Journal: BMC Cancer

Article Title: Intratumoral delivery of IL-18 naked DNA induces T-cell activation and Th1 response in a mouse hepatic cancer model

doi: 10.1186/1471-2407-7-87

Figure Lengend Snippet: Immune response is enhanced by a single injection of naked IL-18 plasmid DNA . Each group (consisting of 4 mice) was administered an intratumoral injection of pCEP4 or naked mIL-18 DNA. Spleens of CT26 tumor-bearing mice treated with plasmids were harvested at 7 days after plasmid injection, and cells analyzed by flow cytometry using the corresponding FITC- or PE- or PE-Cy5-labeled antibodies and isotype control.

Article Snippet: The 11 kb mIL-18 DNA expression plasmid vector, pCEP4-mIL18, was constructed using a CMV early enhancer/promoter/EBNA-1-based pCEP4 plasmid vector (Invitrogen, San Diego, CA) with an ampicillin selection gene.

Techniques: Injection, Plasmid Preparation, Flow Cytometry, Labeling

Journal: BMC Cancer

Article Title: Intratumoral delivery of IL-18 naked DNA induces T-cell activation and Th1 response in a mouse hepatic cancer model

doi: 10.1186/1471-2407-7-87

Figure Lengend Snippet: IL-18 gene transfer results in regression of CT26 liver tumors . CT26 cells (1.0 × 10 5 ) were injected into a left lobe of the livers of 6-8-week-old BALB/c mice. pCEP4 or mIL-18 plasmid DNA was injected directly into the tumors on day 7. Tumor growth was measured on days 1, 4, 7, and 14 after plasmid implantation. Data are presented as the mean of tumor weights of 8 mice/group.

Article Snippet: The 11 kb mIL-18 DNA expression plasmid vector, pCEP4-mIL18, was constructed using a CMV early enhancer/promoter/EBNA-1-based pCEP4 plasmid vector (Invitrogen, San Diego, CA) with an ampicillin selection gene.

Techniques: Injection, Plasmid Preparation

Journal: BMC Cancer

Article Title: Intratumoral delivery of IL-18 naked DNA induces T-cell activation and Th1 response in a mouse hepatic cancer model

doi: 10.1186/1471-2407-7-87

Figure Lengend Snippet: Intracellular IFN-γ production by CT26-specific T cells from pmIL-18-treated mice . Splenocytes from CT26-tumor bearing mice treated with three rounds of pCEP4 or mIL-18 were pooled and incubated in medium alone or that containing CT26 lysates. Numbers within the gates in the FACS plots depict the percentage of CD69 + IFN-γ + CD4 + cells. Data are presented from experiments that were repeated at least twice.

Article Snippet: The 11 kb mIL-18 DNA expression plasmid vector, pCEP4-mIL18, was constructed using a CMV early enhancer/promoter/EBNA-1-based pCEP4 plasmid vector (Invitrogen, San Diego, CA) with an ampicillin selection gene.

Techniques: Incubation

Journal: Nature Communications

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1038/s41467-021-23372-w

Figure Lengend Snippet: Left: Super‐resolution PALM intensity images of β1-WT-mEos2 ( a ), β1-Y795A-mEos2 ( b ), β3-WT-mEos2 ( c ), β3-Y759A-mEos2 ( d ) in MEFs obtained from sptPALM sequences (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labeling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labeled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). e Distributions of the diffusion coefficient D computed from the trajectories of β1-WT-mEos2 (black), β1-Y795A-mEos2 (pink), β1-Y783A-mEos2 (orange) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The gray area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. f Same as e , but with β3-WT-mEos2 (black), β3-Y759A-mEos2 (pink), β3-Y747A-mEos2 (orange). g Fraction of β1-WT and mutants undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right) with or without manganese activation (Mn 2+ ). Values represent the average of the fractions obtained from different cells (error bars: SEM). h Same as g , but for β3-WT and mutants. Super-resolution PALM intensity image of β1-WT ( i ) and β1-Y795A ( j ) density in a FA (inset: labeled with GFP–paxillin) obtained from a sptPALM sequence. k Kymographs generated from a sptPALM time-lapse image along the length of a FA as shown by the dashed lines encompassing the FA in i and j . l Box plots displaying the median (notch) and mean (square) ± percentile (box: 25–75%, whiskers: 10–90%) of immobilization time inside FAs of β1-WT and mutants (left) and of β3-WT and mutants (right). a – h : results for β1-WT-mEos2 without (17 cells) and with Mn 2+ (15 cells), β1-Y795A-mEos2 without (22 cells) and with Mn 2+ (22 cells), β1-Y783A-mEos2 without (16 cells) and with Mn 2+ (20 cells), β3-mEos2 without (16 cells) and with Mn 2+ (13 cells), β3-Y759A-mEos2 without (20 cells) and with Mn 2+ (11 cells, 2 ind. exp.) and β3-Y747A-mEos2 without (16 cells) and with Mn 2+ (10 cells, 2 ind. exp.) correspond to pooled data from three independent experiments unless indicated. For (l), 6 (β1-Y795A, β1-Y783A, β3-WT), 5 (β3-Y759A, β3-Y747A), or 3 cells (β1-WT) corresponding to pooled data from three independent experiments were analysed. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions without Mn 2+ were compared to the corresponding β-integrin-WT condition; with Mn 2+ , each given condition was compared with the value obtained without Mn 2+ . Otherwise, a black line indicates which conditions were compared. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Beta1-integrin-Y795A-mEos2 construct (human, vector: pFB) was kindly provided by C. Albiges-Rizo (IAB, U. Grenoble Alpes). mEos2-paxillin construct (chicken, promotor: CMV, vector: pmEos2) was obtained from Addgene (Plasmid #57409).

