egfp centrin2  (Addgene inc)

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, C2C12 cells treated for 30 min with DMSO (0.1%, vehicle control), Yoda1 (Piezo1 activator, 10 μM) or GsMTx4 (mechanosensitive channel inhibitor, 5 μM) after RO-3066 release. Cells were imaged by IF for α-Tubulin (green), γ-Tubulin (magenta) and DNA (Hoechst dye, blue). b, Quantitative analysis for percentage of C2C12 cells with supernumerary centrosomes from IF images as in (a) following treatment with DMSO (0.1%), Yoda1 (10 μM), Yoda1 plus Dooku1 (10 μM each), Dooku1 (10 μM), GsMTx4 (5 μM), Jedi1 (200 μM), Jedi2 (200 μM), ionomycin (10 μM) or BAPTA-AM (40 μM). c , Phase images of C2C12 cells 30 min after treatment with DMSO, ionomycin, Yoda1, Jedi1 and Jedi2 under the same condition as in (b). Yoda1, Jedi1 or Jedi2 treatment, but not DMSO or ionomycin treatment, led to cell surface protrusions due to plasma membrane Piezo1 activation. d, C2C12 cells stably expressing GFP-Centrin2 treated for 30 min with DMSO, Yoda1, GsMTX4 or DooKu1 after RO-3066 release, showing supernumerary centrosomes and centriole disengagement. Cells were imaged by IF for γ-Tubulin (magenta), Centrin2 (GFP fluorescence) and DNA (Hoechst dye, blue). Rectangular boxes encircle centrosomes, and their zoom-in views are displayed. e, EM gallery of centrosomes, imaged in thin plastic sections of embedded C2C12 cells 1 h after treatment with of DMSO (top) or Yoda1 (bottom). Yellow and magenta circles mark pairs of mother and daughter centrioles, and separated centrioles, respectively. f, g, Live fluorescent and phase images of mitotic C2C12 cells stably expressing GFP-Piezo1 (green) (f) or GFP-Centrin2 (g). Cells were treated with DMSO or Yoda1 together with SiR-Tubulin for staining microtubules (magenta) after release of RO-3306-mediated cell cycle synchronization, and imaged 30 min later. Misaligned spindles following Yoda1 treatment are apparent from SiR-Tubulin staining. Rectangular boxes in (g) encircle centrosomes, and their zoom-in views are displayed. h, Piezo1 and 2 pKO C2C12 cells stably expressing GFP-Centrin2 after day 1 post-KO selection and RO-3306 synchronization. Cells were washed out and visualized by IF for Centrin2 (GFP fluorescence), γ-Tubulin (magenta) and DNA (Hoechst, blue). All images are maximum intensity Z projections. All scale bars are 10 μm except in (e) which is 200 nm. Data are represented by mean ± SEM from three independently quantified experiments counting 100-250 cells each. Statistical significance was assessed between an experimental group and a control group by 2-tailed t-test with ***, ** and * for p < 0.0001, 0.001 and 0.01, respectively.

Article Snippet: The eGFP fused Centrin2 (GFP-Centrin2, Addgene) and Lenti-H2B-mCherry (GeneCopoeia) plasmids were purchased.

Techniques: Control, Clinical Proteomics, Membrane, Activation Assay, Stable Transfection, Expressing, Fluorescence, Staining, Selection

