Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A) Single MDCK cells were cultured in Matrigel for 12 h. Immunostaining was performed with the following markers: apical membrane marker glycoprotein 135 (Gp135, green), centrosome marker γ-tubulin (magenta), acetyl-tubulin (white), and DAPI for nuclei (blue). Single confocal sections through the middle of a cyst are shown. The order of polarization is arranged from single cell (1-cell), metaphase (Meta), telophase (Telo), cytokinetic pre-abscission (Pre-Abs), post-cytokinesis (Post-CK), to lumen open (LO). Arrows indicate Gp135 clusters around centrosomes, while arrowheads point to centrosome positions. Scale bar: 5 μm. (B) Illustration showing the use of a fixed-size oval tool to select and measure Gp135 intensity in the chosen region. 1. Cytosol. 2. Cytokinesis (CK) bridge. 3. Centrosome (Cent.). (C–D) Boxplots of Gp135 intensity surrounding centrosomes at different stages normalized to the mean in metaphase (C) and at different cellular regions at the cytokinetic pre-abscission stage normalized to the mean in the cytosol (D). We analyzed >15 cells for each stage or region in three independent experiments. Statistical analyses were performed via one-way ANOVA and Dunn’s multiple comparisons (****p<0.0001, ***p<0.001, **p<0.01, ns: not significant). The midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values (a.u., arbitrary units). (E) Illustration showing how the polarity index ( L N-C [nucleus–centrosome distance] divided by r N [average nuclear radius]) and θ N-C (angle between the N-C and N-N axes) are calculated. (F–G) Boxplots depicting centrosome positions at different stages, shown by the polarity index (F) and θ N-C (G). We analyzed >16 cells per measurement in three independent experiments. Statistical analyses used one-way ANOVA and Dunn’s multiple comparisons (*p<0.05, **p<0.01, ****p<0.0001, ns: not significant). Midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values. (H) Localization overview summarizing the localization of the centrosome (magenta) and recycling endosome/apical membrane components (green) during cell-division-directed polarization in Matrigel culture. White lines depict intercellular bridge microtubules, and nuclei are shown in blue. Arrows indicate apical membrane component localization around centrosomes and their transport from the centrosome to the AMIS.

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Cell Culture, Immunostaining, Membrane, Marker

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A, F) Single MDCK cells were cultured in Matrigel for 12 h with labeled markers: Gp135 (green), γ-tubulin (magenta), and Rab11a (white) in (A), and aPKC (green), γ-tubulin (magenta), and Gp135 (white) in (F). DAPI staining was applied for nuclei (blue). Single confocal sections are shown through the middle of the cells. The centrosome region during telophase (Telo) and the AMIS region in post-cytokinesis (Post-CK) cells, as shown in the yellow box, are enlarged. Yellow arrows indicate Rab11a and aPKC around centrosomes or at the AMIS. White arrows indicate Gp135. Arrowheads point to the centrosome position. LO: lumen open. Scale bar: 5 μm. (B, D) Illustration showing L O-C (origin to centrosome distance) and L IC (inter-centrosomal distance) for cell doublets. The center point between the two nuclei is taken as the origin (O). (C, E) Boxplots depicting centrosome positions at different stages, shown by L O-C (C) and L IC (E). We analyzed >15 cells per measurement in three independent experiments. Statistical analyses used one-way ANOVA and Dunn’s multiple comparisons (*p<0.05, **p<0.01, ****p<0.0001). Midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values.

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Cell Culture, Labeling, Staining

