Characterization of Blebbistatin Inhibition of Smooth Muscle Myosin and Nonmuscle Myosin-2

Zhang, Hai-Man; Ji, Huan-Hong; Ni, Tong; Ma, Rong-Na; Wang, Aibing; Li, Xiangdong

doi:10.1021/acs.biochem.7b00311

Cited by 26 publications

(25 citation statements)

References 27 publications

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“…, 2016). To examine whether NMMII contraction played a role in AJR expansion during bladder filling, we preincubated bladders with blebbistatin (Bleb; 10 μM), which specifically inhibits the ATPase activity of the myosin heavy chains associated with vertebrate NMMIIA, NMMIIB, and NMMIIC complexes (Zhang et al. , 2017).…”

Section: Resultsmentioning

confidence: 99%

Expansion and contraction of the umbrella cell apical junctional ring in response to bladder filling and voiding

Eaton

Clayton²,

Ruiz³

et al. 2019

MBoC

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The epithelial junctional complex, composed of tight junctions, adherens junctions, desmosomes, and an associated actomyosin cytoskeleton, forms the apical junctional ring (AJR), which must maintain its continuity in the face of external mechanical forces that accompany normal physiological functions. The AJR of umbrella cells, which line the luminal surface of the bladder, expands during bladder filling and contracts upon voiding; however, the mechanisms that drive these events are unknown. Using native umbrella cells as a model, we observed that the umbrella cell’s AJR assumed a nonsarcomeric organization in which filamentous actin and ACTN4 formed unbroken continuous rings, while nonmuscle myosin II (NMMII) formed linear tracts along the actin ring. Expansion of the umbrella cell AJR required formin-dependent actin assembly, but was independent of NMMII ATPase function. AJR expansion also required membrane traffic, RAB13-dependent exocytosis, specifically, but not trafficking events regulated by RAB8A or RAB11A. In contrast, the voiding-induced contraction of the AJR depended on NMMII and actin dynamics, RHOA, and dynamin-dependent endocytosis. Taken together, our studies indicate that a mechanism by which the umbrella cells retain continuity during cyclical changes in volume is the expansion and contraction of their AJR, processes regulated by the actomyosin cytoskeleton and membrane trafficking events.

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Section: Resultsmentioning

confidence: 99%

Expansion and contraction of the umbrella cell apical junctional ring in response to bladder filling and voiding

Eaton

Clayton²,

Ruiz³

et al. 2019

MBoC

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“…Although all three isoforms likely share essential roles in both furrow ingression and abscission, our localization data suggest that IIA might be more specialized for furrow ingression and IIB and IIC are more directly involved in abscission. This notion is supported by the following observations: (1) both the endogenous IIA and IIB localize to the division site during furrow ingression, but IIA disappears from the division site at the midbody stage, whereas IIB remains associated with the midbody and the SOC (Figure 5B); (2) knockdown of IIC by isoform-specific siRNA in human lung tumor cells A549 results in a significant delay at the midbody stage, although these cells eventually underwent abscission (Jana et al., 2006); and (3) IIA translocates actin filaments much faster than IIB or IIC, and the half maximal inhibitory concentration (IC 50 ) of Blebbistatin for IIA is significantly higher than that for IIB or IIC (Zhang et al., 2017).…”

Section: Discussionmentioning

confidence: 99%

Non-muscle Myosin-II Is Required for the Generation of a Constriction Site for Subsequent Abscission

Wang

Wloka

2019

iScience

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Summary It remains unknown when, where, and how the site of abscission is generated during cytokinesis. Here, we show that the sites of constriction, i.e., the sites of future abscission, are initially formed at the ends of the intercellular bridge during early midbody stage, and that these sites are associated with the non-muscle myosin-IIB (not myosin-IIA), actin filaments, and septin 9 until abscission. The ESCRT-III component CHMP4B localizes to the midbody and “spreads” to the site of abscission only during late midbody stage. Strikingly, inhibition of myosin-II motor activity by a low dose of Blebbistatin completely abolishes the formation of the constriction sites, resulting in the localization of all the above-mentioned components to the midbody region. These data strongly suggest that a secondary actomyosin ring provides the primary driving force for the thinning of the intercellular bridge to allow ESCRT-mediated membrane fission.