Techniques: Labeling, Diffusion-based Assay, Activation Assay, Sequencing, Generated, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1038/s41467-021-23372-w

Figure Lengend Snippet: Left: Super‐resolution PALM intensity images of mEos2‐kindlin-2 ( a ) and talin-1-tdEos ( b ) in MEFs obtained from a sptPALM sequence (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labeling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labeled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). c Distributions of the diffusion coefficient D computed from the trajectories of talin-1-tdEos (yellow) and mEos2‐kindlin-2 (blue) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The gray area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. d Fraction of mEos2‐kindlin-2 (blue) and talin-1-tdEos (yellow) undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right). Values represent the average of the fractions obtained from different cells (error bars: SEM). e Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of diffusion coefficients corresponding to the free diffusion trajectories inside (left) and outside FAs (right). Super-resolution PALM intensity image of mEos2-kindlin-2 ( f ) and talin-1-tdEos ( g ) density in a FA (inset: labeled with GFP–paxillin) obtained from a sptPALM sequence. h Kymographs generated from a sptPALM time-lapse image along the length of a FA as shown by the dashed lines encompassing the FA in f and g . i Box plots displaying the median (notch) and mean (square) ± percentile (box: 25–75%, whiskers: 10–90%) of immobilization time inside FAs for kindlin-2 and talin-1. j Distribution of rearward speed (mean ± s.d. for cells) for kindlin-2 and talin-1. k Fraction of rearward speed equal or superior to 2 nm.s −1 (mean ± s.d. for cells) for kindlin-2 and talin-1. Corresponding data points are overlaid on the bar chart as dot plots. Results for mEos2‐kindlin-2 ( a – e : 13 cells; f – k : 1127 immobilization events in 12 cells) correspond to pooled data from three independent experiments. Results for β1-WT-mEos2 (dark gray) and β3-WT-mEos2 (light gray) corresponds to data shown in Fig. and Supplementary Fig. . Results for talin-1-tdEos ( a – e : 8 cells; f – k : 584 immobilization events in 7 cells) correspond to pooled data from two independent experiments. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions were compared to the corresponding mEos2‐kindlin-2 condition Otherwise, a black line indicates which conditions were compared. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Beta1-integrin-Y795A-mEos2 construct (human, vector: pFB) was kindly provided by C. Albiges-Rizo (IAB, U. Grenoble Alpes). mEos2-paxillin construct (chicken, promotor: CMV, vector: pmEos2) was obtained from Addgene (Plasmid #57409).

Techniques: Sequencing, Labeling, Diffusion-based Assay, Generated, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1038/s41467-021-23372-w

Figure Lengend Snippet: Left: Super‐resolution PALM intensity images of mEos2-kindlin-2-WT in Mn 2+ -stimulated MEFs ( a ) and mEos2-kindlin-2-QW614/615AA in MEFs ( b ) obtained from a sptPALM sequence (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labeling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labeled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). c Distributions of the diffusion coefficient D computed from the trajectories of mEos2-kindlin-2-WT (blue), mEos2-kindlin-2-WT in Mn 2+ -stimulated MEFs (black), mEos2-kindlin-2-QW614/615AA (red) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The gray area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. d Fraction of proteins undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right) for mEos2-kindlin-2-WT, mEos2-kindlin-2-WT in Mn 2+ -stimulated MEFs, mEos2-kindlin-2-QW614/615AA (same color-code). Values represent the average of the fractions obtained from several cells (error bars: SEM). e Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of diffusion coefficients corresponding to the free diffusion trajectories inside (left) and outside FAs (right). Results for mEos2-kindlin-2-WT (17 cells) and mEos2-kindlin-2- QW614/615AA (33 cells) correspond to pooled data from five independent experiments. Results for mEos2-kindlin-2-WT + integrin activation by Mn 2+ (17 cells) correspond to pooled data from four independent experiments. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions were compared to the corresponding mEos2-kindlin-2-WT condition. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Beta1-integrin-Y795A-mEos2 construct (human, vector: pFB) was kindly provided by C. Albiges-Rizo (IAB, U. Grenoble Alpes). mEos2-paxillin construct (chicken, promotor: CMV, vector: pmEos2) was obtained from Addgene (Plasmid #57409).