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, Unsynchronized C2C12 cells expressing GFP-Centrin2 (green), fixed 30 minutes after introducing Yoda1 (10 μM) or DMSO (0.1%, vehicle control) and stained for γ-Tubulin (magenta) and DNA (Hoechst, blue). Yoda1 treatment resulted in centriole disengagement. b, Supernumerary centrosomes in RO-3306-synchronized C2C12 cells expressing GFP-Centrin2 (green) fixed 30 minutes after introducing 0.1% DMSO and stained for γ-Tubulin (magenta) and DNA (Hoechst, blue). The low percentage of supernumerary centrosomes in DMSO control was resulted from centriole duplication . c, Analysis of the measured disengagement distance between centrioles upon Piezo1 activation using IF experiments as in (a). d, C2C12 cells expressing GFP-Centrin2 (green), fixed 30 minutes after introducing Yoda1 (10 μM) and stained for γ-Tubulin (magenta), Pericentrin (cyan) and DNA (Hoechst, blue), confirming the centrosomal defect. e, A negative-stain EM image of separated centrioles in a thin plastic section of embedded C2C12 cells 30 min after Yoda1 treatment (left) and its zoom-ins (right). Two centrioles are in a cross-sectional view with the normal 9-fold symmetry and a longitudinal view, respectively, at a disengagement distance of 3.5 μm. f, C2C12 cells expressing GFP-Centrin2 (green), fixed 30 minutes after introducing Yoda1 (10 μM) and stained for γ-Tubulin (magenta), Cep164 (cyan) and DNA (Hoechst, blue). Cep164 marks mother centrioles, and upon Yoda1 addition, the daughter centriole specifically separated from the mitotic spindle, shown in a white circle. g, Fields of view of G2/M synchronized IMCD3 cells 30 min after treatment with DMSO (0.1%), Yoda1 (10 μM) or GsMTx4 (5 μM), imaged by IF for α-Tubulin (green), γ-Tubulin (magenta) and DNA (blue). Synchronization was done using RO-3306 and drugs were added after RO-3306 washout. Cells containing supernumerary centrosomes are marked with white arrows. h, Detailed views of IMCD3 cells treated as in (g) ( left ) and quantitative analysis of cells with supernumerary centrosomes ( right ). i, Quantitative analysis of mitotic Piezo1 and 2 pKO cells at day 1 post-KO selection with supernumerary centrosomes that contain either separated or duplicated centrioles visualized by IF. Among cells with supernumerary centrosomes, significantly more of them contained separated centrioles in Piezo pKO cells compared to Rosa26 control cells. j , Supernumerary centrosomes with duplicated centrioles in GFP-Centrin2 C2C12 cells after 24 h treatment with Yoda1 (10 μM), imaged for GFP-Centrin2 fluorescence (green), γ-Tubulin IF (magenta) and DNA (blue). k, Quantitative analysis of mitotic cells with supernumerary centrosomes that contain either separated or duplicated centrioles upon different Yoda1 treatments, visualized by IF as in (j). The percentage of supernumerary centrosome cells with duplicated centrioles is higher after a 24 h recovery period post the 30 min Yoda1 treatment in comparison with either 30 min or 24 h Yoda1 treatment alone. By contrast, the percentage of supernumerary centrosome cells with separated centrioles is lower after a 24 h recovery period post the 30 min Yoda1 treatment. Scale bars are 10 μm for all IF images, 1 μm for the left panel in (e), and 200 nm for the right zoom-ins in (e). All IF mages are maximum intensity Z projections and data are represented by three independently quantified experiments counting 50-250 cells each. Statistical significance between an experimental group and the control was assessed by two-tailed t-test with ***, ** and * for p < 0.0001, 0.001 and 0.01, respectively.

Article Snippet: The eGFP fused Centrin2 (GFP-Centrin2, Addgene) and Lenti-H2B-mCherry (GeneCopoeia) plasmids were purchased.

Techniques: Expressing, Control, Staining, Activation Assay, Selection, Fluorescence, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a-c, Live time-lapse images from mitotic or interphase C2C12 cells stably expressing GFP-Centrin2 (green) and H2B-mCherry (magenta) treated with Yoda1 (10 μM). Cells were synchronized to G2/M by RO-3306 (a, b) or interphase by double thymidine block (c). Yoda1 was introduced 15 min and 45 min after RO-3306 washout to capture cells at prophase (a) and cytokinesis (b), respectively, and to unsynchronized interphase C2C12 cells at G1 and G2 (c). Phase images are shown for (b) to emphasis the mitotic stage. Distances (μm) between mother and daughter centriole pairs are marked, and lagging chromatin in (a) is labeled by white arrows. Rapid centriole disengagement was observed at prophase (a), cytokinesis (b) and interphase (c). However, cells at metaphase did not exhibit centriole disengagement upon Yoda1 introduction . d, Quantitative analysis of IF images of C2C12 cells for supernumerary centrosomes following treatment with DMSO (0.1%) or Yoda1 (10 μM) at different stages of the cell cycle, captured largely as in (a-c) and in for metaphase cells. All images are maximum intensity Z projections. All scale bars are 10 μm. Data are represented by mean ± SEM from three independently quantified experiments counting 100-250 cells each. Statistical significance between an experimental group and a control group was assessed by 2-tailed t-test with *** and * for p < 0.0001 and 0.01, respectively.

Article Snippet: The eGFP fused Centrin2 (GFP-Centrin2, Addgene) and Lenti-H2B-mCherry (GeneCopoeia) plasmids were purchased.

Techniques: Stable Transfection, Expressing, Blocking Assay, Labeling, Control

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a-d, Movie snapshots of imaged C2C12 cells stably expressing GFP-Centrin2 (green) and H2B-mCherry (magenta) treated with DMSO (0.1%, vehicle control) (a), Yoda1 (10 μM) at metaphase 30 minutes after RO-3306 release (b, top) or after MG132 synchronization (b, bottom), Dooku1 (10 μM) at metaphase or GsMTx4 (5 μM) at both early mitosis and interphase (d). Distances between centrioles are labeled in μm. The DMSO-treated cell contained centrosomes with two centrioles separated by < 0.6 μm. The Yoda1 or Dooku1-treated cell at metaphase did not show separated centrioles. The GsMTx4-treated cells at early mitosis and interphase both exhibited centriole disengagement. e , Representative field views of C2C12 cells treated with DMSO (top row) or Yoda1 (bottom row) for 10 min at 15 min (prophase and prometaphase), 30 min (metaphase) or 45 min (telophase and cytokinesis) after RO-3306 release, after MG132 synchronization (metaphase) or with double thymidine block (interphase). Cells were imaged by IF for α-Tubulin (green), γ-Tubulin (magenta) and DNA (blue). Centriole disengagement was observed in interphase, prophase and prometaphase, and telophase and cytokinesis, but not in metaphase. All images are maximum intensity Z projections and all scale bars are 10 μm.