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A) Time-lapse snapshots of MDCK cells expressing EGFP-Gp135 (green) and PACT-mKO1 (magenta, centrosome marker) in Matrigel. Nuclei were labeled with SiR-DNA (blue) before live imaging. Z-projection images of a dividing cell are shown. Time stamps show hours and minutes, with 0:00 set at the first frame of anaphase onset. Scale bar: 10 μm. (B–D) Change in polarity index, θ N-C , and L O-C over time. Each data point represents the average at a given time (>10 cells in Matrigel culture from three independent experiments). The lines show the means, and the shaded regions indicate SD values. (E) Fluorescent profiles of EGFP-Gp135 along the line connecting the two nuclei in a cell doublet. White arrows indicate the central value used to indicate the level of Gp135 on the AMIS (a.u., arbitrary units). Time stamps show hours and minutes, with 0:00 set to the first frame after anaphase onset. Scale bar: 10 μm. (F) Change in the central value in the EGFP-Gp135 fluorescent profile over time. The value of each time point was normalized to the value at 0:00. Each data point represents the average fluorescent intensity at a given time (10 cells in Matrigel culture from three independent experiments). The line shows the mean, and the shaded region indicates SD values (a.u., arbitrary units).

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Expressing, Marker, Labeling, Imaging

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A) Time-lapse snapshots of MDCK cells expressing EGFP-Gp135 (green) and PACT-mKO1 (magenta, centrosome marker) on a cover glass. Nuclei were labeled with SiR-DNA (blue) before live imaging. Z-projection images of a dividing cell are shown. Time stamps show hours and minutes, with 0:00 set at the first frame of anaphase onset. Scale bar: 10 μm. (B–C) Change in θ N-C and L O-C over time. Each data point represents the average at a given time (6 cells on cover glass from three independent experiments). The lines show the means, and the shaded regions indicate SD values.

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Expressing, Marker, Labeling, Imaging

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A) Time-lapse snapshots of p53-KO and centrinone (CN)-treated cells expressing EGFP-Gp135 (green) and PACT-mKO1 (magenta, centrosome marker) in Matrigel. Nuclei were labeled with SiR-DNA (blue) before live imaging. Z-projection images of dividing cells are shown. Time stamps show hours and minutes, with 0:00 set at the first frame of anaphase onset. Scale bar: 10 μm. (B–C) Change in θ N-C and L O-C over time. Each data point represents the average at a given time (>3 p53-KO cells in Matrigel culture from three independent experiments). The lines show the means, and the shaded regions indicate SD values. (D) Change in the central value of the EGFP-Gp135 fluorescent profile over time. The value of each time point was normalized to the value at 0:00. Each data point represents the average fluorescent intensity at a given time (n = 3 [p53-KO; olive] and 3 [p53-KO + CN; gray]). The lines show the means, and the shaded regions indicate SD values (a.u., arbitrary units). (E) Single MDCK WT and p53-KO cells, with or without CN treatment, after 72 h of Matrigel culture. Single confocal sections through the middle of cysts are shown with immunofluorescent signals of indicated markers: apical membrane Gp135 (green), centrosome marker γ-tubulin (magenta), and DAPI for nuclei (blue). “L” denotes the lumen. WT cells treated with CN remain at the single-cell stage. The insets display enlarged images of the region in the yellow box. Arrowheads indicate the presence of centrosomes. Scale bar: 10 μm. The bottom panels represent a larger view of the 3D culture. Scale bar: 50 μm. (F) Quantification of the proportion of MDCK cysts with a single central lumen after being cultured for different durations (24, 48, 72 h). The lumen structure was identified by Gp135 staining. n = 92 (24 h), 231 (48 h), 78 (72 h) cysts (MDCK WT); n = 22 (24 h), 133 (48 h), 59 (72 h) cysts (WT + CN); n = 154 (24 h), 170 (48 h), 204 (72 h) cysts (p53-KO); n = 105 (24 h), 128 (48 h), 149 (72 h) cysts (p53-KO + CN) analyzed for each measurement in three independent experiments. Statistical analyses used two-way ANOVA and Tukey multiple comparisons (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant). Values represent the mean ± SD. (G) Boxplot of Gp135 intensity on the apical membrane normalized to the mean in WT cells. n = 25 (MDCK WT), 26 (p53-KO), 25 (p53-KO + CN) cysts were analyzed in three independent experiments. Statistical analyses were performed via one-way ANOVA and Dunn’s multiple comparisons (ns: not significant, ****p<0.0001). The midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values (a.u., arbitrary units).