Techniques: Sequencing, Labeling, Diffusion-based Assay, Activation Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1038/s41467-021-23372-w

Figure Lengend Snippet: Left: Super‐resolution PALM intensity images of mEos2‐kindlin-2-ΔPH ( a ) and mEos2‐PH-domain of kindlin-2-WT ( b ) in MEFs obtained from a sptPALM sequence (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labeling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labeled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). c Distributions of the diffusion coefficient D computed from the trajectories of mEos2‐kindlin-2-WT (blue), mEos2‐kindlin-2-ΔPH (orange) and mEos2‐PH-domain of kindlin-2-WT (black) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The gray area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. d Fraction of proteins undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right) for mEos2‐kindlin-2-WT, mEos2‐kindlin-2-ΔPH, and mEos2‐PH-domain of kindlin-2-WT (same color-code). Values represent the average of the fractions obtained from different cells (error bars: SEM). e Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of diffusion coefficients corresponding to the free diffusion trajectories inside (left) and outside FAs (right). Results of sptPALM (in a – e ) for mEos2‐kindlin-2-WT (15 cells), mEos2‐kindlin-2-ΔPH (28 cells) and mEos2‐PH-domain of kindlin-2-WT (18 cells) correspond to pooled data from three independent experiments. f Fraction of proteins recruited at the membrane defined as the ratio of the membrane-level fluorescence signal (TIRF) to the total fluorescence signal of the cell (epifluorescence). Results of membrane fraction (in f ) for mEos2‐kindlin-2-WT (28 cells), mEos2‐kindlin-2-ΔPH (27 cells) correspond to pooled data from two independent experiments, whereas results for β3-integrin-mEos2 (17 cells), cytosolic mEos2 (13 cells), mEos2-kindlin-2-QW (15 cells), mEos2-kindlin-2-K390A (15 cells) and mEos2-kindlin-2-L357A (15 cells) correspond to one experiment. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions were compared to the corresponding mEos2‐kindlin-2-WT condition. Otherwise, a black line indicates which conditions were compared. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Beta1-integrin-Y795A-mEos2 construct (human, vector: pFB) was kindly provided by C. Albiges-Rizo (IAB, U. Grenoble Alpes). mEos2-paxillin construct (chicken, promotor: CMV, vector: pmEos2) was obtained from Addgene (Plasmid #57409).

Techniques: Sequencing, Labeling, Diffusion-based Assay, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1038/s41467-021-23372-w

Figure Lengend Snippet: Top panel: Epifluorescence images of RFP-paxillin showing the localization of FAs. Bottom panel: 3D super-resolution images by DONALD displaying the axial localization of kindlin-2-WT ( a ), kindlin-2-QW ( b ), kindlin-2-ΔPH ( c ), paxillin ( d ) and kindlin-2-ΔPH-CAAX ( e ); scale bar, 3 µm. For each pixel, the average axial localization of detected single molecules is color-coded. f Distributions of the single molecule axial localizations of kindlin-2-WT (blue) and paxillin (brown) inside (top) and outside FAs (bottom). Values represent the average of the distributions obtained from different cells. Positions of occurrence maxima (z peak ) were estimated by Gaussian fitting. g Same as f , but with kindlin-2-WT (blue), kindlin-2-QW (red), kindlin-2-ΔPH (orange) and kindlin-2-ΔPH-CAAX (green). h Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of single molecule localizations inside (top) and outside FAs (bottom). Micrographs ( a – h ) correspond to a single experiment, but are representative of kindlin-2-WT: 6 cells, kindlin-2-QW: 9 cells, kindlin-2-ΔPH: 7 cells, paxillin: 6 cells, kindlin-2-ΔPH-CAAX: 7 cells. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. The different conditions were compared to the mEos2‐kindlin-2-WT condition. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Beta1-integrin-Y795A-mEos2 construct (human, vector: pFB) was kindly provided by C. Albiges-Rizo (IAB, U. Grenoble Alpes). mEos2-paxillin construct (chicken, promotor: CMV, vector: pmEos2) was obtained from Addgene (Plasmid #57409).

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1038/s41467-021-23372-w

Figure Lengend Snippet: Top panel: TIRF images of photoconverted mEos2 coupled to kindlin-2-WT ( a ), kindlin-2-QW ( b ), kindlin-2-ΔPH ( c ), kindlin-2-ΔPH-CAAX ( d ), or photoconverted mEos2 alone ( e ). Bottom panel: TIRF images of GFP-paxillin showing the localization of FAs. Scale bar, 2 µm. f Enrichment inside FAs defined as the ratio of the average fluorescence intensity inside FAs to average fluorescence intensity outside FAs. Black bars represent medians and interquartile ranges. Displayed results correspond to pooled data from three independent experiments (kindlin-2-WT: 36 cells, kindlin-2-QW: 24 cells, kindlin-2-ΔPH: 27 cells, kindlin-2-ΔPH-CAAX: 50 cells, kindlin-2-L357A: 34 cells, kindlin-2-K390A: 30 cells, mEos2: 38 cells). Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. The different conditions were compared to the mEos2‐kindlin-2-WT condition. Otherwise, a black line indicates which conditions were compared. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Beta1-integrin-Y795A-mEos2 construct (human, vector: pFB) was kindly provided by C. Albiges-Rizo (IAB, U. Grenoble Alpes). mEos2-paxillin construct (chicken, promotor: CMV, vector: pmEos2) was obtained from Addgene (Plasmid #57409).

