled fibers (green, n=32 in 5 cells) and kinetochores (magenta, n=16 in 5 cells) did not move together.

D. Correlation coefficients between trajectories (with respect to the spindle’s center of the mass) of a kinetochore pair and the trajectories of the PRC1-labeled fiber coupled to the kinetochore pair (green), the nearest neighbor PRC1-labeled fiber (blue), the next nearest neighbor (black) and a randomly chosen PRC1-labeled fiber (grey); n, the number of trajectories from 3 cells.

E. Examples of scenarios observed in dynamic interplay of PRC1-labeled bundles (green) and kinetochores (magenta) from the spindle in A. Row 1: PRC1-labeled bundle and kinetochore move together until they separate at 130 s. Row 2: kinetochore is free of any PRC1-labeled bundle until it appears at 130 s. Row 3: kinetochore and PRC1-labeled bundle move together. Row 4: kinetochore moves with PRC1-labeled bundle which merges with neighboring bundle at 169 s. Row 5: two kinetochores move together with a single PRC1-labeled bundle.

Scale bars, 2 µm.
PRC1-labeled microtubule bundles and kinetochore pairs show one-to-one association in metaphase (2016)
Figure 3

Figure 3. Endogenous PRC1 localizes to the metaphase spindle midzone.

A. Top row: MG132 arrested HeLa cells stably expressing tubulin-GFP (green) and immunostained for endogenous PRC1 (AlexaFluor555 shown in magenta) in 5 individual z-images. Enlargements of the boxed region (top: merge, middle: GFP, bottom: AlexaFluor555) below the first image show the bridging fiber determined as the line connecting two ends of the sister k-fibers in green channel and localization of endogenous PRC1 (magenta). Corresponding maximum projections are shown to the right (max z, left: merge, middle: GFP, right: AlexaFluor555).

B. Quantification of tubulin-GFP bridging fibers (green bar) immunostained for endogenous PRC1 (magenta bar).

C. Images show pole-to-pole tracking (white curve) of tubulin signal (top, green), which was used to measure the endogenous PRC1 signal along the same contour (middle, magenta), and the corresponding scheme (bottom) from a HeLa cell expressing tubulin-GFP and immunostained for PRC1. Images of the same spindle without (left) and with the tracked contour (right) are shown. The graph shows normalized pole-to-pole intensity profiles (each intensity profile was scaled so that one pole is at x=0 and the other at x=1) of endogenous PRC1 (grey lines) acquired in tubulin-GFP HeLa cells immunostained for PRC1. Black line shows the mean value.

D. Images show pole-to-pole tracking (white curve) of PRC1-GFP signal (top, merge; middle, GFP; bottom, scheme) from a HeLa cell expressing PRC1-GFP and mRFP-CENP-B. Images of the same spindle without (left) and with the tracked contour (right) are shown. The graph shows normalized pole-to-pole intensity profiles (legend as in C) of PRC1-GFP acquired in HeLa cells expressing PRC1-GFP and mRFP-CENP-B.

E. Example of length measurement of the immunostained PRC1 signal (LPRC1) in HeLa cell expressing tubulin-GFP and immunostained for PRC1.

F. Comparison of the length LPRC1 between PRC1-GFP signal (green, corresponds to the PRC1 fused to GFP from a PRC1-GFP cell line) and PRC1-AlexaFluor555 (magenta, corresponds to the endogenous immunostained PRC1) (p=0.10).

G. Cross section signal intensity of the immunostained endogenous PRC1 (magenta line) from a HeLa cell expressing tubulin-GFP and immunostained for PRC1. Area under the peak is defined as Icross, measured at the position of the blue line as in scheme. Horizontal lines mark the background signal (black line), vertical lines delimit the area (grey) where the signal was measured.

Scale bar, 2 µm; numbers in bars are number of cells; n, number of bridging fibers; error bars, s.e.m.; ns, not significant difference (p≥ 0.05, t-test).
PRC1-labeled microtubule bundles and kinetochore pairs show one-to-one association in metaphase (2016)
Figure 4

Figure 4. PRC1 silencing reduces bridging fiber thickness and interkinetochore distance.

A. Images of HeLa cells stably expressing tubulin-GFP (green) and transiently mRFP-CENP-B (magenta) treated with control siRNA (left images) and siRNA targeting PRC1 (right images). Enlargements of the boxed region, shown to the right of the image of the whole spindle, are focused on kinetochores and bridging fiber (top: merge; middle: GFP, bottom: scheme).

