Journal: International Journal of Biological Sciences

Article Title: MRCK as a Potential Target for Claudin-Low Subtype of Breast Cancer

doi: 10.7150/ijbs.88285

Figure Lengend Snippet: Focal adhesion - MRCK - YAP/TAZ is particularly important for the Claudin-low subtype. (A and B) Using TCGA breast cancer gene expression data, we examined the YAP/TAZ target scores and sum of YAP1 and WWTR1 in ER+ (ER), ER-/HER2+ (HER), and TNBC (TN) breast cancers. *TN shows the significantly higher YAP/TAZ target score as well as YAP1 + WWTR1 expression than other subtypes. (C) Breast cancer patients with Basal subtype were divided into two groups based on the high and low total YAP1 / WWTR1 expression levels, and their survival curves were compared (Logrank P=0.021). (D) We calculated the YAP/TAZ target scores for six intrinsic subtypes using the METABRIC dataset. *Claudin-low is significantly higher than any other subtype, and ¶ Basal is significantly higher than HER2, Luminal A (LumA) and Luminal B (LumB) subtypes. (E) We calculated the Focal adhesion scores for six intrinsic subtypes using the METABRIC dataset. *Claudin-low is significantly higher than any other subtype, and ¶ Basal is significantly higher than HER2, Luminal A (LumA), Luminal B (LumB) and Normal subtypes. (F and G) The relationship between the CLDN score and susceptibility to MRCKi in breast cancer cell lines is shown in scatterplots with regression lines. The CLDN scores are plotted on the X-axis and IC50 against BDP-9066 (F) or BDP-8900 (G) are plotted on the Y-axis. There is a positive correlation between the CLDN score and IC50 of MRCKi.

Article Snippet: Anti-MRCKα (sc-374568) and anti-MRCKβ (sc-374597) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Gene Expression, Expressing

Journal: bioRxiv

Article Title: MRCK controls myosin II activation in the polarized cortex of mouse oocytes and promotes spindle rotation and male pronucleus centration

doi: 10.1101/2022.09.25.509421

Figure Lengend Snippet: (A) Immunofluorescence detection of activated myosin II (P-MRLC) in MII oocytes treated for 1 h with DMSO, ML-141 (5 μM), BDP-9066 (1 μM) or a combination of BDP-9066 (1 μM) and calyculin A (CalA; 0.5 nM). Images are single confocal frames taken across metaphase chromosomes. (B) Immunofluorescence detection of endogenous MRCKbeta in MII oocytes. The top row shows a control oocyte treated with DMSO. An enlarged view of the polarized cortex is shown on the right. The bottom row shows an oocyte treated with ML-141 (5 μM). (C) Bar graph depicting the fraction of oocytes showing polarized accumulation of MRCKbeta, in MII oocytes treated with DMSO, ML-141 or BDP-9066. P values were calculated using Fisher’s exact test. (D) Immunofluorescence detection of MRCKα in a mouse MII oocyte. The dotted line denotes the oocyte perimeter. No cortical staining could be detected. The image is representative of 33 similar observations. (E) Localization of MRCKβ-EGFP (left) and MRCKβ H1593/1596A-EGFP (right) in MII oocytes. Note the absence of cortical localization for the Cdc42 binding-deficient MRCKβ. Images are representative of over 40 similar observations. (F) Immunofluorescence detection of activated myosin II (P-MRLC) in MII oocytes expressing MRCKβ-K105A. A majority of oocytes showed a complete loss of the P-MRLC ring (62.5%; top row), while the remaining oocytes showed incomplete inhibition (37.5%; bottom row, white arrow). (G) Bar graph depicting the percentage of MII oocytes showing a P-MRLC ring, in various experimental conditions as shown in (A), (E) and (F). P values were calculated using Fisher’s exact test. (H) Immunofluorescence detection of tubulin showing spindle localization in MII oocytes treated with DMSO (left), CK-666 (100 μM) for 3h (middle), and CK-666 (100 μM) and BDP-9066 (1 μM) for 3h (right). The yellow line shows the distance (d) separating the maternal chromosomes from the nearest cortex. (I) Box plot showing the distance between maternal chromosomes and the nearest cortical region in oocytes treated with DMSO (controls), CK-666 alone, CK-666 and BDP-9066, and in oocytes expressing MRCKβ-K105A treated with CK-666. P values were calculated using Student’s t -test. NS: non significant. F-actin was labeled with Alexa Fluor 568-phalloidin. DNA was stained with TO-PRO-3. The number of oocytes scored is indicated above each bar/boc (C,G,I). Scale bars represent 10 μm.