Techniques: Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1038/s41467-021-23372-w

Figure Lengend Snippet: a Phase contrast images showing cell spreading of Kind Ko (kindlin-1, kindlin-2 knock-out) cells after 4 h on fibronectin. Two days before the experiment, the cells were transfected to induce the expression of GFP-paxillin (leftmost), or GFP-paxillin along with mEos2-kindlin-2-WT (middle left), mEos2-kindlin-2-ΔPH (middle right), or mEos2-kindlin-2-ΔPH-CAAX (rightmost). Images representative of 3 independent experiments. b Top: Phase contrast images showing the morphological features of cells classified as non-spread, partially spread, or spread. Bottom: Relative fraction of non-spread, partially spread, and spread Kind Ko cells 2 days after re-expression of kindlin-2-WT or mutated variants and 4 h after seeding on fibronectin. Values represent the average of fractions from 3 independent experiments (error bars: SEM). Results for GFP-paxillin (141 cells), GFP-paxillin + mEos2-kindlin-2-WT (312 cells), GFP-paxillin + mEos2-kindlin-2-ΔPH (311 cells), GFP-paxillin + mEos2-kindlin-2-ΔPH-CAAX (309 cells), GFP-paxillin + mEos2-kindlin-2-QW614/615AA (284 cells), GFP-paxillin + mEos2-kindlin-2-WT-CAAX (289 cells) correspond to pooled data from three independent experiments. Between 13 and 41 fields of view (20x objective) per condition and per experiment were used to quantify cell spreading, except for the cells expressing GFP-paxillin alone (negative control: between 8 and 17 fields per experience). Between 84 and 107 cells per condition and per experiment were included (except for the GFP-paxillin condition: between 31 and 58 cells per experiment). c TIRF images of GFP-paxillin showing adhesion sites of Kind Ko cells 2 days after re-expression of mEos2-kindlin-2-WT (top) and mEos2-kindlin-2-ΔPH-CAAX (bottom) and 4 h after seeding on fibronectin. For each image, the upper-right panel shows the outlined region at higher magnification. Quantification of number of adhesions per cell ( d ) and total cell area ( e ) of Kind Ko cells (4 h after seeding on fibronectin) re-expressing for 2 days mEos2-kindlin-2-WT (light blue), mEos2-kindlin-2-ΔPH (orange), mEos2-kindlin-2-ΔPH-CAAX (green), mEos2-kindlin-2-QW614/615AA (red) or mEos2-kindlin-2-WT-CAAX (dark blue). Each point in the distribution represents the value obtained from a single cell. FAs were drawn manually and cell boundaries were determined by manually setting a threshold on the pixel intensity values using the TIRF GFP-paxillin images as shown in (c). Black bars represent medians and interquartile ranges. GFP-paxillin + mEos2-kindlin-2-WT: 84 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH: 74 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH-CAAX: 62 cells; GFP-paxillin + mEos2-kindlin-2-QW614/615AA: 30 cells; GFP-paxillin + mEos2-kindlin-2-WT-CAAX: 63 cells. The results correspond to pooled data from three independent experiments with at least 8 cells per experiment. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file. f Schematic model of integrin activation by kindlin and talin in mature FAs. Integrin and kindlin enter independently in FAs by membrane free diffusion whereas talin and paxillin reach FAs by a cytosolic free diffusion. In a FA, integrins display cycles of free-diffusion and immobilizations triggered by binding to kindlin and talin. The probability of freely diffusing integrin, kindlin and talin to meet simultaneously with the correct orientation is extremely low. Our results favor a model where the encounter of freely diffusing integrin and kindlin at the plasma membrane triggers the formation of an immobile integrin-kindlin complex (i). This complex could constrain the integrin β-tail orientation and favor the binding of talin to the proximal NPxY motif, leading to the formation of a transient tripartite integrin-kindlin-talin complex (ii). Then, kindlin could intermittently dissociate (iii) and re-associate (ii) with the longer-lived integrin-talin complex. The role of transient tripartite integrin-kindlin-talin complex, could extend the duration of integrin/talin interactions. Dissociation of both talin and kindlin will end this cycle of integrin immobilization before its next encounter with kindlin.