B. Western blot showing PRC1 protein from unlabeled cells, tubulin-GFP HeLa cell line and PRC1-GFP HeLa cell line. Detailed measurements are in Expanded View 4.

C. Mean ratio of signal intensities of the bridging fiber (Ib, measured at the position of a blue line as in scheme) and sum of the bridging and k-fiber (Ibk, measured at the position of the orange line as in scheme), measured in control cells (grey bar) and PRC1 siRNA treated cells (black bar) (p=0.0003).

D. Mean interkinetochore distance (dk, see scheme) measured in control cells (grey bar) and PRC1 siRNA treated cells (black bar) (p=0.0001).

E. Images of individual spindles of HeLa cells expressing tubulin-GFP and immunostained for PRC1 obtained from control cells (top) and PRC1 siRNA treated cells (bottom). All cells were imaged with the same imaging parameters. Only AlexaFluor555 channel is shown.

F. Mean signal intensity I of the immunostained PRC1 in control cells (grey bar) and PRC1 siRNA treated cells (black bar) in HeLa cells expressing tubulin-GFP and immunostained for PRC1 (p=0.0001).

G. Images of HeLa cells stably expressing PRC1-GFP treated with control (left image) and siRNA targeting PRC1 (right image) immunostained for PRC1 (magenta). Graph showing the length of the immunostained PRC1 signal, LPRC1 (p=0.5167), signal intensity, I (p=0.0003) and Icross (p=0.0001) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1-GFP and immunostained for PRC1.

H. Images of HeLa cells stably expressing PRC1-GFP (green) treated with control (left image) and siRNA targeting PRC1 (right image) immunostained for PRC1. Graph showing the length of the PRC1-GFP signal, LPRC1 (p=0.3407), signal intensity, I (p=0.0001), and Icross (p=0.0001) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1-GFP and immunostained for PRC1.

Scale bar, 2 µm; numbers in bars are number of cells; n, number of bridging fibers; error bars, s.e.m, p-value legend: p=0.0001 to 0.001 (***), p=0.001 to 0.01 (**), p=0.01 to 0.05 (*), p≥ 0.05 (ns).
Single-cell transcriptomics uncovers an instructive T-cell receptor role in adult γδ T-cell lineage commitment (2022)
Figure 5

Figure 5. Single-cell RNAseq analysis of DN3 γδ- and DN3 γδint cells from Lat-/- thymus.

A, B UMAP plots of Dataset 3 which contains five WT (DN3a, DN3b γδ+, DN4 γδ+, DN3b γδ- and DN4 γδ-) and two Lat-/- (DN3 γδ- and DN3 γδint) scRNAseq datasets. The UMAP plot is colored according to sorted samples (A), and to expression levels of the specified genes (B).

C Heatmap showing scaled expression of top 25 DEG characterizing each of the seven sorted samples of Dataset 3. The names of the sorted samples are specified at the top of the heatmap, and 3,000 cells were randomly selected for each sample. Each column represents the expression profile of a single cell. The sample from which a DEG is derived is indicated by colored bars on the left. Bar superpositions specify DEG that are found in more than one sample. The genes displayed in panel B are highlighted with a star. Gene expression is color-coded with a scale based on z‐score distribution, from −2 (purple) to 2 (yellow).
An actin remodeling role for Arabidopsis processing bodies revealed by their proximity interactome (2023)
Figure 3

Fig 3 DCP1 protein interfaces with the plasma membrane.

A Diagram of a root showing the three developmental regions (1-3) under examination in this study: stem cell niche (SCN; region 1), meristematic zone (MZ; region 2) and transition zone (TZ)-differentiation zone (DFZ; region 3). The different cell types are color-coded. The region used in TIRF-M experiments is highlighted by the dashed magenta rectangle (region 3, see also B).

B Representative TIRF-M of DCP1-GFP (DCP1pro:DCP1-GFP transgene) in the lateral PM of epidermal cells showing the transient attachment of DCP1-positive puncta to the PM. Yellow arrowheads denote a PB that shows motility at the PM focal plane; blue arrowheads show a PB that transiently localizes to the PM. Scale bars: 2 μm. The corresponding kymographs are shown to the right. Right: distribution of immobile and mobile DCP1 molecules relative to the motility log(D) value of -0.75 (threshold; see Material and methods for details), in NS or HS conditions (D, diffusion coefficient). Inset: individual trajectories of mobile DCP1-GFP in NS and HS conditions (500 frames, n = 120), showing a combination of directional and Brownian motion for both NS/HS. The green arrowheads denote the beginning of the NS and HS tracks for DCP1-GFP.