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“…NM2A and NM2B isoforms are key components of stress fibers as they assemble into minifilaments connecting and moving actin bundles. The ATPase activity of NM2 can be inhibited by the ROCK-specific inhibitor Y-27632 33 , 34 and blebbistatin 35 , 37 – 39 , although with different molecular mechanisms. ROCK inhibition leads to decreased phosphorylation levels of the regulatory MLC, which facilitates the formation of the 10S state 29 .…”

Section: Discussionmentioning

confidence: 99%

“…In this state myosin heads are weakly bound to actin 36 . Blebbistatin inhibits the ATPase and actin filament sliding activities of all stress fiber-forming NM2 isoforms 35 , 37 – 39 . However, adverse properties of blebbistatin have hindered its application in cell biological and in vivo experiments.…”

Section: Introductionmentioning

confidence: 99%

Effect of allosteric inhibition of non-muscle myosin 2 on its intracellular diffusion

Horváth

Gyimesi

Várkuti

et al. 2020

Sci Rep

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Subcellular dynamics of non-muscle myosin 2 (NM2) is crucial for a broad-array of cellular functions. To unveil mechanisms of NM2 pharmacological control, we determined how the dynamics of NM2 diffusion is affected by NM2′s allosteric inhibitors, i.e. blebbistatin derivatives, as compared to Y-27632 inhibiting ROCK, NM2′s upstream regulator. We found that NM2 diffusion is markedly faster in central fibers than in peripheral stress fibers. Y-27632 accelerated NM2 diffusion in both peripheral and central fibers, whereas in peripheral fibers blebbistatin derivatives slightly accelerated NM2 diffusion at low, but markedly slowed it at high inhibitor concentrations. In contrast, rapid NM2 diffusion in central fibers was unaffected by direct NM2 inhibition. Using our optopharmacological tool, Molecular Tattoo, sub-effective concentrations of a photo-crosslinkable blebbistatin derivative were increased to effective levels in a small, irradiated area of peripheral fibers. These findings suggest that direct allosteric inhibition affects the diffusion profile of NM2 in a markedly different manner compared to the disruption of the upstream control of NM2. The pharmacological action of myosin inhibitors is channeled through autonomous molecular processes and might be affected by the load acting on the NM2 proteins. Stress fibers are contractile actomyosin bundles in non-muscle cells, playing fundamental roles in diverse biological functions 1-4. Stress fibers and stress fiber-like structures play central roles in cell division as key components of the cleavage furrow 5 , determine cancer cell motility during metastasis 6 , influence cancer cell growth 7 , and they have been demonstrated as crucial elements in neuronal plasticity including neurite outgrowth 4,8-11. These force-generating structures consist of actin filaments and different co-assemblies of non-muscle myosin 2 (NM2) isoforms 12,13. The two major stress fiber-forming myosin isoforms in human cells are NM2A and NM2B, that together build up mixed minifilaments of various length and thickness 14. Stress fibers may be categorized as central and peripheral, based on their subcellular localization. Central stress fibers include those localized ventrally, dorsally, in transverse arcs and those associated around the nucleus. Their main roles are in cell adhesion, motility, cell division and cell shape determination 15,16. Peripheral stress fibers are located at the edge of the cell. Laser nanosurgery experiments have shown that incision of peripheral stress fibers causes them to retract significantly, suggesting that these structures are under larger mechanical strain than their central counterparts 17. Resisting loads on NM2 heads have been shown to significantly decrease the rate of ADP release from actin-bound myosin heads, resulting in slower actomyosin dissociation 18-20. NM2 minifilaments are also activated or repressed by the phosphorylation or dephosphorylation of the myosin light chains (MLC), respectively 21-24. Rho-dependent kinase (ROCK) positively regulates...

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Characterization of Blebbistatin Inhibition of Smooth Muscle Myosin and Nonmuscle Myosin-2

Cited by 26 publications

References 27 publications

Expansion and contraction of the umbrella cell apical junctional ring in response to bladder filling and voiding

Expansion and contraction of the umbrella cell apical junctional ring in response to bladder filling and voiding

Non-muscle Myosin-II Is Required for the Generation of a Constriction Site for Subsequent Abscission

Effect of allosteric inhibition of non-muscle myosin 2 on its intracellular diffusion

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