Article Snippet: Beta1-integrin-Y795A-mEos2 construct (human, vector: pFB) was kindly provided by C. Albiges-Rizo (IAB, U. Grenoble Alpes). mEos2-paxillin construct (chicken, promotor: CMV, vector: pmEos2) was obtained from Addgene (Plasmid #57409).

Techniques: Knock-Out, Transfection, Expressing, Negative Control, Two Tailed Test, MANN-WHITNEY, Activation Assay, Diffusion-based Assay, Binding Assay

Journal: Communications Biology

Article Title: Architectural basis for cylindrical self-assembly governing Plk4-mediated centriole duplication in human cells

doi: 10.1038/s42003-023-05067-8

Figure Lengend Snippet: a , b 3D-SIM analyses for U2OS cells stably expressing the indicated Cep63 or Cep152 constructs after depleting respective endogenous Cep63 or Cep152 by RNAi. a Representative images displaying the PCM-localized mCherry-Cep63-mGFP or mGFP-Cep152-mCherry signals are shown with two surface-rendered models (top and side views). Original fluorescence images are provided in Supplementary Fig. . Boxes, areas of enlargement; double arrows, the diameters and heights measured for quantification. b Quantification of the mCherry and mGFP fluorescent signals in ( a ) to determine the peak-to-peak diameters (top) and heights (bottom) for the cylindrically localized mCherry-Cep63-mGFP (total n = 53 and n = 48, respectively) and mGFP-Cep152-mCherry (total n = 49 and n = 48, respectively) obtained from three independent experiments. Error bars, mean of n ± s.d. *P < 0.05, ****P < 0.0001 (unpaired two-tailed t test). Detailed methods employed for quantification are described in Supplementary Fig. . c (left) A schematic showing the structures of Cep63 and Cep152 with various lengths of CCs (round bars) predicted by the COILS server . c (right) The organization of Cep63 and Cep152 around a centriole, illustrated based on prior observations , , , , , . Numbers (red and green) indicate the diameters from ( b ).

Article Snippet: The pShuttle-CMV vector-based constructs expressing mGFP-Cep152-mCherry (pKM7710), mGFP-Cep152∆(1273–1295) (i.e., ∆23)-mCherry (pKM7737), and mGFP-Cep152 (I1279D, L1286D) (i.e., 2D)-mCherry (pKM7755) were generated by inserting corresponding BglII-NotI fragments into the pShuttle-CMV vector (Addgene) digested with the same enzymes.

Techniques: Stable Transfection, Expressing, Construct, Fluorescence, Two Tailed Test

Journal: Communications Biology

Article Title: Architectural basis for cylindrical self-assembly governing Plk4-mediated centriole duplication in human cells

doi: 10.1038/s42003-023-05067-8

Figure Lengend Snippet: a A schematic and the SAXS envelope for the heterotetrameric Cep63 (424–541)•Cep152 (1205–1295) complex are shown with the embedded crystal structure of Cep63 (502–541)•Cep152 (1205–1250) (PDB: 6CSU; red and blue helices). Averaged χ 2 (the difference between actual and expected data) and normalized spatial discrepancy (NSD) values calculated from 32 independent DAMMIN reconstructions and Rc (cross-sectional radius) calculated from low q Guinier fit are shown. Various physical parameters of the complex calculated from its respective SAXS curve are provided (table, right). a,b,c,d , MWs determined from the SAXS data using four different methods (see Methods for details). Raw data are provided in Supplementary Fig. . A 3D-rendered envelope is provided as Supplementary Movie . b SEC of Cep63 (424–541)•Cep152 (1205–1295) performed at the indicated pH. Black arrow, the heterotetrameric complex; blue arrows, higher-MW complexes. c Sedimentation velocity c(s) profiles for the Cep63 (424–541)•Cep152 (1205–1295) complex under the indicated conditions. Colored arrows, faster-sedimenting, higher-MW species detected under the respective concentrations. Samples were analyzed in 3 mm pathlength cells. d Sedimentation equilibrium absorbance data collected for the same complex in ( c ) at 7,000 (blue), 11,000 (red), and 20,000 (green) rpm at the loading concentrations indicated. Data collected at pH 5.5 were analyzed globally in terms of a tetramer–octamer–hexadecamer reversible self-association model. For clarity, only every third experimental data point is shown. Best fits are represented by a solid line through the experimental points, and the combined residuals are shown above the plots. The table (bottom) shows concentrations for the tetramer, octamer, and hexadecamer (in tetramer units) calculated based on the best-fit reversible self-association model. e Histograms showing the particle distribution of the same complex in ( c ) as a function of molecular weight. The y -axis (Incidence) denotes the number of particles. The yellow histogram with the red dotted line was generated by reanalyzing the data after discarding a third of the particles, as described previously .