Article Snippet: The eGFP fused Centrin2 (GFP-Centrin2, Addgene) and Lenti-H2B-mCherry (GeneCopoeia) plasmids were purchased.

Techniques: Stable Transfection, Expressing, Control, Labeling, Blocking Assay

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, Live images of NLS-GCaMP6-C2C12 cells stably expressing an NLS-tagged GCaMP6 Ca 2+ -responsive fluorescence reporter construct. In interphase cells, the NLS-GCaMP6 reporter demonstrates the expected fluorescence in the nucleus (left) . Following nuclear envelope breakdown during early mitosis, concentrated fluorescence at the centrosomes was observed ( middle ). In metaphase, the concentrated fluorescence localized to the mitotic spindles, visualized also by SiR-Tubulin-staining of microtubules (magenta) (right). b, Live images of Piezo1 and 2 pKO NLS-GCaMP6-C2C12 cells compared to an off target Rosa26 pKO control cell. c, Live images of NLS-GCaMP6-C2C12 cells, imaged just prior to Yoda1 addition (left), 5 min after Yoda1 (10 μM, middle) or 5 min after GsMTx4 (5 μM, right) addition. In the left and middle panels, the same cell was imaged before and after Yoda1 activation. d, Left: live cell imaging of an unsynchronized C2C12 cell (at G1) stably expression GCaMP6-γ-Tubulin (GCaMP6-GTU) after addition of DMSO (top) or Yoda1 (bottom). Upon Yoda1 addition, Ca 2+ levels at centrosomes increased by ∼ 3 fold, followed by centriole disengagement. Right: average maximal centrosomal Ca 2+ intensities marked by GCaMP6-GTU (line) as a function of time upon addition of Yoda1 or DMSO. Individual values for the two centrosomes are shown separately as dots. While intensity levels stayed constant with DMSO, they increased with Yoda1 addition and decreased at around the same time when centrioles were separated. e, Caged-Yoda1 design that allows photoactivation. The caged group added to Yoda1 is labeled in green. Upon photo uncaging, a hydroxyl group, also marked in green, is formed on the Yoda1 scaffold, generating hydroxy-Yoda1. f, NLS-GCaMP6 fluorescence intensities upon addition of hydroxy-Yoda1 (10 μM) as a function of time in comparison to Yoda1 (10 μM) and DMSO. Hydroxy-Yoda1 retained ∼50% of the Yoda1 activity. g, Quantitative analysis of percentage of cells with supernumerary centrosomes upon treatment with DMSO or hydroxy-Yoda1 on G2/M synchronized C2C12 cells from IF experiments (h), or upon treatment with caged-Yoda1 followed by light-mediated uncaging on unsynchronized C2C12 cells in comparison with DMSO control from live cell experiments (i). For IF experiments, data are represented by mean ± SEM from three independently quantifications counting 50-200 cells each. The live cell experiments for centriole disengagement by uncaging caged-Yoda1 were performed 36 times with observed 16.7% centriole disengagement. Statistical significance was assessed between an experimental group and the control group by 2-tailed t-test with ***, ** and * for p < 0.0001, 0.001 and 0.01, respectively. h, C2C12 cells stably expressing GFP-Centrin2 treated with hydroxy-Yoda1 after release of RO-3306, imaged by IF after 30 min for Centrin2 (GFP fluorescence), γ-Tubulin (magenta) and DNA (Hoechst dye, blue). Supernumerary centrosomes and centriole disengagement are shown in mitotic and interphase cells. i, An example of centriole disengagement upon uncaging caged-Yoda1 at the centrosome (bottom row) in comparison with controls. C2C12 cells stably expressing GFP-Centrin2 and treated with 10 μM caged-Yoda1 were imaged after application of photolytic light at centrosomes (bottom row), away from centrosomes (middle row) or no application (top row) at T = 0 min. Selective snapshots are shown with 1 min intervals. The cells at the top and bottom rows are from the same movie (Supplementary Video 15), but only the light-treated centrosome underwent prompt centriole disengagement.

Article Snippet: The eGFP fused Centrin2 (GFP-Centrin2, Addgene) and Lenti-H2B-mCherry (GeneCopoeia) plasmids were purchased.

Techniques: Stable Transfection, Expressing, Fluorescence, Construct, Staining, Control, Activation Assay, Live Cell Imaging, Labeling, Comparison, Activity Assay