Article Snippet: The following primary antibodies were used : Phospho-myosin light chain 2 (Ser19) (1:200; Cell Signaling Technology #3671), Non-muscle myosin IIA (1:100; Abcam ab24762), Myosin IIB (D8H8) (1:100; Cell Signaling Technology #8824), MRCKα (B-3) (1:100; Santa Cruz Biotechnology sc-374568), MRCKβ (A-2) (1:100; Santa Cruz Biotechnology sc-390127), Tubulin (1:200; Abcam ab6161), p34-ARC (1:100; Santa Cruz Biotechnology sc-515754), Rock-1 (K-18) (1:100; Santa Cruz Biotechnology sc-6056), Trimethylhistone H3 (Lys4) (1:100; Merck Millipore 04-745).

Techniques: Immunofluorescence, Control, Staining, Binding Assay, Expressing, Inhibition, Labeling

Journal: bioRxiv

Article Title: MRCK controls myosin II activation in the polarized cortex of mouse oocytes and promotes spindle rotation and male pronucleus centration

doi: 10.1101/2022.09.25.509421

Figure Lengend Snippet: (A) Schematic depicting the primary structure of full-length human MRCKβ and its functional domains. C1: protein kinase C conserved region 1. PH: pleckstrin homology. CH: citron homology. CRIB: Cdc42/Rac interactive binding. (B) Schematic outlining the different constructs generated through stepwise truncation to obtain a Cdc42·GTP biosensor, using full-length MRCKβ-GFP as a template. All constructs were expressed in MII oocytes via cRNA injection, and examined for polarized localization in the cortex overlying maternal chromosomes. The total number of oocytes scored is indicated in parentheses for each construct, with an indication of cortical polarization (+) or absence of (-). While the CRIB domain alone was not sufficient for cortical localization in vivo, stepwise extensions toward the N-terminus allowed us to define a minimal Cdc42·GTP-binding fragment encompassing the PH, CH and CRIB domains. In this study, this minimal fragment was used as a biosensor for Cdc42 activation, designated as MRCK-derived Cdc42·GTP Biosensor (MCB). Mutation of the two key histidine residues in the CRIB domain to alanine, corresponding to H1593/1596A in the full-length sequence, and indicated by a double asterisks (**), abolished polarized localization. aa: amino acids. (C) Confocal image of a live MII oocyte expressing the MCB-EGFP biosensor. (D) Confocal image of a live MII oocyte expressing the MCB-EGFP biosensor, and treated with ML-141 (5 μM, 1h). (E) Confocal image of a live MII oocyte expressing the MCB(2A)-EGFP biosensor, bearing the H1593/1596A substitution. Chromosomes were stained with SiR-DNA.

Article Snippet: The following primary antibodies were used : Phospho-myosin light chain 2 (Ser19) (1:200; Cell Signaling Technology #3671), Non-muscle myosin IIA (1:100; Abcam ab24762), Myosin IIB (D8H8) (1:100; Cell Signaling Technology #8824), MRCKα (B-3) (1:100; Santa Cruz Biotechnology sc-374568), MRCKβ (A-2) (1:100; Santa Cruz Biotechnology sc-390127), Tubulin (1:200; Abcam ab6161), p34-ARC (1:100; Santa Cruz Biotechnology sc-515754), Rock-1 (K-18) (1:100; Santa Cruz Biotechnology sc-6056), Trimethylhistone H3 (Lys4) (1:100; Merck Millipore 04-745).

Techniques: Functional Assay, Binding Assay, Construct, Generated, Injection, In Vivo, Activation Assay, Derivative Assay, Mutagenesis, Sequencing, Expressing, Staining

Journal: bioRxiv

Article Title: MRCK controls myosin II activation in the polarized cortex of mouse oocytes and promotes spindle rotation and male pronucleus centration

doi: 10.1101/2022.09.25.509421

Figure Lengend Snippet: (A) Box plot showing the width of the actin cap in control MII oocytes treated with DMSO and MII oocytes treated with BDP-9066 (1 μM). P value was calculated using Student’s t -test. The number of oocytes scored is indicated above each box. (B) Immunofluorescence detection of activated myosin II (P-MRLC) and the Arp2/3 complex (p34ARC) in MII oocytes treated for 1 h with DMSO (top row) or BDP-9066 (1 μM; bottom row). White arrows point to the co-localization of P-MRLC and p34ARC at the shoulders of the actin cap. (C) Immunofluorescence detection of activated myosin II (P-MRLC) and MRCKβ in MII oocytes treated for 1 h with DMSO (top row) or BDP-9066 (1 μM; bottom row). (D) Detection of Cdc42·GTP in a MII oocyte expressing the Cdc42 biosensor MCB-EGFP. (E) Immunofluorescence detection of MRCKβ and F-actin in MII oocytes treated for 3 h with 100 μM CK-666. In (B-E), fluorescence intensity profiles are shown, corresponding to the polarized cortex region delinated by the two grey arrowheads in the merge images. F-actin was labeled with Alexa Fluor 568-phalloidin. DNA was stained with TO-PRO-3. Scale bars represent 10 μm.