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Expressing, Marker, Labeling, Imaging, Membrane, Cell Culture, Staining

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A) Single MDCK WT and p53-KO cells, with or without centrinone (CN) treatment, after 8–12h of Matrigel culture. Single confocal sections through the middle of cysts are shown with immunofluorescent signals of indicated markers: Gp135 (green), γ-tubulin (magenta), acetyl-tubulin (white), and DAPI for nuclei (blue). Acentrosomal cells fail to cluster Gp135-positive vesicles and affect lumen formation in the two-cell stage (Pre-Abs, pre-abscission; LO, lumen open). Scale bar: 5 μm. (B) Boxplot of Gp135 intensity at the AMIS normalized to the mean in WT cells. n = 22 (MDCK WT), 26 (p53-KO), 28 (p53-KO + CN) post-CK cells were analyzed in three independent experiments. Statistical analyses were performed via one-way ANOVA and Dunn’s multiple comparisons (ns: not significant, ***p<0.001, ****p<0.0001). The midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values (a.u., arbitrary units). (C) Proliferation curves of MDCK WT and p53-KO cells, with or without CN treatment. The cells with CN treatment underwent a 3-day pre-treatment before examination. Values are presented as the mean ± SD. (D) Proportion of lumen opening in MDCK cell doublets after 24 h of Matrigel culture based on Gp135 staining. n = 17 (MDCK WT), 30 (p53-KO), and 45 (p53-KO + CN) cell doublets were analyzed in three independent experiments. Statistical analyses used two-way ANOVA and Tukey multiple comparisons (ns: not significant, ****p<0.0001). Values represent the mean ± SD.

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Staining

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A) Illustration depicting individual centrosomal structures (DA, SDA, and PCM proteins) and the state of microtubules on the centrosome in cells with knockout (KO) of CEP164 , ODF2 , p53/CEP120 , or PCNT genes. (B, F) Single MDCK cells of different genotypes were cultured in Matrigel for 12 h. Images show post-cytokinesis (CK) cells (B) or cells during pre-abscission (pre-Abs, F) with labeled markers: Gp135 (green), γ-tubulin (magenta), acetyl-tubulin (white), and DAPI for nuclei (blue). Images shown are single confocal sections through the middle of cells and side-view x-z cross-sections. Scale bar: 5 μm. (C–D, G–H) Boxplots of centrosome positions, represented by θ N-C and L O-C , for post-CK cells (C– D, n = 16 [WT], 10 [CEP164-KO], 15 [ODF2-KO], 20 [CEP120-KO], 14 [PCNT-KO]) and pre-Abs cells (G–H, n = 36 [WT], 14 [CEP164-KO], 16 [ODF2-KO], 12 [CEP120-KO], 14 [PCNT-KO]). Cells were analyzed in three independent experiments. Statistical analyses used one-way ANOVA and Dunn’s multiple comparisons (ns: not significant). Midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values. (E) Boxplot of Gp135 intensity at the AMIS normalized to the mean in WT cells. n = 25 (WT), 10 (CEP164-KO), 12 (ODF2-KO), 10 (CEP120-KO), and 10 (PCNT-KO) cell doublets were analyzed in three independent experiments. Statistical analyses were performed via one-way ANOVA and Dunn’s multiple comparisons (ns: not significant). The midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values (a.u., arbitrary units).

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Knock-Out, Cell Culture, Labeling

