Figure 1. MRCK promotes myosin II... | Rockefeller University Press

Figure 1.

$MRCK promotes myosin II activation in the polarized cortex. (A) Immunofluorescence detection of activated myosin II (P-MRLC) in MII oocytes treated for 1 h with DMSO, ML-141 (5 µM), BDP9066 (1 µM), or a combination of BDP9066 (1 µM) and calyculin A (CalA; 0.5 nM). Images are single confocal frames taken across metaphase chromosomes. (B) Immunofluorescence detection of endogenous MRCKβ 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 MRCKβ in MII oocytes treated with DMSO, ML-141, or BDP9066. P values were calculated using Fisher’s exact test. (D) Localization of MRCKβ-EGFP (left) and MRCKβ H1593/1596A-EGFP (right) in live MII oocytes. Note the absence of cortical localization for the Cdc42 binding-deficient MRCKβ. Images are representative of >40 similar observations. (E) Immunofluorescence detection of activated myosin II (P-MRLC) in MII oocytes expressing MRCKβ-K105A. A majority of oocytes (62.5%) showed a complete loss of the P-MRLC ring (top row), while the remaining oocytes (37.5%) showed incomplete inhibition (bottom row; white arrow). (F) Bar graph depicting the percentage of MII oocytes showing a P-MRLC ring in various experimental conditions as shown in A, D, and E. P values were calculated using Fisher’s exact test. (G) Immunofluorescence detection of tubulin showing spindle localization in MII oocytes treated with DMSO (left), CK-666 (100 µM) for 3 h (middle), and CK-666 (100 µM) and BDP9066 (1 µM) for 3 h (right). The yellow line shows the distance (d) separating the maternal chromosomes from the nearest cortex. (H) 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 BDP9066, as illustrated in G, and in oocytes expressing MRCKβ-K105A treated with CK-666. P values were calculated using two-tailed Student’s t test. F-actin was labeled with Alexa Fluor 568–phalloidin. Chromosomes were stained with TO-PRO-3 (A, B, E, and G; magenta) or with SiR-DNA (D; magenta). The number of oocytes scored is indicated above each bar/box (C, F, and H). Scale bars are 10 µm. In A, D, and E, scale bars apply to the entire panel. NS: nonsignificant.$

MRCK promotes myosin II activation in the polarized cortex. (A) Immunofluorescence detection of activated myosin II (P-MRLC) in MII oocytes treated for 1 h with DMSO, ML-141 (5 µM), BDP9066 (1 µM), or a combination of BDP9066 (1 µM) and calyculin A (CalA; 0.5 nM). Images are single confocal frames taken across metaphase chromosomes. (B) Immunofluorescence detection of endogenous MRCKβ 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 MRCKβ in MII oocytes treated with DMSO, ML-141, or BDP9066. P values were calculated using Fisher’s exact test. (D) Localization of MRCKβ-EGFP (left) and MRCKβ H1593/1596A-EGFP (right) in live MII oocytes. Note the absence of cortical localization for the Cdc42 binding-deficient MRCKβ. Images are representative of >40 similar observations. (E) Immunofluorescence detection of activated myosin II (P-MRLC) in MII oocytes expressing MRCKβ-K105A. A majority of oocytes (62.5%) showed a complete loss of the P-MRLC ring (top row), while the remaining oocytes (37.5%) showed incomplete inhibition (bottom row; white arrow). (F) Bar graph depicting the percentage of MII oocytes showing a P-MRLC ring in various experimental conditions as shown in A, D, and E. P values were calculated using Fisher’s exact test. (G) Immunofluorescence detection of tubulin showing spindle localization in MII oocytes treated with DMSO (left), CK-666 (100 µM) for 3 h (middle), and CK-666 (100 µM) and BDP9066 (1 µM) for 3 h (right). The yellow line shows the distance (d) separating the maternal chromosomes from the nearest cortex. (H) 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 BDP9066, as illustrated in G, and in oocytes expressing MRCKβ-K105A treated with CK-666. P values were calculated using two-tailed Student’s t test. F-actin was labeled with Alexa Fluor 568–phalloidin. Chromosomes were stained with TO-PRO-3 (A, B, E, and G; magenta) or with SiR-DNA (D; magenta). The number of oocytes scored is indicated above each bar/box (C, F, and H). Scale bars are 10 µm. In A, D, and E, scale bars apply to the entire panel. NS: nonsignificant.

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