Model of myosin II dynamics within cells regulated by phosphorylation. For details, see text.

Recruitment of myosin II to the purse string at wound closure and MRLC phosphorylation. A wound was made by laser irradiation in WT-MRLC-EGFP- or its mutant-expressing cell sheets during time-lapse imaging. Arrows indicate edges of the wounds. Bar, 10 μm.

Localizations of introduced MRLC-EGFP and its mutants. Each clone was fixed with 4% FA and processed for confocal microscopy to detect EGFP fluorescence. White arrows indicate apical junction regions. Black arrows indicate apical direction along apicobasal axis in vertical sections of cells constructed from serial optical sections. Bar, 10 μm.

Changes in local fluorescence intensity of time-lapse images of cells expressing MRLC-EGFP or its mutant. Average fluorescence intensity of the same areas indicated by numbered circles of each frame (left) at every 30 s was measured for 10 min and average intensity of the same area of all frames was calculated. Fluorescence intensity of the same area at each frame relative to the average intensity was plotted as a function of time (right). Bar, 5 μm.

Myosin II-ATPase activity is involved in stress fiber-localization of MYPT1 and ZIP kinase. MDCK II cells expressing AD-MRLC-EGFP were treated with control medium (H2O/DMSO), 30 μM Y27632 (Y27632 DMSO), 100 μM blebbistatin [H2O (±)Blebb.], or both [Y27632 (±) Blebb.] for 30 min. (A) Each sample was fixed with 1% FA on ice and stained for MYPT1 and ZIP kinase. Bar, 10 μm. (B) Each sample was processed for immunoblotting to detect MYPT1, MYPT1 phosphorylated at Thr850, and MRLC.

Establishment of MDCK II clones stably expressing MRLC-EGFP and its mutants. (A) Scheme of recombinant MRLCs; WT-MRLC, AA-MRLC, AS-MRLC, AD-MRLC, and DD-MRLC. EGFP-tag was added to the C-terminal of each recombinant MRLC. (B) Expression of endogenous and introduced EGFP-tagged MRLCs in each clone. Parental, GFP-expressing or MRLC-EGFP or its mutant-expressing MDCK II cells were processed for immunoblotting using anti-MRLC antibody. Equivalent amounts of proteins were loaded. (C) Mosaic cultures consisting of parental MDCK II cells and WT-MRLC-EGFP (green in merge)-expressing cells were fixed with 1% FA and stained for MRLC (magenta in merge). Bar, 20 μm.

FRAP analysis of MRLC-EGFP or its mutants. (A) Time-lapse images of WT-, AA-, AD-, and DD-MRLC before and during FRAP analysis. Arrowheads indicate bleached areas in stress fibers. Bar, 5 μm. (B) Top graph shows FRAP of each MRLC. Bottom graph shows FRAP of WT-MRLC in control cells and cells treated with 5 nM calyculin A for 1 h. (C) Effects of calyculin A on MRLC phosphorylation of MDCK II cells. Control cells and cells treated with 5 nM calyculin A for 1 h were immunoblotted for detection of total, 1P-, and 2P-MRLC. The amount of actin was used as a loading control.

MRLC-diphosphorylation and stress fiber contraction. (A) MDCK II cells expressing WT-MRLC-EGFP were fixed with 1% FA immediately after time-lapse imaging and stained for 1P- and 2P-MRLC. Bar, 5 μm. (B) Kymograph of the boxed area in (A) constructed from time-lapse images of MRLC-EGFP showing stress fiber contraction and stretching during 540 s immediately before fixation and images of the fixed sample showing MRLC-EGFP, 1P-MRLC, and 2P-MRLC localizations. Arrowheads indicate positions of several bright points along the stress fiber. Fluorescence intensity of every point along the length of the stress fiber of each image of fixed cells was measured and ratios of the intensity, 1P-MRLC/EGFP and 2P-MRLC/EGFP were plotted along the length of the stress fiber. Areas where 1P-MRLC/EGFP > 2P-MRLC/EGFP are shown in green. Areas where 2P-MRLC/EGFP > 1P-MRLC/EGFP are shown in magenta. Bar, 2 μm.

Localization of NMHC isoforms and phosphorylated myosin II. (A) Expression level of NMHC-IIA, -IIB or -IIC was compared among MDCK II, MTD-1A and EpH4 cells by immunoblotting. The amount of MRLC was used as a loading control. (B) Localization of NMHC-IIA and -IIB in MDCK II cells fixed with methanol and processed for confocal microscopy. At free edges of cells, both isoforms make thick bundles (white arrows). NMHC-IIB accumulates at apical junction regions (arrowheads). Black arrows indicate apical direction along apicobasal axis in vertical sections of cells constructed from serial optical sections. (C) Localizations of 1P-, 2P-myosin II, and F-actin of MDCK II cell fixed with 1% FA and processed for confocal microscopy. Arrows indicate apical direction along apicobasal axis in vertical sections of cells constructed from serial optical sections. Insets indicate magnified views of boxed areas. Bars, 10 and 5 μm (insets).

MRLC dephosphorylation with inhibitors and myosin II organization. (A) Effects of several reagents for cytoskeletons on MRLC phosphorylation. MDCK II cells were treated with each reagent for 30 min at indicated concentration and processed for immunoblotting to detect total, 1P- and 2P-MRLC. (B) MDCK II cells expressing WT-MRLC or DD-MRLC were treated with 30 μM Y27632 for 20 min. Live images before and after treatment show that stress fibers in DD-mutants are resistant for Y27632. AD-mutant showed similar results. (C) MDCK II cells treated with 30 μM Y27632 for 30 min and control cells were fixed with 1% FA, stained for 1P-MRLC, and processed for confocal microscopy. Vertical sections constructed from serial optical sections are shown. Arrowheads indicate stress fibers. Arrows indicate apical direction along apicobasal axis. (D) MDCK II cells treated with 30 μM Y27632 and control cells were processed for immunoblotting to detect total cofilin and phosphorylated cofilin. (E) MDCK II cells treated with 30 μM ML-7 were processed for immunoblotting at indicated time to detect total, 1P-, and 2P-MRLC. The relative amounts of 1P- and 2P-MRLC to total MRLC are shown (n = 3, error bars indicate SD). (F) Effects of ML-7 on myosin II organization in WT- or DD-MRLC–expressing cells. Live images at indicated time after ML-7 treatment are shown. Bars, 10 μm.