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A) Single MDCK cells, stably expressing pLKO-GFP-shRNA to knock down specified genes, cultured in Matrigel for 12 h. Images show post-cytokinesis cells with labeled markers: γ-tubulin (magenta), Gp135 (white), pLKO-GFP (green), and DAPI for nuclei (blue). Z-projection images between two centrosomes and the side-view x-z cross-section (bottom) are shown. “Ctrl” indicates the expression of scrambled-sequence shRNA. Arrowheads point to mislocalized centrosomes. Scale bar: 5 μm. (B–D) Boxplots of centrosome positions, represented by θ N-C , L O-C (B–C, n = 22 [sh-Ctrl], 20 [sh-Rab11a], 20 [sh-Sec15a], 20 [sh-MyoVb], 44 [sh-Par3], 20 [sh-Crb3], 20 [sh-Cdc42]), and L IC (D, n = 10 [sh-Ctrl], 10 [sh-Rab11a], 10 [sh-Sec15a], 10 [sh-MyoVb], 16 [sh-Par3], 10 [sh-Crb3], 10 [sh-Cdc42]), in post-cytokinesis cells. Cells were analyzed in three independent experiments. Statistical analyses used one-way ANOVA and Dunn’s multiple comparisons (ns: not significant, ***p<0.001, **p<0.01, *p<0.05). Midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values. (E) Single WT and Rab11a-KO MDCK cells were cultured in Matrigel for 12 h with labeled markers: Par3 (green), γ-tubulin (magenta), acetyl-tubulin (white), and DAPI for nuclei (blue). Single confocal sections of pre-abscission cells are shown. The bridge region is enlarged from the yellow box. Scale bar: 5 μm. (F) Single confocal sections of p53-KO cells, with or without centrinone (CN) treatment, cultured in Matrigel for 12 h with labeled markers: Par3 (green), γ-tubulin (magenta), acetyl-tubulin (white), and DAPI for nuclei (blue). The bridge region shown in the yellow box is enlarged. The bottom panels display Z-projected images to demonstrate the complete depletion of centrosomes. Scale bar: 5 μm.

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Stable Transfection, Expressing, shRNA, Knockdown, Cell Culture, Labeling, Sequencing

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: Centrosome position relative to the cytokinesis site in polarized epithelial sheets (A) Experimental design: Aphidicolin (Aphi)-treated cells (synchronized at the S phase) were seeded into Matrigel and observed within 8 h to exclude the possibility of cell doublet formation through cell division. (B) Bright-field images of MDCK cells cultured in low-attachment microwells. The images are merged with the DAPI (cyan) signal to show cell division occurring over time. (C–D) Polarized MDCK cyst and epithelial sheet with labeled markers: the centrosome markers PACT-mKO1 and γ-tubulin, acetyl-tubulin, F-actin, and DAPI. Insets show enlargements of the image in the boxed area. “L” indicates the lumen in (C). The z-projection of the top view and the X-Z cross-section of the side view (bottom) are shown in (D). Telo, telophase; Pre-Abs, pre-abscission. Arrowheads point to the centrosome position. Scale bar: 10 μm. (E) Time-lapse snapshots of MDCK cells expressing EGFP-Gp135 and PACT-mKO1 cultured on a Transwell insert to form polarized epithelial sheets. The microtubule probe SiR-tubulin (white) was applied before live cell imaging. The X-Y top view and the X-Z cross-section of the side view (bottom) are shown. Time stamps show hours and minutes, with 0:00 set to the first frame after anaphase onset. Arrowheads point to the centrosomes. The yellow arrow indicates the abscission of the cytokinesis bridge. Scale bar: 10 μm. A schematic depicts the centrosomes moving to the apical membrane before bridge abscission. (F) Change in distance from the centrosome to the apical membrane, as measured in each frame (data from three independent experiments, n = 5 cells). The graph presents the mean ± SD.

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Cell Culture, Labeling, Expressing, Live Cell Imaging, Membrane