Article Snippet: The following primary antibodies were used : Phospho-myosin light chain 2 (Ser19) (1:200; Cell Signaling Technology #3671), Non-muscle myosin IIA (1:100; Abcam ab24762), Myosin IIB (D8H8) (1:100; Cell Signaling Technology #8824), MRCKα (B-3) (1:100; Santa Cruz Biotechnology sc-374568), MRCKβ (A-2) (1:100; Santa Cruz Biotechnology sc-390127), Tubulin (1:200; Abcam ab6161), p34-ARC (1:100; Santa Cruz Biotechnology sc-515754), Rock-1 (K-18) (1:100; Santa Cruz Biotechnology sc-6056), Trimethylhistone H3 (Lys4) (1:100; Merck Millipore 04-745).

Techniques: Control, Immunofluorescence, Expressing, Fluorescence, Labeling, Staining

Journal: bioRxiv

Article Title: MRCK controls myosin II activation in the polarized cortex of mouse oocytes and promotes spindle rotation and male pronucleus centration

doi: 10.1101/2022.09.25.509421

Figure Lengend Snippet: (A) Detection of Cdc42·GTP (green) and RhoA·GTP (red) in live oocytes at the metaphase-II (MII, left) and anaphase-II (AII, right) stages. Oocytes were injected at the MII stage with cRNAs encoding the MCB-EGFP and mcherry-AHPH biosensors. Images are Z-compressions of 3 consecutive confocal frames. (B) Immunofluorescence detection of MRCKβ in fixed MII (left) and AII (right) oocytes. (C) Immunofluorescence detection of ROCK1 in an AII oocyte. (D) Immunofluorescence detection of activated myosin II (P-MRLC) and MRCKβ in an AII oocyte. (E) Immunofluorescence detection of activated myosin II (P-MRLC) in the cortex of activated oocytes undergoing anaphase II. Oocytes were treated with DMSO (top row), BDP-9066 (middle row) or a combination of BDP-9066 and Y-27632 (bottom row). Open arrowheads point to the P-MRLC rings overlying the chromatid clusters in the control (DMSO) oocyte. White arrows point to cytokinetic P-MRLC in the control (DMSO) and BDP-9066-treated oocytes. Images are z-compression of 33 (DMSO), 15 (BDP-9066) and 30 (BDP-9066 + Y-27632) consecutive confocal frames. F-actin was labeled with Alexa Fluor 568-phalloidin. DNA was stained with SiR-DNA (panel A) or TO-PRO-3 (panels BE). Fluorescence intensity profiles correspond to the cortical regions delineated by the grey arrowheads in the merge images. In (D), the white arrow points to the ingressing furrow region that is enriched in P-MRLC but devoided of MRCKβ.

Article Snippet: The following primary antibodies were used : Phospho-myosin light chain 2 (Ser19) (1:200; Cell Signaling Technology #3671), Non-muscle myosin IIA (1:100; Abcam ab24762), Myosin IIB (D8H8) (1:100; Cell Signaling Technology #8824), MRCKα (B-3) (1:100; Santa Cruz Biotechnology sc-374568), MRCKβ (A-2) (1:100; Santa Cruz Biotechnology sc-390127), Tubulin (1:200; Abcam ab6161), p34-ARC (1:100; Santa Cruz Biotechnology sc-515754), Rock-1 (K-18) (1:100; Santa Cruz Biotechnology sc-6056), Trimethylhistone H3 (Lys4) (1:100; Merck Millipore 04-745).