C Representative confocal micrographs from lines co-expressing RPS5apro:HF-mScarlet-DCP1 and PIN2pro:PIN2-GFP (epidermal cells, region 3, Scale bars: 5 μm). Bottom: polarity index of DCP1 in root meristematic cells (compared to propidium iodide (PI) and tubulin staining of root cells; N, biological replicates = 3 roots, n = 13 cells). Polarity index calculated as the ratio of average of apical and basal VS lateral side of fluorescence signal intensity of the root epidemies cells. The arrowhead in PIN2 indicates the cell plate or PM in DCP1. Right: representative confocal micrographs showing that PM localization is independent of the promoter used (DCP1pro, 35Spro; region 2, epidermal cells, or RPS5apro on the lower right). The details from the inset show increased localization at the cell edge (discussed later). mSc, mScarlet. Scale bars: 7 μm.

Data information: In C, P values were determined by Wilcoxon. Upper and lower lines in the violin plots when visible, represent the first and third quantiles, respectively, horizontal lines mark the median and whiskers mark the highest and lowest values.
An actin remodeling role for Arabidopsis processing bodies revealed by their proximity interactome (2023)
Figure 4

Fig 4 DCP1 protein accumulates at edges and then vertices during development.

A Left: Gradual edge or vertex accumulation of DCP1-GFP in three different root regions. DCP1 signal intensity among the three different regions, at the edge/vertex (epidermis). Right: z cross-sectional images of DCP1-GFP (green) in the whole root compared to FM4-64 staining (magenta, staining membranes). The circular plots indicate the average DCP1 localization (regions 1-3; N, biological replicates = 3, n = 10 cells; comparing regions 1-2 and 2-3). The 3D-rendered images (PIN2-GFP vs. mSCarlet-DCP1) show the localization of mScarlet-DCP1 at edges/vertices in two different regions (in comparison to PIN2-GFP signal which decorated almost evenly the PM). Scale bars: 20 μm. Arrowhead denotes the edge (region 2) and the vertex (region 3) decorated by mScarlet-DCP1 (also in the z cross-sectional image). Note in a single cell file, how the localization from the PM changes to the edge, as indicated, along the proximodistal axis.

B Representative confocal micrograph of DCP1-GFP (DCP1pro:DCP1-GFP, transgene) in root meristematic cells under NS/HS conditions (region 2). Scale bars, 5 μm. Note the depletion of DCP1 from the PM upon HS, but the increased edge/vertex signal (yellow arrowheads denote the vertex signal). Right: DCP1 signal intensity at the PM or edge/vertex (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 18-23 PMs or edges/vertices).

C Representative confocal images showing fusion (coarsening), fission, and growth of PBs (DCP1-positive) at the PM (region 3). Right: states of PBs (dynamic: fusion and fission and non-dynamic: stable; N, biological replicates = 2, n (pooled data of 3 biological replicates) = 6-8 PBs). As a cautionary note, the "stable" PBs may not show dynamicity in the imaging time used (~3-5 min) but later, may do.

Data information: In A, * P<0.05 was determined by a nested t-test. In B, P values were determined by the Kolmogorov-Smirnoff, while in C by one-way ANOVA. Upper and lower lines in the violin plots when visible, represent the first and third quantiles, respectively, horizontal lines mark the median and whiskers mark the highest and lowest values.
An actin remodeling role for Arabidopsis processing bodies revealed by their proximity interactome (2023)
Figure 5

Fig 5 DCP1 interacts with DCP2 in a PLA assay occasionally in the cytoplasm but not at the PM.

Confocal micrographs showing single optical sections PLA-assays producing signal that resembles spots. The antibodies used were anti-FLAG/anti-GFP detecting the HF-mScarlet-DCP1/ DCP2-YFP, respectively (RPS5apro:HF-mScarlet-DCP1 and 35Spro:DCP2-YFP). Inset 1: magnification showing the colocalization of PLA spots with DCP1 or DCP2 signals (colocalization and Pearson's correlation coefficient PCC value for the spots shown). The dotted white line in the PLA channel corresponds to the PM plane. Inset 2: positive PLA signal for DCP1 and DCP2 in the nucleus. "n1-n3" correspond to nuclei regions (green circles). On the right, note the PLA spot nearby the nucleus ("detail of PLA signal"). The chart shows the quantification of PLA spots per cell at puncta (cytoplasm) or on the PM (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 16-33 cells). As a cautionary technical note, the cytoplasmic, nuclear or PM "spots" do not connote physiologically relevant puncta, condensates or PM clusters. Scale bar: 20 μm / 10 μm for the insets.