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: (A) Time-lapse snapshots of MDCK cells expressing EGFP-Gp135 (green) and PACT-mKO1 (magenta, centrosome marker) in Matrigel. Nuclei were labeled with SiR-DNA (cyan) before live imaging. Z-projection images of an aggregated cell doublet are shown. Arrows indicate Gp135 on the outer surface of the cell aggregate. Arrowheads point to mislocalized centrosomes that did not move over 3 h. Time stamps show hours and minutes, with 0:00 set at the beginning of live cell imaging. Scale bar: 10 μm. (B, E) MDCK cells, with or without aphidicolin (Aphi) synchronization, were cultured in Matrigel for 6h with labeled markers: Rab11a (B, green), Par3 (E, green), γ-tubulin (magenta), and DAPI for nuclei (blue). Single confocal sections are shown. Two types of cell doublets, formed by cell division (post cytokinesis [CK]) or aggregation, were compared. Arrows indicate Gp135 clusters around centrosomes. Arrowheads point to mislocalized centrosomes. Scale bar: 5 μm. (C–D) Boxplots of centrosome positions, represented by θ N-C (C) and L IC (D), in cell doublets (n = 15 [unsynchronized control], 20 [Aphi-synchronized cells]). Cells were analyzed in three independent experiments. Statistical analyses used an unpaired two-tailed Mann–Whitney U test (*p<0.05, ****p<0.0001). Midlines and boxes show the mean ± SD, with whiskers indicating minimum and maximum values. (F) MDCK cells were cultured in low-attachment microwells for 24h with labeled markers: Gp135 (green), γ-tubulin (magenta), Par3 (white), and DAPI for nuclei (blue). A single confocal section through the middle of a cell doublet is shown. Yellow arrows indicate the Par3signal at the edge of cell-cell contacts. The 3D reconstruction reveals Par3 forming a ring around the edge between cell doublets. Scale bar: 10 μm. (G) Time-lapse snapshots of MDCK cells expressing EGFP-Gp135 (green) and PACT-mKO1 (magenta, centrosome marker) in low-attachment microwells. Nuclei were labeled with SiR-DNA (cyan) before live imaging. Z-projection images of a dividing cell are shown. White and yellow arrows indicate Gp135 on the outer surface of the cell doublet and around the centrosome, respectively. Arrowheads point to the centrosome position. Time stamps show hours and minutes, with 0:00 set at the first frame of anaphase onset. Scale bar: 10 μm. (H–J) Change in polarity index (H), θ N-C (I), and L O-C (J) over time. Each data point represents the average at a given time (three cells in low-attachment microwells from three independent experiments). The lines show the means, and the shaded regions indicate SD values.

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Expressing, Marker, Labeling, Imaging, Live Cell Imaging, Cell Culture, Control, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

doi: 10.1101/2024.06.17.598507

Figure Lengend Snippet: Summary of centrosome migration, apical membrane component trafficking, and Par3 recruitment in different experiments and culture conditions (A) In conventional Matrigel culture, centrosomes move directionally toward AMIS following cell division (magenta arrow). Apical membrane components like Gp135, Crb3, and Cdc42 follow the centrosomes (green arrow). Par3 first emerges at the cytokinesis site, which regulates centrosome positioning and polarized vesicle trafficking during polarization. (B) Loss of centrosomes diminishes the effectiveness of apical membrane component trafficking (smaller green arrow), yet Par3 still localizes to the cytokinetic bridge. Additionally, centrosome loss mainly impacts the initial stages of epithelial polarization rather than the later stages of lumen formation. (C) Loss of Par3 results in randomized centrosome positioning, obstructs polarized trafficking, and traps apical membrane components around mislocalized centrosomes. (D) Two-cell aggregates without cell division cannot effectively guide centrosome migration and polarized vesicle trafficking. Despite Par3 being recruited to the cell-cell interface, apical membrane components remain trapped around mislocalized centrosomes. (E) MDCK cells suspended in the ECM-free condition. Centrosomes migrate to the center of cell doublets during cytokinesis (magenta arrow), while Apical membrane components transport in the opposite direction (green arrow). After mitosis, Par3 exhibits a pattern distinct from that observed in Matrigel culture.