Article Snippet: The pShuttle-CMV vector-based constructs expressing mGFP-Cep152-mCherry (pKM7710), mGFP-Cep152∆(1273–1295) (i.e., ∆23)-mCherry (pKM7737), and mGFP-Cep152 (I1279D, L1286D) (i.e., 2D)-mCherry (pKM7755) were generated by inserting corresponding BglII-NotI fragments into the pShuttle-CMV vector (Addgene) digested with the same enzymes.

Techniques: Sedimentation, Molecular Weight, Generated

Journal: Communications Biology

Article Title: Architectural basis for cylindrical self-assembly governing Plk4-mediated centriole duplication in human cells

doi: 10.1038/s42003-023-05067-8

Figure Lengend Snippet: a , b SEC elution and circular dichroism profiles of the Cep63 (424–541)•Cep152 (1205–1295), Cep63 (424–541)•Cep152 (1205–1272) truncation, and Cep63 (424–541)•Cep152 (1205–1295) 2D complexes. c , d Sedimentation velocity c(s) profiles for Cep63 (424–541)•Cep152 (1205–1272) ( c ) and Cep63 (424–541)•Cep152 (1205–1295) 2D ( d ) mutants at various loading concentrations. Samples were analyzed in 3 or 12 mm pathlength cells as indicated. Arrows in ( c ), higher-MW complexes and/or aggregates observed at high concentrations. e Schematics and the ab-initio-reconstructed envelopes of the Cep63 (424–541)•Cep152 (1205–1272) truncation (left, green) and the Cep63 (424–541)•Cep152 (1205–1295) 2D mutants (right, green). The envelope of the Cep63 (424–541)•Cep152 (1205–1295) complex (magenta) and the crystal structure (PDB: 6CSU; red and blue helices) of the Cep63 (502–541)•Cep152 (1205–1250) heterotetramer docked in position are overlaid for morphological comparisons. Averaged χ 2 and NSD values calculated from 32 independent DAMMIN reconstructions, along with the Rc calculated from the SAXS data, are shown. Calculated physical parameters from the respective SAXS data of the complexes are given (table). a,b,c,d , MWs calculated from the SAXS data using four different methods (see Methods for details). Note that due to the aggregative nature of the Cep63 (424–541)•Cep152 (1205–1272) truncation mutant, the SAXS-estimated MWs are somewhat overestimated. 3D-rendered envelopes are provided as Supplementary Movie .

Article Snippet: The pShuttle-CMV vector-based constructs expressing mGFP-Cep152-mCherry (pKM7710), mGFP-Cep152∆(1273–1295) (i.e., ∆23)-mCherry (pKM7737), and mGFP-Cep152 (I1279D, L1286D) (i.e., 2D)-mCherry (pKM7755) were generated by inserting corresponding BglII-NotI fragments into the pShuttle-CMV vector (Addgene) digested with the same enzymes.

Techniques: Circular Dichroism, Sedimentation, Mutagenesis

Journal: Communications Biology

Article Title: Architectural basis for cylindrical self-assembly governing Plk4-mediated centriole duplication in human cells

doi: 10.1038/s42003-023-05067-8

Figure Lengend Snippet: a Surface topographic rendering of the His-Cep63 (424–541)•Cep152 (1205–1295) (left) and His-Cep63 (424–541)•Cep152 (1205–1295) 2D (right) complexes on APS-mica surface. b Quantification of the dimension of the two complexes in ( a ) from two independent experiments [for His-Cep63 (424–541)•Cep152 (1205–1295): average length = 14.74 ± 1.25 nm and average height = 1.58 ± 0.18 nm (average volume = 123.32 ± 17.47 nm 3 ); for His-Cep63 (424–541)•Cep152 (1205–1295) 2D: average length = 18.35 ± 1.73 nm and average height = 2.16 ± 0.21 nm (average volume = 280.01 ± 45.38 nm 3 ]. Bars, mean of n ± s.d. ****P < 0.0001 (unpaired two-tailed t test). No t e that the lateral dimensions of the particles are tip-broadened, while their height is a precise value. Examples of unprocessed “raw” images are provided in Supplementary Fig. .

Article Snippet: The pShuttle-CMV vector-based constructs expressing mGFP-Cep152-mCherry (pKM7710), mGFP-Cep152∆(1273–1295) (i.e., ∆23)-mCherry (pKM7737), and mGFP-Cep152 (I1279D, L1286D) (i.e., 2D)-mCherry (pKM7755) were generated by inserting corresponding BglII-NotI fragments into the pShuttle-CMV vector (Addgene) digested with the same enzymes.