Data information: In A, * P<0.05 was determined by one-way ANOVA. Upper and lower lines in the violin plots when visible, represent the first and third quantiles, respectively, horizontal lines mark the median and whiskers mark the highest and lowest values.
An actin remodeling role for Arabidopsis processing bodies revealed by their proximity interactome (2023)
Figure 6

Fig 6 DCP1 cooperates with the SCAR-WAVE complex.

A DCP1 colocalizes with the SCAR-WAVE components SCAR2 and BRK1. Representative confocal micrographs showing colocalization between DCP1 and SCAR2 or DCP1 and BRK1 (in lines coexpressing DCP1pro:DCP1-GFP and SCAR2pro:SCAR2-mCherry or RPS5apro:HF-mScarlet-DCP1 and BRK1pro:BRK1-YFP; arrowheads indicate colocalization at cell edges or vertices) in root epidermal cells (regions 2-3). Right: relative signal intensity profiles of DCP1 or SCAR2 at vertices. Colocalization at these regions was also calculated (PCC; N, biological replicates = 2, n = 4-10 edges/vertices in regions 2-3; a slight increase was observed in HS, 0.78 in NS vs. 0.87 in HS). Scale bars: 10 μm.

B Representative confocal micrographs with acceptor photobleaching-FRET efficiency between SCAR2-mCherry (up) or BRK1-mRuby (down) and DCP1-GFP (epidermal cells, regions 1 and 3). Right: normalized FRET efficiency between SCAR2 or BRK1 and DCP1, respectively, among the different developmental root regions (N, biological replicates = 2, n = 16 cells). Scale bars: 3 μm.

C Representative confocal micrographs showing PLA spots produced by α-GFP/α-RFP in DCP1-GFP/SCAR2-mCherry lines and DCP1-GFP/BRK1-mRuby lines, α-FLAG/α-GFP in HF-mScarlet-DCP1/SPI-YPet (SPIRRIG [SPI] is a negative control as it localizes to PBs only during salt stress and was not found in the APEAL). In the SPI PLA, a high contrast inset is presented. Right: number of PLA spots per cell (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 14-33 cells). In the merged images, the cell contours are shown (light green transparent). Scale bars: 5 μm.

D Representative confocal micrographs showing DCP1 localization detected by α-DCP1 in the wild type (WT) or the scar1 scar2 scar3 scar4 (scar1234) quadruple mutant or in live-cell imaging of mScarlet-DCP1 (RPS5apro:HF-mScarlet-DCP1) in WT or brk1 mutant (bottom right; epidermal cells, region 3 for α-DCP1 and 2 for live-cell imaging). The arrowheads denote the lack of robust DCP1 localization in scar1234 at the edge/vertex. Small panels (insets) at right show details corresponding to the regions delineated by dashed lines, where arrowhead denotes the edge signal of DCP1 in WT; scale bars, 1 μm. Bottom: percentage of cells with proper edge/vertex localization and quantification of PB numbers (DCP1-foci; N, biological replicates = 3, n (pooled data of 3 biological replicates) = 18-35 cells). Scale bars, 5 μm.

Data information: In B, P values were determined by Kruskal-Wallis, and the comparisons are among the scar1234 to the corresponding WT samples. In C and D, P values were determined by Wilcoxon. PCCs are means ± s.d.. Upper and lower lines in the violin plots when visible, represent the first and third quantiles, respectively, horizontal lines mark the median and whiskers mark the highest and lowest values.
An actin remodeling role for Arabidopsis processing bodies revealed by their proximity interactome (2023)
Figure 7

Fig 7 DCP1 phosphostatus defines its localization at the edge or vertex where it regulates actin remodeling

A Representative confocal micrographs showing colocalization between DCP1 or two DCP1 phosphovariants with LifeAct-mCherry in lines co-expressing DCP1pro:DCP1-GFP (or variants) and UBQ10pro:LifeAct-mCherry (cell edges are indicated by yellow arrowheads). Right: relative signal intensity of actin at cell edges/vertices (normalized to the PM) in epidermal cells (N, biological replicates = 3, n = 15-30, regions 2-3). Scale bars: 7 μm.