Article Snippet: We used rabbit polyclonal antibodies against Gp135 (IF: 1:400, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , Rab11a (IF: 1:400, WB: 1:2000, 20229-1-AP, Proteintech, Rosemont, IL), aPKC (IF: 1:200, 610175, BD Biosciences, Franklin Lakes, NJ), CEP120 (IF: 1:1000, WB: 1:2000, human residues 639–986, described in our previous paper) , p53 (IF: 1:100, 9282, Cell Signaling Technology, Danvers, MA), p21 (WB: 1:2000, 10355-1-AP, Proteintech), CEP164 (IF: 1:400, NBP1-81445, NOVUS, Centennial, CO), PCNT (IF: 1:200, ab4448, Abcam, Cambridge, UK), Par3 (IF: 1:200, WB: 1:1000, 07-330, EMD Millipore, Burlington, MA), and beta-actin (IF: 1:200, A2066, Sigma-Aldrich, St. Louis, MO); rabbit monoclonal antibodies against E-cadherin (IF: 1:200, 24E10, #3195, Cell Signaling Technology); and mouse monoclonal antibodies against γ-tubulin (IF: 1:400, GTU-88, T6557, Sigma-Aldrich), acetylated tubulin (IF: 1:400, T6793, Sigma-Aldrich), Gp135 (IF: 1:10, the culture medium of hybridoma clone 3F2D8, a kind gift from Dr. Jou Tzuu-Shuh, NTU, Taiwan) , polyglutamylated tubulin (IF: 1:100, GT335, AG-20B-0020, Adipogen, San Diego, CA), ODF2 (IF: 1:400, H00004957-M01, NOVUS), α-tubulin (WB: 1:5000, DM1α, T9026, Sigma-Aldrich), CDC42 (WB: 1:1000, 610928, BD Biosciences), and ZO-1 (IF: 1:400, 33-9100, ThermoFisher Scientific, Waltham, MA).

Techniques: Migration, Membrane

Journal: Nature

Article Title: Capillary cell-type specialization in the alveolus.

doi: 10.1038/s41586-020-2822-7

Figure Lengend Snippet: Fig. 1 | Two stable, intermingled alveolar capillary cell types. a, Alveolar capillaries in adult mouse lung immunostained for PECAM1. b, t-distributed stochastic neighbour embedding (t-SNE) plot of endothelial cell populations annotated in scRNA-seq data for adult mouse lung13. c, Heat map of expression of capillary subset markers and the general endothelial marker Cldn5 in individual capillary cells. CPM, counts per million. d–f, Single-molecule fluorescent in situ hybridization (smFISH) for the capillary subset markers Apln (d, f) or Ednrb and Car4 (aCap) (e), and Aplnr (gCap) (d–f), in adult mouse lung. Images in d (right) and f show individual aCap and gCap cells. g, Relative abundance of aCap cells, gCap cells and cells that co-express aCap and gCap markers (intermediate (IM) cells) in lungs from 3-month-old (young) and 24-month-old (aged) mice (data shown as mean; n = 500 cells scored per mouse; 2 mice per group). h, i, Co-expression of tdTomato lineage label (asterisks) and aCap marker Ednrb but not gCap marker Aplnr (h), or gCap marker Aplnr but not aCap marker Apln (i), in lungs collected six months after mature aCap (h) or gCap (i) cells were lineage-labelled. Blue, DAPI. Scale bars, 10 μm.

Article Snippet: CD34 (BD Biosciences, 347660):https://www.bdbiosciences.com/eu/applications/research/clinical-research/oncology-research/ 3 nature research | reporting sum m ary O ctober 2018 blood-cell-disorders/surface-markers/human/purified-mouse-anti-human-cd34-my10/p/347660 Endomucin (Invitrogen, eBioV.7C7, 14-5851-82): https://www.thermofisher.com/antibody/product/Endomucin-Antibody-cloneeBioV-7C7-V-7C7-Monoclonal/14-5851-82 Integrin alpha8 (R&D, AF4076): https://www.rndsystems.com/products/mouse-rat-integrin-alpha8-antibody_af4076 Pecam1 (rat anti-mouse; BD Biosciences, 553370): https://www.bdbiosciences.com/ds/pm/tds/553370.pdf Pecam1 (mouse anti-human; R&D, BBA7): https://www.rndsystems.com/products/human-cd31-pecam-1-antibody-9g11_bba7 tdTomato (Rockland, 600-401-379): https://rockland-inc.com/store/Antibodies-to-GFP-and-Antibodies-to-RFP-600-401-379O4L_24299.aspx VE-Cadherin (R&D, AF938): https://www.rndsystems.com/products/human-ve-cadherin-antibody_af938 The following primary antibodies were used to stain turtle and/or alligator tissue.

Techniques: Expressing, Marker, In Situ Hybridization