Techniques: Two Tailed Test

Journal: Communications Biology

Article Title: Architectural basis for cylindrical self-assembly governing Plk4-mediated centriole duplication in human cells

doi: 10.1038/s42003-023-05067-8

Figure Lengend Snippet: a Cylindrical self-assemblies were generated by placing 10 µL of Cep63 (424–541)•Cep152 (1205–1295) (left), Cep63 (424–541)•Cep152 (1205–1272) lacking the 23 basic CC residues (∆23) (middle), and Cep63 (424–541)•Cep152 (1205–1295) (2D) (right) (5 µM) on a poly- l -lysine-coated coverslip. The assemblies on the coverslip were reacted with FITC, washed, and subjected to 3D-SIM as described in the Methods. Boxes, areas of enlargement; arrows, incomplete cylindrical assemblies. b The total number of cylindrical self-assemblies present in the surface area of 1 mm 2 was estimated from 15 randomly chosen fields (6813 µm 2 /field) obtained from three independent experiments ( n = 5 fields/sample/experiment). ****P < 0.0001 (unpaired two-tailed t test). Bars, mean of n ± s.d. c The percentage of cylindrical assemblies with a complete circumferential wall quantified from ( b ). *P < 0.05, ****P < 0.0001 (unpaired two-tailed t test). Bars, mean of three expriments ± s.d.

Article Snippet: The pShuttle-CMV vector-based constructs expressing mGFP-Cep152-mCherry (pKM7710), mGFP-Cep152∆(1273–1295) (i.e., ∆23)-mCherry (pKM7737), and mGFP-Cep152 (I1279D, L1286D) (i.e., 2D)-mCherry (pKM7755) were generated by inserting corresponding BglII-NotI fragments into the pShuttle-CMV vector (Addgene) digested with the same enzymes.

Techniques: Generated, Two Tailed Test

Journal: Communications Biology

Article Title: Architectural basis for cylindrical self-assembly governing Plk4-mediated centriole duplication in human cells

doi: 10.1038/s42003-023-05067-8

Figure Lengend Snippet: a – f U2OS cells stably expressing endogenous promoter (P endo )-controlled siCep152-insensitive constructs (shown in Supplementary Fig. ) were analyzed after depleting endogenous Cep152. a Confocal microscopy analyses for anti-Cep152-immunostained cells obtained from three independent experiments [per experiment, n ≥ 42 for Vec/siGL (total n = 140); n ≥ 50 for Vec/siCep152 (total n = 150); n ≥ 81 for Cep152/siCep152 (total n = 256); n ≥ 86 for Cep152 (∆23)/siCep152 (total n = 273), n ≥ 108 for Cep152 (2D)/siCep152 (total n = 328)]. b – f 3D-SIM analyses for immunostained pericentriolar Cep152, Plk4, and CP110 signals. b Representative images showing the localization patterns of Cep152 WT and its respective mutant forms. Cep152’s diameters ( c ) and heights ( d ) were determined from the same images obtained from two independent experiments [per experiment, n ≥ 33 for Vec/siGL (total n = 70); n ≥ 27 for Vec/siCep152 (total n = 55); n ≥ 30 for Cep152/siCep152 (total n = 68); n ≥ 31 for Cep152 (Δ23)/siCep152 (total n = 63), n ≥ 30 for Cep152 (2D)/siCep152 (total n = 63)]. Plk4’s ring versus dot state ( e ) was quantified from three independent experiments [per experiment, n ≥ 76 for Vec/siGL (total n = 258); n ≥ 66 for Vec/siCep152 (total n = 215); n ≥ 65 for Cep152/siCep152 (total n = 234); n ≥ 67 for Cep152 (Δ23)/siCep152 (total n = 244), n ≥ 71 for Cep152 (2D)/siCep152 (total n = 235)]. CP110 dot numbers ( f ) were quantified from three independent experiments [per experiment, n ≥ 44 for Vec/siGL (total n = 144); n ≥ 50 for Vec/siCep152 (total n = 150); n ≥ 40 for Cep152/siCep152 (total n = 129); n ≥ 43 for Cep152 (Δ23)/siCep152 (total n = 136), n ≥ 54 for Cep152 (2D)/siCep152 (total n = 163)]. Bars for ( a , c , d ), mean of n ± s.d. Bars for ( e , f ), mean of three experiments ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., not significant (unpaired two-tailed t test). g A summary proposing that a defect in the formation of the Cep63•Cep152 self-assembly (as demonstrated in Fig. 6) results in a reduced level of pericentriolar Cep152 that leads to improper Plk4’s ring-to-dot conversion and Plk4-mediated centriole duplication.

Article Snippet: The pShuttle-CMV vector-based constructs expressing mGFP-Cep152-mCherry (pKM7710), mGFP-Cep152∆(1273–1295) (i.e., ∆23)-mCherry (pKM7737), and mGFP-Cep152 (I1279D, L1286D) (i.e., 2D)-mCherry (pKM7755) were generated by inserting corresponding BglII-NotI fragments into the pShuttle-CMV vector (Addgene) digested with the same enzymes.