B Representative high-resolution confocal micrographs showing the localization of DCP1-GFP or phosphovariants (regions 2-3, epidermal cells). Left: signal at the vertex, expressed as a color-coded edge/cytoplasmic signal ratio. Right: representative 3D projection from super-resolution (120 nm axial, FM4-64 counterstaining of PM) images of root meristematic cells captured from DCP1pro:DCP1-GFP and phosphovariants (N, biological replicates = 4, n = 8). Circular insets show the differential vertex localization of DCP1 (absent in DCP1S237D-GFP line), and arrowheads denote the vertex. Note the enhanced accumulation of DCP1S237A-GFP at the vertex. Scale bars: 15 μm.

Data information: In A, P values were determined by Kruskal-Wallis, while in B, *P<0.005 by ordinary one-way ANOVA relative to unmutated DCP1. Upper and lower lines in the violin plots when visible, represent the first and third quantiles, respectively, horizontal lines mark the median and whiskers mark the highest and lowest values.
An actin remodeling role for Arabidopsis processing bodies revealed by their proximity interactome (2023)
Figure 8

Fig 8 DCP1 phosphostatus regulates actin remodeling at the edge.

A Representative confocal 3D rendering micrographs of root meristematic cells from the WT or the dcp1-3 mutant stained with phalloidin for actin visualization (N, biological replicates = 3, n = 4). Scale bars: 5 μm (z-scale is 4 μm). Upper middle: the "Spectrum" micrographs indicate the maximum color-coded signal intensity (scale on the right, middle inset). Note that the signal is evenly distributed in region 1 (left upper micrograph), whereas it mostly accumulates at the edge or vertex in regions 2 and 3 (see arrowheads; images on top). Lower middle: a detail of the higher actin accumulation at edges/vertices in region 2 (compare regions 1 and 2, Scale bars: 7 µm). Insets (details, Scale bars: 2 µm) indicate the loss of vertex actin accumulation in dcp1-3. Right: plot profile from the actin signal in the WT or dcp1-3. The vertices are indicated.

B Representative confocal micrographs showing actin localization in WT, dcp1-1, dcp1-3, scar1234, dcp2-1, dcp5 and pat1 upon phalloidin staining and graph (right) indicating the percentage of cells in region 3 with an accumulation of actin at edges in various genotypes (N, biological replicates = 3, n = 7-9 roots, bars show means + s.d.). Scale bars: 7 μm.

Data information: In A, P values were determined by Kruskal-Wallis, while in B, the exact P values were determined by Brown-Forsythe and Welch ANOVA.
An actin remodeling role for Arabidopsis processing bodies revealed by their proximity interactome (2023)
Figure 9

Fig 9 Mutual potentiation of DCP1 and SCAR-WAVE localization at the edge/vertex.

A SE-FRET efficiency between DCP1-GFP or its phosphovariants with SCAR2-mCherry (among the three different root regions; mainly epidermal cells). Scale bar: 50 μm. The arrowhead denotes high FRET efficiency at edges/vertices of region 3. Right: signal quantification of SE-FRET efficiency between the indicated combinations at the epidermis of 3 regions (N, biological replicates = 6, n (pooled data of 3 biological replicates) = 10).

B Actin nucleation site at an edge/vertex, as indicated by DCP1-GFP and LifeAct-mCherry localization. Right: correlation between DCP1 intensity, ACTIN, SCAR2, BRK1 and ARPC5 intensities (simple regression model). The R2 values are shown, along with representative micrographs for DCP1/SCAR2 (N, biological replicates = 3, n = 6 for each point).

C Representative confocal micrographs showing SCAR2-mCherry localization in WT and dcp1-3 mutant, respectively (root region 2, epidermal cells) and quantification of edge/vertex with SCAR2 a confined signal (N, biological replicates = 3, n = 5-8 cells).

D Representative confocal micrographs showing the colocalization between DCP1-GFP or phosphovariants and SCAR2-mCherry in root meristematic cells (root region 2, epidermal cells). Scale bars: 10 μm. The insets show details of colocalization; the white arrowhead denotes the expansion of the SCAR2/DCP1 domain, while the yellow arrowheads the restricted edge/vertex SCAR2/DCP1 domains. Scale bars: 3 μm. The graph indicates the relative signal intensity for the indicated combinations (as Pearson's correlation coefficient; N, biological replicates = 3, n = 5 at edges/vertices: spread edges were not considered in calculations).

Data information: In A and B, *P<0.005 were determined by nested one-way ANOVA relative to the WT in the respective region. In B, a simple linear regression (best-fitted model) with a 95% confidence interval is shown with dashed lines. In C and D, P values were determined by an unpaired t-test. Upper and lower lines in the violin plots when visible, represent the first and third quantiles, respectively, horizontal lines mark the median and whiskers mark the highest and lowest values.