Techniques: Injection, Immunofluorescence, Control, Labeling, Staining, Fluorescence

Journal: bioRxiv

Article Title: MRCK controls myosin II activation in the polarized cortex of mouse oocytes and promotes spindle rotation and male pronucleus centration

doi: 10.1101/2022.09.25.509421

Figure Lengend Snippet: (A) Immunofluorescence detection of activated myosin II (P-MRLC) in fertilized oocytes. Top row: control oocyte treated with DMSO. Bottom row: oocyte fertilized in the presence of BDP-9066. Note the P-MRLC ring at the base of the fertilization cone (FC) in the control oocyte, and absence of in the MRCK-inhibited oocyte. Oocytes were fixed 3 hours postinsemination. PB2: second polar body. Sp: sperm chromatin. Fluorescence intensity profiles correspond to the cortical regions delineated by the grey arrowheads in the merge images. (B) Bar graph depicting the percentage of fertilized oocytes showing a P-MRLC ring at the base of the FC. The fertilization medium was supplemented with DMSO (control) or BDP-9066. P value was calculated using Fisher’s exact test. (C) Schematic illustrating the experimental protocol for monitoring male pronucleus migration. (D) Detection of male and female pronuclei in zygotes fixed 8h after insemination. The left image shows a control zygote treated with DMSO after PB2 emission. The yellow line denotes the distance (d) of the male pronucleus to the nearest cortex. The right image shows a zygote treated with BDP-9066 after PB2 emission. Note the expanded male pronucleus still apposed to the FC cortex. Images are z-compressions of two consecutive confocal frames. (E) Scatter plot of the male pronucleus distance to the nearest cortex, as depicted in (D). Red and blue horizontal bars show the median values. P value was calculated using a two-tailed Mann-Whitney U test. DNA was labeled with TO-PRO-3. F-actin was labeled with Alexa Fluor 568-phalloidin. Female pronuclei were immuno-stained for H3K4me3 (green).

Article Snippet: The following primary antibodies were used : Phospho-myosin light chain 2 (Ser19) (1:200; Cell Signaling Technology #3671), Non-muscle myosin IIA (1:100; Abcam ab24762), Myosin IIB (D8H8) (1:100; Cell Signaling Technology #8824), MRCKα (B-3) (1:100; Santa Cruz Biotechnology sc-374568), MRCKβ (A-2) (1:100; Santa Cruz Biotechnology sc-390127), Tubulin (1:200; Abcam ab6161), p34-ARC (1:100; Santa Cruz Biotechnology sc-515754), Rock-1 (K-18) (1:100; Santa Cruz Biotechnology sc-6056), Trimethylhistone H3 (Lys4) (1:100; Merck Millipore 04-745).

Techniques: Immunofluorescence, Control, Fluorescence, Migration, Two Tailed Test, MANN-WHITNEY, Labeling, Staining

Journal: bioRxiv

Article Title: MRCK controls myosin II activation in the polarized cortex of mouse oocytes and promotes spindle rotation and male pronucleus centration

doi: 10.1101/2022.09.25.509421

Figure Lengend Snippet: (A) Immunofluorescence detection of activated myosin II (P-MRLC) in a fertilized oocyte. Images are z-compressions across the entire zygote volume, for 3d rendition, highlighting the FC area. (B) Detection of Cdc42·GTP in the FC. The image shows a fertilized oocyte expressing the Cdc42·GTP biosensor MCB-EGFP. The oocyte was fertilized in vitro after zona pellucida removal. (C) Immunofluorescence detection of MRCKβ in a fertilized oocyte. Images are z-compressions across the entire zygote volume, for 3d rendition, highlighting the FC area. (D) Immunofluorescence detection of activated myosin II (P-MRLC) and MRCKβ in a fertilized oocyte. Fluorescence intensity profiles correspond to the cortical regions delineated by the grey arrowheads in the merge images. FC: fertilization cone. Sp: sperm chromatin. PB2: second polar body. F-actin was labeled with Alexa Fluor 568-phalloidin. DNA was stained with TO-PRO-3.

Article Snippet: The following primary antibodies were used : Phospho-myosin light chain 2 (Ser19) (1:200; Cell Signaling Technology #3671), Non-muscle myosin IIA (1:100; Abcam ab24762), Myosin IIB (D8H8) (1:100; Cell Signaling Technology #8824), MRCKα (B-3) (1:100; Santa Cruz Biotechnology sc-374568), MRCKβ (A-2) (1:100; Santa Cruz Biotechnology sc-390127), Tubulin (1:200; Abcam ab6161), p34-ARC (1:100; Santa Cruz Biotechnology sc-515754), Rock-1 (K-18) (1:100; Santa Cruz Biotechnology sc-6056), Trimethylhistone H3 (Lys4) (1:100; Merck Millipore 04-745).

Techniques: Immunofluorescence, Expressing, In Vitro, Fluorescence, Labeling, Staining