Techniques: Stable Transfection, Expressing, Construct, Confocal Microscopy, Mutagenesis, Two Tailed Test

Journal: Communications Biology

Article Title: Architectural basis for cylindrical self-assembly governing Plk4-mediated centriole duplication in human cells

doi: 10.1038/s42003-023-05067-8

Figure Lengend Snippet: a – e 3D MINFLUX data for U2OS cells expressing the indicated RNAi-insensitive mGFP-Cep152 constructs (WT, ∆23, and 2D) and depleted of endogenous Cep152 by RNAi. Raw fluorescence signals acquired with an anti-GFP nanobody fused to a single Alexa Fluor 647 (i.e., one fluorophore per nanobody) were filtered and processed as shown in Supplementary Fig. (see also Methods). Representative images in ( a ) are shown with dotted boundaries [the maximum (excluding outliers; white dotted line) and the 5th – 95th percentiles of the entire minimum – maximum values (yellow dotted lines) of Cep152 WT (see Supplementary Fig. )] for easy comparison. Numbers, the radial width and height of the mGFP-Cep152 WT. Concentrations of mGFP-Cep152 in ( b ) were calculated from Supplementary Fig. . Rendered 3D images (top, generated by binning localizations into 0.5 × 0.5 × 0.5 nm voxels, also see Methods), and all localizations plotted in 3D (bottom; respective movies in Supplementary Movie ) are provided. To reveal mislocalized mGFP-Cep152 signals ( c – e ), the entire min – max range data points shown in Supplementary Fig. were plotted for WT, ∆23, and 2D ( c , d ), and the population outside of the 5th – 95th percentiles of the WT radius was determined ( e ). Note in ( d ) that, unlike Cep152 WT (gray dots), both ∆23 an 2D mutants are spread out over a larger area, yielding a greater mislocalized population. All quantifications in ( a – e ), n = 15 centriole images for each group obtained from three independent experiments. Bars in ( b , e ), mean of n ± s.d. *P < 0.05, ****P < 0.0001 (unpaired two-tailed t test). Vertical lines in ( c , d ), median with 5th – 95th (thin red lines) and 1st – 99th percentiles (thin blue lines). f FRAP analysis for the mGFP-Cep152-mCherry fluorescence localized around a centriole. Images were acquired for 42 minutes at 3-minute intervals. Representative confocal images acquired before and after photobleaching are shown in Supplementary Fig. . Relative signal intensities were quantified from three independent experiments [ n = 18 for Cep152/siCep152 ( n = 6/experiment); n = 17 for Cep152 (Δ23)/siCep152 ( n ≥ 5/experiment); n = 18 for Cep152 (2D)/siCep152 ( n = 6/experiment)]. Bars, mean of n ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (unpaired two-tailed t test). n.s., not significant.

Article Snippet: The pShuttle-CMV vector-based constructs expressing mGFP-Cep152-mCherry (pKM7710), mGFP-Cep152∆(1273–1295) (i.e., ∆23)-mCherry (pKM7737), and mGFP-Cep152 (I1279D, L1286D) (i.e., 2D)-mCherry (pKM7755) were generated by inserting corresponding BglII-NotI fragments into the pShuttle-CMV vector (Addgene) digested with the same enzymes.

Techniques: Expressing, Construct, Fluorescence, Comparison, Generated, Two Tailed Test

Journal: Communications Biology

Article Title: Architectural basis for cylindrical self-assembly governing Plk4-mediated centriole duplication in human cells

doi: 10.1038/s42003-023-05067-8

Figure Lengend Snippet: a In a concentration-dependent manner, the Cep63 (424–541)•Cep152 (1205–1295) heterotetramer generates octameric and hexadecameric complexes, leading to the formation of a cylindrical self-assembly. iSCAMS was used to determine the K d value for forming the heterotetrameric building block, while the K d values for tetramer–octamer and octamer–hexadecamer equilibria were determined from sedimentation equilibrium analyses (Fig. ). Formation of the heterotetrameric building block is presumably almost irreversible (dotted arrow), hardly dissociating its components under various conditions. Cylindrical self-assemblies are very stable . The two Cep152 mutants either lacking the (1205–1295) region (∆23) or containing the I1279D, L1286D mutations (2D) fail to form the heterotetrameric building block and a higher-order self-assembly. b Cep152 WT, which localizes within a defined region of the PCM space, properly recruits and promotes Plk4-dependent centriole duplication and cell proliferation. In contrast, its respective ∆23 and 2D mutants misorganize their pericentriolar platform, displaying a broader distribution and lower density of the Plk4-binding Cep152 N-terminus (see text for details). Consequently, these mutants fail to properly promote Plk4’s ring-to-dot conversion and centriole duplication. This defect could ultimately lead to various human disorders.

Article Snippet: The pShuttle-CMV vector-based constructs expressing mGFP-Cep152-mCherry (pKM7710), mGFP-Cep152∆(1273–1295) (i.e., ∆23)-mCherry (pKM7737), and mGFP-Cep152 (I1279D, L1286D) (i.e., 2D)-mCherry (pKM7755) were generated by inserting corresponding BglII-NotI fragments into the pShuttle-CMV vector (Addgene) digested with the same enzymes.

Techniques: Concentration Assay, Blocking Assay, Sedimentation, Binding Assay